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If a cell has two different GPCRs, how does the cell differentiate between the phosphorylation cascade caused by each?

If a cell has two different GPCRs, how does the cell differentiate between the phosphorylation cascade caused by each?


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In my biochem course, we learned that GPCR receptors trigger a phosphorylation cascade, with the end result being a large amplification of the signal in the form of cAMP. We never studied any particular GPCR individually, but we were told that GPCRs always end in the formation of a large amount of cAMP, which will go on to phosphorylate key targets that achieve the end result.

My question is as follows: If there are two GPCRs on the same cell membrane (let's say GPCR-A and GPCR-B triggered by substrates A and B respectively), how does the cell "know" which GPCR triggered the phosphorylation cascade if the end result is the same (large amount of cAMP). In theory, couldn't substrate B bind to GPCR-B and cause a phosphorylation cascade identical to that of GPCR-A, thus triggering GPCR-A's cellular response?

Is there some deeper specialization/uniqueness to each type of GPCR receptor that we didn't cover in my course? Does each GPCR produce a slightly different cascade that ends in something similar, but not exactly cAMP? Or is there some restriction, like each cell limited to only one GPCR (I would find this surprising, if this is the case)?


First, it would be an oversimplification to say that GPCRs act only through increases in cytosolic cAMP. This is true for receptors coupled to Gs proteins, but there are other G proteins like Gi, Go and Gq which act differently [1].

Now, a cell can have receptors coupled to many different G proteins. Vascular smooth muscle, for instance, has Gs, Gi and Gq-coupled receptors [2]. Further, a cell can have different receptors coupling to G proteins of the same type (e.g., hepatocytes have glucagon and beta adrenergic receptors [3], which both couple to Gs).

In such situations, the effects of different hormones add up. The final effect is a function of the total cytosolic concentration of second messenger (e.g., cAMP). Thus glycogen breakdown in a hepatocyte depends on the overall effects of glucagon and epinephrine [3]. The hepatocyte has no way of 'knowing' which cAMP molecule was formed by which hormone.

References

  1. https://en.wikipedia.org/wiki/Heterotrimeric_G_protein

  2. https://cvphysiology.com/Blood%20Pressure/BP026

  3. https://www.nature.com/articles/emm2015122


Chapter Four - Spatiotemporal Modulation of ERK Activation by GPCRs

ERK1/2 (extracellular signal-regulated protein kinases) are the nodal proteins that regulate diverse cellular functions primarily in response to activation from receptor tyrosine kinases (RTKs). Not only is ERK activated through a variety of RTKs, but noncanonical signaling through GPCRs also activates them. Such multimodal activation allows appropriate integration of many inputs to critical cell fate decisions such as proliferation and differentiation that MAP kinases typically regulate. MAP kinases also regulate many polar responses such as apoptosis and proliferation, dedifferentiation–differentiation, and the diversity in the outcomes though the same terminal molecule can be explained based on differences in the activation dynamics and rates. However, two processes have now been established as drivers for most of the diversity recorded in the outcomes of MAP kinase signaling. These parameters are cellular compartmentalization, i.e., spatial confinement of the molecules participating in a pathway and changes in the kinetics of the activation–deactivation, i.e., temporal regulation. While phosphorylation is the key to activating responses, specifically for ERK, the terminal MAP kinase, it is the spatiotemporal dynamics that governs the outcome generated by it. This chapter reviews our present understanding of the spatial and temporal regulation of MAP kinase cascade and the ERK activity, specifically through GPCRs.


MINI REVIEW article

Xinming Wang 1,2 , Abishek Iyer 3 , A. Bruce Lyons 4 , Heinrich Körner 1,5 * † and Wei Wei 1 * †
  • 1 Key Laboratory of Anti-inflammatory and Immune Medicine, Anhui Collaborative Innovation Center of Anti-inflammatory and Immune Medicine, Institute of Clinical Pharmacology, Ministry of Education, Anhui Medical University, Hefei, China
  • 2 Department of Pharmacy, First Affiliated Hospital of Anhui Medical University, Hefei, China
  • 3 Institute for Molecular Bioscience, University of Queensland, Brisbane, QLD, Australia
  • 4 School of Medicine, University of Tasmania, Hobart, TAS, Australia
  • 5 Menzies Institute for Medical Research, University of Tasmania, Hobart, TAS, Australia

Macrophages have emerged as a key component of the innate immune system that emigrates to peripheral tissues during gestation and in the adult organism. Their complex pathway to maturity, their unique plasticity and their various roles as effector and regulatory cells during an immune response have been the focus of intense research. A class of surface molecules, the G-Protein coupled receptors (GPCRs) play important roles in many immune processes. They have drawn attention in regard to these functions and the potential for therapeutic targets that can modulate the response of immune cells in pathologies such as diabetes, atherosclerosis, and chronic inflammatory diseases. Of the more than 800 GPCRs identified, 񾄀 are currently targeted with drugs which have had their activity investigated in vivo. Macrophages express a number of GPCRs which have central roles during cell differentiation and in the regulation of their functions. While some macrophage GPCRs such as chemokine receptors have been studied in great detail, the roles of other receptors of this large family are still not well understood. This review summarizes new insights into macrophage biology, differences of human, and mouse macrophages and gives details of some of the GPCRs expressed by this cell type.


Conclusion

It is proven that fluorescence techniques are powerful tools for investigation of the very dynamic family of GPCRs to understand their subcellular localisation and to further elucidate key elements in GPCR trafficking and interaction with other signal pathways.

However, to obtain physiologically relevant results, some considerations have to be made. First, it is of utmost importance that the investigated GPCR, ligand or interacting protein is not influenced in its functionality by the fluorescent modification. Therefore careful characterizations are needed and to exclude interferences it might be helpful to apply different labels at different sites of the protein for data evaluation [212]. Additionally, the label should be as small as possible, the recent and ongoing development and optimization of self-labeling tags will be advantageous in this field. Using unnatural amino acid mutagenesis the site-specific incorporation of reactive keto groups, such as p-benzoyl-L-phenylalanine (Bzp) or p-acetyl-L-phenylalanine (Acp), into functional GPCRs and their ability to react with a variety of spectroscopic and other probes was previously described [213]. Because of their excellent fluorescent properties quantum dots are very attractive for labeling, however the full potential of QDs for cellular imaging has not yet been realized because of problems with large QD size, QD multivalency and the difficulty of delivering QDs into the cytosol. Recently, monovalent and reduced-size quantum dots were generated and successfully applied for receptor imaging in living cells [214].

Fluorescent antibodies provide a powerful tool for examining the cellular distribution of GPCRs. However, quantification is highly depended on the accessibility of – in most cases – the small epitope by the large antibody. The challenge is to develop even more high-affinity fluorophore- or enzyme-conjugated primary antibodies for one-step labeling assays on living cells. The generation of bright and stable dyes as well as pH sensitive ones, such as CypHer 5 [34], will lead to further insights into the life of GPCRs and will enable high-throughput screening applications. A new group of molecules, called affibody molecules, is especially interesting for imaging applications because of their small size (7–15 kDa) compared to antibodies. These proteins are composed of a three-helix bundle of 58 amino acids and are derived from the scaffold of one of the IgG-binding domains of the staphylococcal protein A [215]. The binding site is equivalent to an antibody with respect to the surface area. The size, the simple structure, the specific target recognition, the ease of production and the high stability give affibody molecules significant advantages over antibodies. These molecules can be labeled with fluorophores but also with radionuclides which make them promising candidates for GPCRs associated tumor diagnosis and therapy [216].

Recombinant DNA technologies have highly advanced fluorescence labeling as well as transfection and transgenic techniques that enable simple DNA delivery to cells that results in covalent labeling by using the protein expression system of the cell. However, expression levels in cell cultures may significantly differ from those in natural systems. Concerning the signaling and trafficking behavior of GPCRs the relationship between the occupation of the receptor by physiological levels of agonists and the initiation of translocation is an important issue. The general use of very high concentrations of agonist leaves open the possibility that the investigated processes are more pharmacological than physiological.

A major criticism of FRET/BRET studies used to investigate protein-protein-interactions, is that the required protein overexpression can result in RET attributed to a high incidence of random collisions, rather than direct protein-protein-interactions. If low expression levels can not be obtained by varying DNA amounts within transient cell transfections, stable cell transfections will provide an alternative, since there is a homogenous population of cells expressing the protein of interest at the same level. Another possibility is the baculovirus expression system which enables protein expression levels to be controlled more closely than with transient transfection, because protein expression can be titrated by adjusting the multiplicity of viral infection [217].

Since protein co-localization is the first prerequisite for interactions, this should be proven by fluorescence microscopy, and by using parallel labeling strategies to locate subcellularly the interaction of interest. For correct evaluation of FRET and BRET data appropriate controls have to be used to demonstrate the specificity of the interactions and to establish levels of RET considered to be background in any given experiment. The additional application of a biochemical approach might support the results. To validate the physiological role of the detected interaction studies in other, more natural cell systems, e.g. cell lines endogenously expressing one protein of interest, as well as investigations on tissues and animals will be indispensable in proving the relevance of the interactions in the future. For example, the in vivo co-expression of GPCRs has to be demonstrated in the same tissue, and ideally in the same cell for establishing the physiological relevance of receptor oligomerization. Functional cross-talk between the receptor signaling pathways as well as novel pharmacological and/or functional properties will provide evidence for the mechanism by which receptor-receptor-interactions modulate cellular activity [218].

An exciting application of GPCR-GFP chimeras involves their use in genetic screens in genetically tractable organisms such as yeast, e.g. to identify mutant yeast strains in which the receptor is mis-localized. Such strategies contribute greatly to the identification of new components involved in GPCR targeting and trafficking in additional model organisms [219]. New approaches using whole organisms, in which the GFP-chimera can be expressed under the control of the endogenous promoter, e.g. invertebrates as C. elegans or mouse models, allow cell biological, molecular and biochemical results to be interpreted in a physiologically relevant context and to be compared to those observed in cultured cells [220, 221]. GFP and its variants as reporters represent the next step in mouse genome engineering technology by opening up the possibility of combinatorial non-invasive reporter usage within a single animal, e.g. for gene-expression, as well as for co-visualization and FRET assays [222].

In summary, many issues concerning the life of GPCRs can be addressed by fluorescence techniques, however many remain challenging. Further rapid advances in labeling and imaging technology can be expected and their parallel as well as their combined application will provide novel insights that will also broaden the range of new therapeutic interventions.


GPCR oligomers: static or dynamic interactions?

The family C GPCRs, which include the metabotropic glutamate receptors (mGluR1–mGluR5), the calcium-sensing receptor (CaSR) and the γ-aminobutyric acid receptors (GABABR), among others, differentiate themselves from the other GPCR families by large bilobed N-terminal extracellular domains known as the venus flytrap domains (Pin et al., 2003 Pin et al., 2005). These receptors associate with themselves and function as constitutive dimers. In many cases, receptor dimers are stabilized by disulfide bridges between Cys residues located in the venus flytrap domains (Romano et al., 1996 Ray et al., 1999 Ray and Hauschild, 2000). Most oliogomers of class A GPCRs, however, are formed through non-covalent interactions, and recent fluorescence recovery after photobleaching (FRAP) studies support the existence of a dynamic equilibrium between monomer and homodimer states of class A GPCRs, such as the dopamine D2 receptor (D2R) and the β1-AR (Dorsch et al., 2009 Fonseca and Lambert, 2009). The dynamics of a class A GPCR dimer has been further detailed by total internal reflection fluorescence (TIRF) microscopy, revealing that

30% of recombinant muscarinic acetylcholine M1 receptor (M1R) expressed in CHO cells form transient homodimers with a life-time as short as 0.5 seconds (Hern et al., 2010). These studies suggest that at least some class A GPCRs, such as M1R, β1-AR and D2R, can transiently associate with each other, with half-lives presumably long enough to permit G-protein activation.

General principle of the GPCR signaling system. (A) Molecular representation of a GPCR in complex with a Gαβγ, based on crystal structures of rhodopsin (red coordinates from PDB code 1GZM) and the inactive heterotrimeric Gi protein (from PDB 1GG2). (B) Following ligand binding, the receptor undergoes conformational changes, which promote the coupling with heterotrimeric G proteins (Gαβγ), and catalyzes the exchange of GDP for GTP on the α-subunit. This event triggers conformational and/or dissociation events between the α-subunit and βγ-subunit. GαS activates adenylyl cyclases, leading to cAMP synthesis, which in turn activates protein kinase A (PKA). Gαq activates phospholipase C, which cleaves phosphatidylinositol (4,5)-bisphosphate (PIP2) into diacylglycerol (DAG) and inositol (1,4,5)-trisphosphate (IP3). IP3 then diffuses through the cytosol and activates IP3-gated Ca 2+ channels in the membranes of the endoplasmic reticulum, causing the release of stored Ca 2+ into the cytosol. The increase of cytosolic Ca 2+ promotes PKC translocation to the plasma membrane, and then activation by DAG. Activation of Gi blocks adenylyl-cyclase-mediated cAMP synthesis by its α-subunits, whereas Gβγ-mediated signaling processes such as activation of G-protein-regulated inwardly rectifying potassium (GIRK) channels. VSCC, voltage-sensitive Ca 2+ channel.

General principle of the GPCR signaling system. (A) Molecular representation of a GPCR in complex with a Gαβγ, based on crystal structures of rhodopsin (red coordinates from PDB code 1GZM) and the inactive heterotrimeric Gi protein (from PDB 1GG2). (B) Following ligand binding, the receptor undergoes conformational changes, which promote the coupling with heterotrimeric G proteins (Gαβγ), and catalyzes the exchange of GDP for GTP on the α-subunit. This event triggers conformational and/or dissociation events between the α-subunit and βγ-subunit. GαS activates adenylyl cyclases, leading to cAMP synthesis, which in turn activates protein kinase A (PKA). Gαq activates phospholipase C, which cleaves phosphatidylinositol (4,5)-bisphosphate (PIP2) into diacylglycerol (DAG) and inositol (1,4,5)-trisphosphate (IP3). IP3 then diffuses through the cytosol and activates IP3-gated Ca 2+ channels in the membranes of the endoplasmic reticulum, causing the release of stored Ca 2+ into the cytosol. The increase of cytosolic Ca 2+ promotes PKC translocation to the plasma membrane, and then activation by DAG. Activation of Gi blocks adenylyl-cyclase-mediated cAMP synthesis by its α-subunits, whereas Gβγ-mediated signaling processes such as activation of G-protein-regulated inwardly rectifying potassium (GIRK) channels. VSCC, voltage-sensitive Ca 2+ channel.

Modulation of cell signaling by fast inter-conformational switches

During the past decade, studies recently reviewed by Rozenfeld and Devi (Rozenfeld and Devi, 2010) revealed that receptor heterodimerization modulates pharmacological, signaling and trafficking properties of individual parent receptors. Examples of such fascinating aspects of receptor heteromerization are found in the different combinations between the taste receptors T1R1, TIR2 and TIR3, which modulate the sensitivity of taste molecules – the T1R1–T1R3 heterodimer mediates the glutamate umami taste, whereas the T1R2–T1R3 heterodimer mediates the sweet taste (Nelson et al., 2001 Zhao et al., 2003 Mueller et al., 2005). Receptor heterodimers between the adenosine A2A and dopamine D2 receptors is another attractive example of how receptor heterodimerization can modulate receptor function. This receptor dimer is expressed in striatopallidal GABAergic neurons that are present in the basal ganglia, a region of the brain that is involved in sensory–motor integration (Canals et al., 2003). Adenosine inhibits dopamine-induced locomotor activity, and by exerting opposite effects in the basal ganglia, the A2A–D2 receptor heteromer contributes to the fine-tuning of neural activity. At the biochemical level, the activation of A2A receptors by adenosine or adenosine analogs affects the pharmacology of D2 receptors by modulating the binding characteristics of dopamine agonists, and counteracts D2-receptor-mediated intracellular responses (Agnati et al., 2003). The binding of ligand to one receptor altering the activation of the partner receptor in the heterodimer complex led to the hypothesis that conformational changes induced by ligand binding to one receptor might be transmitted to the receptor partner to modulate its function (Zoli et al., 1993).

Kinetics of early reactions in the signaling cascade of GPCRs. Time constants (τ) of (A) ligand (L) association, (B) receptor (R) activation (R*), (C) receptor–G-protein association, and (D) G protein (G) activation (G*) are shown for two distinct GPCR systems: the parathyroid hormone receptor (PTHR) in response to the major endocrine regulator of Ca 2+ homeostasis, PTH, and α2A-AR in response to the principal neurotransmitter of sympathetic nerves, norepinephrine.

Kinetics of early reactions in the signaling cascade of GPCRs. Time constants (τ) of (A) ligand (L) association, (B) receptor (R) activation (R*), (C) receptor–G-protein association, and (D) G protein (G) activation (G*) are shown for two distinct GPCR systems: the parathyroid hormone receptor (PTHR) in response to the major endocrine regulator of Ca 2+ homeostasis, PTH, and α2A-AR in response to the principal neurotransmitter of sympathetic nerves, norepinephrine.

Modulation of G-protein signaling by receptor heterodimers. Representation of the α2A-AR (red)–MOR (blue) heterodimer complex based on the crystal structure of rhodopsin (coordinates from PDB code 1GZM), and chemical structures of norepinephrine and morphine. G-protein signaling mediated by the receptor heterodimer in response to norepinephrine or morphine is modulated by the simultaneous action of both signaling ligands. This modulation proceeds through conformational changes (arrows) that propagate from one receptor to the other with a half-life of 400 mseconds.

Modulation of G-protein signaling by receptor heterodimers. Representation of the α2A-AR (red)–MOR (blue) heterodimer complex based on the crystal structure of rhodopsin (coordinates from PDB code 1GZM), and chemical structures of norepinephrine and morphine. G-protein signaling mediated by the receptor heterodimer in response to norepinephrine or morphine is modulated by the simultaneous action of both signaling ligands. This modulation proceeds through conformational changes (arrows) that propagate from one receptor to the other with a half-life of 400 mseconds.

We confirmed this hypothesis with FRET studies in living cells, by showing that allosteric interactions between the α2A-AR and μ-opioid receptor (MOR) are mediated by direct cross-conformational changes between these receptors (Vilardaga et al., 2008). Norepinephrine and morphine, which are the native ligands for the α2A-AR and MOR, respectively, are well known to stimulate common inhibitory G protein (Gi)-mediated signaling pathways through their respective receptors, but the simultaneous action of norepinephrine and morphine produces a cellular response different from the expected additive effects (Jordan et al., 2003). In this case, morphine binding to the MOR triggers a rapid conformational change in the norepinephrine (NE)-bound α2A-AR that proceeds with a t1/2=400 mseconds, which is faster than the kinetics for G-protein activation (Fig. 3). This fast trans-conformational switch between receptors decreases both activation of Gi signaling and stimulation of MAP kinase phosphorylation. A trans-conformational switch in the reverse direction, from the α2A-AR towards the MOR, was not directly observed because FRET-based MOR biosensors are not functional. The inhibition of morphine-mediated G-protein activation by NE suggests, however, that such a conformational transfer also occurs. Thus, conformational crosstalk between receptors is bidirectional, and rapidly prevents overstimulation of signaling pathways by a combination of ligands acting on a receptor heteromer, by rapidly adjusting the extent of G-protein activation.

A fundamental property of ligands acting at a given GPCR is their capacity to stabilize different active receptor conformations that can each activate a unique signaling pathway. The term ‘functional selectivity’ was introduced as a parameter to express the ability of a ligand to produce a selective response (Kenakin and Miller, 2010). Functional selectivity in agonism is also linked with the GPCR heterodimer. For example, the Gi-coupled cannabinoid receptor (CB1R) forms a heterodimer complex with the GS-coupled A2AR and signals to Gi only when CB1 and A2A agonists are combined (Carriba et al., 2007). These findings suggest that the receptor–receptor interaction constrains the capacity of the CB1R to adopt an active conformation that can stimulate Gi activation in the CB1R–D2R heterodimer, and that this constraint is released only by the action of an A2A agonist. This release might be accomplished by means of inter-conformational switches between these two receptors. This hypothesis needs to be tested, but would indicate that functional selectivity at GPCR heteromers is modulated directly at the level of receptors through conformational crosstalk.


G protein–coupled receptors (GPCRs) play an integral role in the signal transduction of an enormous array of biological phenomena, thereby serving to modulate at a molecular level almost all components of human biology. This role is nowhere more evident than in cardiovascular biology, where GPCRs regulate such core measures of cardiovascular function as heart rate, contractility, and vascular tone. GPCR/ligand interaction initiates signal transduction cascades, and requires the presence of the receptor at the plasma membrane. Plasma membrane localization is in turn a function of the delivery of a receptor to and removal from the cell surface, a concept defined most broadly as receptor trafficking. This review illuminates our current view of GPCR trafficking, particularly within the cardiovascular system, as well as highlights the recent and provocative finding that components of the GPCR trafficking machinery can facilitate GPCR signaling independent of G protein activation.

G protein–coupled receptors (GPCRs) are central mediators of nearly all aspects of cardiovascular biology. GPCRs were originally identified as receptors capable of coupling to specific guanine nucleotide-binding proteins (G proteins), thereby transducing an extracellular signal to an intracellular effector, although more recently, several GPCRs have been demonstrated to signal via G protein–independent mechanisms both in vitro and in vivo. 1 As a family of proteins, GPCRs share common structural features, including seven membrane-spanning domains, and thus are alternatively referred to as 7-transmembrane receptors. GPCRs are the largest superfamily of cell-surface receptors, accounting for approximately 2% of the human genome. 2 Further, ligands directed at GPCRs (primarily agonists and antagonists) represent the largest family of pharmacological agents, accounting for nearly 30% of current clinical pharmaceutical agents available. 3 Both hormones and neurotransmitters exert their effects on the cardiovascular system via GPCRs. Examples of GPCRs with well-ascribed roles in cardiovascular biology include the β1- and β2-adrenergic receptors (ARs), the α1- and α2-ARs, the M2- and M3-muscarinic acetylcholine receptors, the angiotensin II (Ang II) receptors, the endothelin receptors, the adenosine receptor, the thrombin receptor, and the vasopressin receptor.

Over the past nearly 3 decades, a wealth of information has revealed much about the signaling properties of this family of seven membrane-spanning receptors. Much work has focused on revealing the ways in which the GPCRs regulate discrete effector molecules including adenylyl cyclase, phospholipases, and ion channels. Still further work has shed light on mechanisms by which GPCR signaling is regulated and has led to the discovery of additional proteins including the GPCR kinases (GRKs) 4,5 and β-arrestin proteins, 6,7 which respectively phosphorylate agonist-activated GPCRs and bind phosphorylated GPCRs to physically disrupt the receptor/G protein interaction, thereby leading to desensitization of receptor-mediated G protein activation. In addition to its role in GPCR desensitization, β-arrestin binding also promotes the cytosol to cell surface translocation of components of the endocytic machinery, namely adaptor protein-2 (AP-2) 8 and clathrin, 9 thereby facilitating receptor removal from the plasma membrane.

Although still substantial, comparatively less work has focused on GPCR trafficking, much of it related to mechanisms regulating endocytosis of GPCRs from the cellular surface, including the role of β-arrestin in facilitating GPCR endocytosis as above. Indeed, the appropriate delivery of GPCRs to the cell surface to permit receptor/ligand interactions, and their subsequent retrieval from the plasma membrane, are of fundamental importance for the regulation of GPCR activity. This review highlights our current understanding of GPCR movement from synthesis onward, with special emphasis on studies of GPCRs from the cardiovascular system. Lastly, we discuss the importance of the newly recognized role that GPCR trafficking itself can have on cellular signaling, including the recently recognized and expanding role for β-arrestins in GPCR signaling independent of G proteins.

GPCR Trafficking: Posttranslation

Both during and subsequent to synthesis, membrane proteins including GPCRs undergo a continual process of maturation before reaching residence at the plasma membrane. They must be properly inserted into the membrane (a process believed to occur cotranslationally for most membrane proteins), achieve proper folding while still resident in the endoplasmic reticulum, traverse from the cis- to the trans-Golgi while undergoing modification, and finally be targeted to the plasma membrane where they attain residence as mature proteins. This section will discuss various aspects of this maturational process that have been determined for GPCRs (Figure, A).

General model of GPCR trafficking. A, Following synthesis, GPCRs initially reside in the ER, where they undergo processing and folding guided by chaperone and quality-control proteins. Within the ER, many GPCRs likely form either homo- or heterodimeric structures. Following ER exit, GPCRs transit through the Golgi apparatus, where they may undergo additional modifications such as oligosaccharide processing. On the distal edge of the Golgi, GPCRs are packaged in exocytic transport vesicles and enter the endosomal system, where they are subsequently targeted to the plasma membrane. Multiple proteins, as listed, have been identified that affect GPCR stability at the cell surface. ERAD indicates ER-associated degradation. B, Although variations have been described, GPCR endocytosis from the plasma membrane most commonly occurs in a GRK- and β-arrestin–dependent manner. Ligand binding promotes GRK-mediated phosphorylation of the cytoplasmic surface of GPCR and subsequent β-arrestin translocation and binding to the receptor. β-Arrestin binding, in turn, facilitates the subsequent recruitment of AP-2 and clathrin and GPCR inclusion in CCPs before endocytosis via CCVs. C, Following endocytosis, GPCRs may be either recycled to the plasma membrane or sorted for lysosomal degradation. The Rab family of small GTPases is integral in determining the fate of a GPCR, whereas SNX1 has more recently been shown to play a role in endosomal to lysosomal GPCR sorting. Receptor ubiquitination also plays a role in receptor degradation via lysosomes. D, More recently, several GPCRs have been shown to be capable of signaling via β-arrestin–dependent pathways. Well-characterized β-arrestin–dependent signaling cascades that have been described include agonist-dependent nonreceptor tyrosine kinase activation as well as activation of the MAPK signaling pathway.

Folding and Chaperones

Strict quality-control mechanisms within cells ensure that improperly or incompletely folded proteins are targeted for degradation, usually via the proteasome pathway. For most nascent proteins studied, folding into a proper/functional conformation requires the presence of endogenous accessory chaperone proteins. 10 GPCRs are no exception to this quality-control process. As example, the human DnaJ protein HSJ1b, a member of the heat shock protein (HSP) family of cytoplasmic cochaperones, regulates trafficking of rhodopsin from the endoplasmic reticulum (ER) to the cell surface. 11 Alternatively, single-membrane–spanning chaperone proteins can facilitate GPCR exit from the ER, as recently demonstrated for the calcitonin receptor-like receptor, which must form a heterodimeric complex with the receptor activity modifying protein (RAMP) before ER egress (reviewed by Tan et al 12 ). Whether these or similar endogenous chaperone proteins regulate the folding of GPCRs important to some aspects of cardiovascular biology is currently unknown, but certainly possible given the structural conservation across this large protein family.

Despite such checks to ensure proper protein folding errors do, however, occur. As such, a variety of human diseases have been identified in which naturally occurring mutations result in the misfolding and/or mistargeting of a mutant protein. Such “protein conformational” diseases are thus considered to result from mutations that do not affect the functional domain(s) of the mutant protein, but rather interfere with the normal cellular trafficking of the protein (reviewed by Bernier et al 13 ). Such misfolded proteins typically either form aggregates that are deleterious to the cell or are recognized as improperly folded and therefore targeted for degradation by the cellular quality-control mechanisms noted above (reviewed by Sitia and Braakman 14 ). Interestingly, recent work has demonstrated that in some instances, chemical or pharmacological manipulation can rescue misfolded proteins and lead to their proper translocation to the plasma membrane where the proteins are functionally active.

Nephrogenic diabetes insipidus (NDI) is an X-linked disorder 15 with an incidence in the population of approximately 1 per 250 000. NDI is characterized by renal resistance to the posterior pituitary-derived antidiuretic hormone (also called arginine vasopressin), an octapeptide which normally acts at the vasopressin 2 receptor (V2R) present on renal epithelial cells to allow for normal urinary concentration. 15 In patients with NDI and the complete absence of renal epithelial V2R cell surface expression, daily urinary volume can exceed 15 L and lead to rapid death. More than 150 different mutations in the V2R have been described, the majority of which (≈70%) impair V2R cell surface trafficking (reviewed by Bernier et al 13 ). The addition of a cell-permeable V2R antagonist to a subset of mutant V2Rs previously shown to accumulate in the ER resulted in the proper folding, ER exit, correct targeting to the cell surface, and functional rescue of receptor activity of the mutant proteins. 16 This is presumably attributable to the ability of the small molecule ligand to stabilize the native state of the protein, thereby facilitating the proper trafficking of the receptor. Additional mutant 7-transmembrane receptors known to have altered trafficking properties that result in human disease, and for which molecular chaperones have been identified, include rhodopsin, the sulfonylurea receptor 1 (SUR1), smoothened, and the gonadotropin-releasing hormone receptor (reviewed by Bernier et al 13 ). It remains to be seen, however, whether pharmacological treatment of the mistrafficked receptor targets identified above in cellular systems will translate to amelioration of human disease.

Oligomerization

Early studies using rhodopsin, 17,18 muscarinic, 19 and β-adrenergic 20 receptors as model GPCRs suggested that GPCRs exist primarily as monomers, although modification of the detergent extraction systems used for protein purification led early investigators to suggest that a varying fraction of GPCRs may also be present in oligomeric form. 19,20 Much recent work using coimmunoprecipitation and resonance energy-transfer techniques have convincingly demonstrated that dimeric GPCR structures are present at the plasma membrane and are the topic of several recent reviews. 21–24 Indeed, it has been postulated that homo- and heterodimeric GPCRs may represent the basic functional unit necessary for most, if not all, GPCR signaling (reviewed by Park et al 25 ). Whether higher-order oligomeric complexes can form at the plasma membrane has not, however, been clearly demonstrated, although the M2-muscarinic receptor has been suggested to be capable of forming a trimer. 26

Not well appreciated nor understood, however, is the likely important role that oligomerization of GPCRs plays in the biosynthesis and trafficking of nascent GPCRs to the cellular surface. Indeed, multiple GPCRs including the β2-AR 27 and vasopressin receptors 28 undergo constitutive homodimerization early in the biosynthetic pathway, likely occurring in the ER. Expression of mutant β2-ARs constructed to either lack an ER-export motif or to contain a heterologous ER-retention signal led to entrapment of wild-type β2-AR in the ER, likely because of receptor dimerization. 27 Importantly, addition of a peptide corresponding to the putative glycophorin-like dimerization motif in the sixth-transmembrane domain of the β2-AR inhibited both receptor dimerization and transit to the cell surface. 29 Similar results in which mutants of the V2R 30 or the D2 dopamine receptor 31 act as dominant negatives for plasma membrane expression of their respective wild-type receptors suggest that receptor oligomerization before cell surface delivery may be a general mechanism by which multiple members of the GPCR family are regulated.

In addition to homodimerization, early heterodimerization is also like to play an important role in the proper targeting of some GPCRs, as recently demonstrated for the α1D-AR, which required heterodimerization with the closely related α1B-AR for cell surface expression. 32 Immunoprecipitation studies in which epitope-tagged β2-AR and Ang II type 1 (AT1) receptors (AT1Rs) were coexpressed suggested that the receptors are able to form oligomers before their localization on the plasma membrane, as the amount of immunoprecipitated receptor complex was unaffected by exposure to either agonist or antagonist. 33 Further studies have demonstrated the unanticipated finding that heterodimerization between the β2-AR and either an olfactory receptor 34 or the α1D-AR 35 facilitates receptor ER export and cell surface expression. Finally, expression of the β2-AR along with the δ and κ opioid receptors in cultured cells leads to heterooligomerization of the β2-AR with either the δ or κ opioid receptor at the plasma membrane. 36 Interestingly, this association did not affect ligand binding or functional properties of the receptors but did alter the trafficking properties. In the δ-β2 cells, δ receptors underwent β2-AR agonist-stimulated internalization and β2-AR underwent opioid-mediated endocytosis, whereas in κ-β2-AR cells, the β2-AR did not internalize in response to either β2-AR agonist or opioid. 36 Although this is a single example, such results suggest that GPCR heterooligomerization may be an important way of modulating GPCR trafficking and signaling. It is important to note, however, that in each oligomerization study described above, overexpression of the receptor(s) of interest was performed and that such alterations in cellular receptor content may modify the endogenous molecular interactions that occur in the absence of receptor overexpression.

Thus, in addition to the important role that endogenous molecular chaperones such as the HSP and RAMP family proteins play in protein folding and ER export of GPCRs, homo- and heteroreceptor oligomerization also likely play a critical step in the pathway used by at least some GPCRs for cellular trafficking, although it is at present unclear whether oligomerization following protein synthesis is a general pathway used by all GPCRs.

Cell Surface Stability

In order for a GPCR to transduce an extracellular signal, it must both traffic correctly to and be retained at the cellular surface to allow for receptor/ligand interaction. Multiple proteins not directly involved in the signal transduction cascade have been identified which stabilize receptor surface expression. These include spinophilin, Homer, actin-binding protein 280/filamin A, protein 4.1N, muskelin, and postsynaptic density-95 (PSD-95) (reviewed by Tan et al 12 ). Of these, PSD-95, a multiple PDZ domain–containing scaffolding protein, has been most conclusively shown to specifically interact with a GPCR fundamental to cardiovascular biology, namely the β1-AR. This interaction occurs via the third PDZ domain of PSD-95, which interacts with the carboxyl terminus of the β1-AR. Interestingly, overexpression of PSD-95 decreased β1-AR internalization but did not affect agonist-stimulated cAMP production or receptor desensitization, suggesting a role for PSD-95 in maintaining the β1-AR at the cellular surface. 37

GPCR Trafficking: Endocytosis

Much work over the past several decades has illuminated our current understanding of the molecular mechanisms underlying GPCR removal from the cell surface. Fundamental to this process are 2 families of proteins, the GPCR kinases (GRKs) and the β-arrestins, both of which were initially identified in studies of GPCR desensitization and which are involved in removal of ligand-activated GPCRs from the plasma membrane. Additional work has identified an ensemble of accessory proteins, which interact with the GRK and β-arrestin classes of proteins, and much recent effort has been devoted to delineating the details of these multiple interactions that are inherent to the process of GPCR endocytosis. Furthermore, as will be described below, the agonist-induced posttranslational ubiquitination of both receptor and β-arrestin play definitive and discrete roles in regulating the life cycle of GPCRs (Figure, B).

Role of the Lipid Microenvironment in GPCR Trafficking

Understanding of the importance of the membrane lipid microenvironment for GPCR signaling and trafficking is rapidly evolving. As example, it has recently been demonstrated that following translation, the AT1R requires caveolin as an intracellular molecular chaperone for trafficking to the plasma membrane. 38 Moreover, once at the cell surface, it is clear that some subsets of GPCRs are preferentially segregated to discrete regions of the membrane defined as lipid rafts. 39–41 GPCRs of fundamental importance to cardiovascular biology that have been localized to lipid rafts and/or caveolae include the adenosine A1, α1-AR, β1-AR, β2-AR, AT1R, the endothelin (ETA-A and ET-B) receptors, and the M2-muscarinic receptors. 42

Caveolae, a specific type of lipid microdomain, represent for some GPCRs the preferred microenvironment for certain events such as signaling. However, a receptor’s maintenance within a specific microenvironment may be subject to dynamic regulation. Indeed, reversible GPCR modifications have been described, including both covalent attachment of a lipid to the GPCR or GPCR phosphorylation, which can shift the GPCR between different membrane milieus. For example, a reversible lipid modification (eg, palmitoylation/depalmitoylation of cysteine residues) has been demonstrated to target GPCRs such as the 5-HT1a receptor to lipid rafts. 43 Interestingly, agonist-induced endocytosis of the β1-AR via clathrin-coated pits (CCPs) in human embryonic kidney (HEK) 293 cells requires GRK phosphorylation of the receptor, whereas endocytosis of the β1-AR in lipid rafts/caveolae is dependent on the receptor undergoing protein kinase A phosphorylation. 44 Further evidence supporting the importance of GPCR membrane microdomain restriction is that confinement of the β2-AR to caveolae has been reported to be of critical importance for regulation of the intrinsic contraction rate in neonatal cardiac myocyte membrane preparations. 45 The importance of the lipid microenvironment for the assemblage of signaling scaffolds beneath the GPCR/membrane interface is also an area of active investigation and may play a role in multiple aspects of GPCR trafficking but is beyond the scope of this review. For a more complete review of the role of the lipid microenvironment on both GPCR signaling and trafficking, refer to several recent excellent reviews. 39,42

Agonist-Dependent Versus Agonist-Independent GPCR Internalization

Receptor internalization following agonist exposure is a well-documented response for a wide variety of GPCRs important for cardiovascular biology. As example, the prototypic GPCR, the β2-AR, was initially shown to internalize following exposure to agonist, as demonstrated by loss of surface binding of a nonpermeable membrane ligand. 46 Use of a membrane permeant ligand, however, demonstrated that the β2-AR was still ligand accessible, suggesting that the receptor was sequestered in an intracellular compartment following agonist treatment. 46 Alternatively, whereas exposure of the AT1R to Ang II leads to receptor internalization and endosomal sequestration, the Ang II type 2 receptor (AT2R) does not undergo endocytosis with Ang II addition, demonstrating that subtype-specific receptor sorting and internalization can occur within the cardiovascular GPCR system. 47

Internalization for most GPCRs occurs on the order of minutes and correlates with receptor phosphorylation by the GRKs and subsequent β-arrestin translocation, as will be discussed below. Indeed, agonist-induced β2-AR receptor internalization can be inhibited either by mutations of the β2-AR, which inhibit agonist-induced GRK phosphorylation 48 or by mutations in the β-arrestin proteins. 49 Following internalization, receptors may be either recycled to the cell surface or targeted for lysosomal degradation (reviewed by Bohm et al 50 ).

Internalization of GPCRs in the absence of agonist has also been examined. Although mean rates of internalization vary between receptors assayed, rates are in general substantially slowed in the absence relative to the presence of the cognate ligand of a GPCR. The β2-AR, as example, undergoes sequestration from the cell surface with a half-life of approximately 10 minutes in the presence of agonist but remains on the cell surface for greater than 1 hour in the absence of agonist. 51

The Role of Accessory Proteins in the Endocytosis of GPCRs

Endocytosis of GPCRs can occur via caveolae, clathrin-coated vesicles, or uncoated vesicles. 52 Although short linear amino acid stretches in the cytoplasmic domains of GPCRs likely play a role in their endocytosis, the majority of work to date has demonstrated that much of GPCR endocytosis is primarily regulated by GRK and β-arrestin–dependent processes involving clathrin-coated pits.

GPCR Kinases

As shown for multiple GPCRs, the serine/threonine-specific GPCR kinases (GRKs) are recruited following agonist binding to the cytoplasmic surface of the activated receptor, leading to receptor phosphorylation. The phosphorylated surface of the GPCR is then competent to serve as a platform for the cytosol to membrane translocation of the β-arrestin proteins (reviewed by Shenoy and Lefkowitz 53 ).

The GRK family of kinases is composed of 7 members that share significant amino acid and structural homology (reviewed previously 54,55 ). Within this family, 4 kinases (GRKs 2, 3, 5, and 6) are expressed broadly and are believed to play a role in GPCR phosphorylation within the cardiovascular system. GRK2 and GRK3 reside in the cytosol in the absence of agonist and translocate to the membrane following GPCR stimulation. GRK2/3 translocation and membrane localization are mediated in part by their binding to heterotrimeric G protein βγ subunits. 56 GRK5 and GRK6, on the other hand, are constitutively localized to the plasma membrane. Whereas GRK6 palmitoylation is essential for membrane association, 57 localization of GRK5 to the plasma membrane is believed to be attributable to an electrostatic interaction between the highly basic carboxyl terminus of GRK5 and phospholipids at the plasma membrane. 58,59

Although GRK-specific phosphorylation of the cytoplasmic surface of agonist-occupied GPCRs mediates β-arrestin recruitment, the structural features common to activated receptors that are recognized by the GRKs remain largely unknown. Indeed, it is a question of fundamental importance as to how members of this limited group of broadly expressed GRKs are able to phosphorylate such a diverse array of activated GPCRs and thereby lead to β-arrestin recruitment.

Functionally, 2 classes of GPCRs, denoted “class A” and “class B,” can be defined, based on the relative stability of the GPCR/β-arrestin interaction. For the β2-AR (a class A receptor) and the vasopressin receptor (a class B receptor), these determinants appear to be present within the carboxyl termini of the receptors, as the stability of their interaction with β-arrestin, as well as their ability to be dephosphorylated, recycled, and resensitized was completely reversed in mutant receptors in which their carboxyl-terminal tails were switched. 60

Interestingly, whereas in vitro studies have localized GRK2- and GRK5-mediated phosphorylation sites of the β2-AR to distal portion of the cytoplasmic tail of the receptor, 61 more recent studies in intact cells have suggested that agonist induced β2-AR phosphorylation occurs in the proximal portion of the carboxyl terminus of the receptor. 62 Although the observed β2-AR proximal tail phosphorylation was believed to be mediated by GRK rather than protein kinase A, this was not confirmed. Thus although GRK-mediated phosphorylation of agonist-stimulated GPCRs underlies β-arrestin recruitment and thereby initiates GPCR endocytosis in CCPs, many of the molecular details remain to be determined. Notably, a recent study using high-throughput RNA interference implicated GRKs as playing a more general role in the process of clathrin-mediated endocytosis itself. 63

Β-Arrestin As an Endocytic Adaptor Protein

Within humans, there exist 2 isoforms of the nonvisual β-arrestin proteins, namely β-arrestin1 and β-arrestin2, both of which show ubiquitous tissue distribution. In addition to their well-described role in limiting receptor-G protein interaction, the β-arrestin proteins also serve to both recruit and physically bridge the receptor to the endocytic machinery. Experimentally, receptor mutations that impair agonist-induced GPCR phosphorylation limit β-arrestin recruitment and lead to poor receptor internalization, as demonstrated for a β2-AR in which all of the GRK phosphorylation sites had been altered. 48 Further, expression of “dominant-negative” mutant β-arrestin proteins (such as β-arrestin1 V53D or β-arrestin2 V54D) inhibit β2-AR internalization. 64 In addition, the β-arrestin proteins themselves are able to interact directly with the essential components of the clathrin-coated vesicle (CCV) coat machinery, namely the heterotetrameric AP-2 complex, 8 as well as clathrin, 9 and these interactions are critical both for recruitment of the β2-AR into clathrin-coated pits as well as for receptor internalization. Studies with other GPCRs, including the α2-AR 65 and the A2B adenosine receptor 66 have also shown important roles for the β-arrestins in receptor endocytosis.

Interestingly, although the 2 β-arrestin isoforms exhibit nearly 80% amino acid identity, 6 they do not appear to perform redundant biologic roles and, indeed, exhibit differences in their regulation. Whereas β-arrestin1 is phosphorylated by extracellular signal regulated kinase (ERK) enzymes, 67 β-arrestin2 is phosphorylated by casein kinase II. 68 For both β-arrestin proteins, however, the phosphorylation/dephosphorylation status appears to regulate the ability of the β-arrestin protein to promote internalization of the β2-AR via clathrin-coated vesicles.

As noted above, analysis of agonist-stimulated β-arrestin translocation for a variety of GPCRs suggests there exist 2 largely distinct classes receptors with which the β-arrestins associate, denoted class A and class B GPCRs. Following agonist exposure, class A receptors including the β2-AR, endothelin A receptor, and α1b-AR preferentially recruit β-arrestin2. 69 In contrast, class B receptors, including the AT1aR and V2 receptor, are able to bind both β-arrestin isoforms with nearly equal affinity. 70 Whereas the β-arrestin1/2–class A receptor interactions occur solely at the plasma membrane and are lost following GPCR internalization, β-arrestin1/2 interactions with class B receptors are much more stable and can be detected on endosomal structures following receptor endocytosis (reviewed by Pierce and Lefkowitz 71 ). Further, class A receptors generally recycle to the plasma membrane rapidly, whereas class B GPCRs recycle more slowly. The role of ubiquitination in modulating GPCR/arrestin interaction is likely important and is discussed below. Mouse embryonic fibroblasts generated to lack both β-arrestin isoforms showed a marked impairment of agonist-stimulated internalization of either the class A β2-AR or the class B AT1aR, whereas only β2-AR internalization was affected by the single deletion of the β-arrestin2 isoform. 69 Studies using RNA interference technology to selectively ablate the β-arrestin proteins have shown similar results with respect to internalization of the β2-AR and AT1-AR as model class A and B receptors, respectively. 72

Agonist-Induced GPCR Ubiquitination and Sorting

Posttranslational modification of substrate proteins by the covalent attachment of ubiquitin (ubiquitination), originally discovered in the context of cellular protein degradation, has recently been shown to play a noncanonical role in regulating the postendocytic sorting of several membrane proteins including GPCRs. 73,74 Protein ubiquitination is mediated by the concerted action of 3 enzymes. The first 2 enzymes (E1 and E2) are responsible, respectively, for activating ubiquitin and escorting the activated ubiquitin. The third enzyme, E3 ubiquitin ligase (E3), recognizes and modifies the substrate in a timely fashion. 75 For the β2-AR, both GRK-mediated phosphorylation and β-arrestin binding are essential for receptor ubiquitination to occur. 76 Importantly, this agonist-stimulated β2-AR ubiquitination modification is necessary for the receptor to undergo degradation in lysosomes. Further, ubiquitin-dependent lysosomal degradation is applicable to other GPCRs such as V2R and the protease-activated receptor2 (PAR2). 77,78 For both the β2-AR and V2R, receptor ubiquitination requires the β-arrestin proteins. Although the nature of this requirement is not entirely clear, 1 supposition is that the β-arrestins may serve as adaptors to bring as yet unidentified E3 ligase(s) to the receptors in a stimulus dependent fashion. In the case of the PAR2 and the CXCR4 receptors, ubiquitination is mediated by the E3 ligases c-Cbl 78 and AIP4, 79 respectively, but no involvement of the β-arrestins has as yet been demonstrated.

For the above mammalian GPCRs, as well as for others such as the chemokine receptor CXCR4, 80 receptor ubiquitination is not required for internalization per se but is crucial for the sorting of ubiquitinated receptors to lysosomes. A recent study of the β1-AR in a heterologous cellular system reported the resistance of the receptor protein to ubiquitination as well as agonist-mediated degradation, suggesting a strong relationship between this receptor modification and downregulation pathways. 81

Whereas the degradation of GPCRs and other membrane proteins is known to occur in lysosomes, that of some membrane receptors such as the single-membrane spanning erythropoietin receptor involve both lysosomes and 26S proteasomes, the megaprotease complexes that degrade most cellular proteins. 82 Interestingly, however, a recent report suggests that ubiquitination and proteasomal degradation of newly synthesized intracellular A2A adenosine receptors serves as a method of ER quality control (Figure, A). Importantly, this degradation could be overcome by the coexpression of USP4, a deubiquitinating enzyme belonging to a family of enzymes that catalyze removal of ubiquitin from the modified substrates. 83 USP4 expression led to more robust functional expression of the A2A receptor at the plasma membrane, suggesting that deubiquitination can facilitate cell-surface targeting of membrane proteins (Figure, A).

On the other hand, GPCR internalization can be regulated by the agonist-dependent ubiquitination of β-arrestin by the E3 ligase Mdm2, as demonstrated for the β2-AR. 76 Moreover, the stability of β-arrestin/GPCR binding that defines GPCRs as class A or B (as described above) also correlates with the ubiquitination status of the β-arrestin proteins. The separation of β-arrestin from class A GPCRs results from rapid β-arrestin deubiquitination, whereas the more stable β-arrestin interaction with class B receptors is caused by the sustained ubiquitination of β-arrestin. 84 As will be described below, ubiquitination of β-arrestin appears to not only be capable of regulating GPCR trafficking properties but also likely plays an important role in directing downstream signaling events.

Sorting Signals Used for GPCR Intracellular Trafficking and Endocytosis

The identification of short, linear amino acid signals present in the intracellular domains of transmembrane proteins responsible for mediating the intracellular sorting and endocytosis of a transmembrane protein from the plasma membrane has been the focus of much work over the past 2 decades. Such sequences are believed to act as recognition sites for components of the cellular adaptor protein machinery necessary for intracellular protein trafficking. The importance of such signals contained within the intracellular domains of GPCRs, however, is less well described than that for other transmembrane proteins. Albeit limited in number, exceptions to the general paradigm of GRK/β-arrestin–mediated endocytosis have been delineated for 7-membrane-spanning receptors important to the cardiovascular system. Interestingly, the best-described motifs are analogous to those used by non-GPCR transmembrane receptors and include di-leucine–based (LL or LXL) and tyrosine-based (NPXY or NPXXY, or YXXO) motifs.

As a prototypic GPCR, the β2-AR contains a di-leucine motif within its carboxyl terminus, alteration of which does not affect the ability of the receptor to traffic correctly to the cell surface, bind agonist, or to activate adenylyl cyclase. Agonist addition, however, does not lead to internalization of this mutant β2-AR, 85 demonstrating a role for di-leucine motif of the β2-AR in agonist-induced receptor endocytosis. Similarly, mutations introduced into the di-leucine motif in the cytoplasmic tail of the vasopressin 1a receptor (V1aR) significantly impaired agonist-induced receptor internalization. 86 Interestingly, however, mutation of the analogous di-leucine motif in the V2R resulted in a receptor that was unable to escape from the ER, suggesting a role for this motif in V2R maturation. 87

Tyrosine-based sorting signals have also been shown to be necessary in the trafficking of the β2-AR. Mutation of Y326 in the human β2-AR, located at the proposed junction of the seventh-transmembrane domain and the proximal portion of the carboxyl terminus and conserved in position across many members of the large superfamily of GPCRs, does not affect the ability of the receptor to traffic correctly to the cell surface, bind agonist, or to activate adenylyl cyclase when the receptor is overexpressed. 88 The mutation, however, did completely abolish β2-AR agonist-induced internalization. Complicating this interpretation, however, was the finding that lower expression levels of mutant β2-AR resulted in the loss of ligand binding and adenylyl cyclase coupling, likely because of intracellular retention of the mutant receptor. 89 More recently, a highly conserved tyrosine-based motif (YXXO) in the cytoplasmic tail of the protease-activated receptor-1 (PAR1), a GPCR for thrombin, which has previously been shown to undergo β-arrestin–independent internalization, was shown to be necessary for agonist-mediated but not constitutive internalization. 90 Finally, more recent studies have shown a direct interaction between a stretch of 8 arginines contained in the carboxyl terminus of the α1b-AR and the AP-2 μ subunit. 91 Whether the alternative tyrosine-based motif found in PAR1 or the nonclassical arginine motif identified in the α1b-AR plays a role in the endocytosis and intracellular trafficking of other GPCRs classically identified as fundamentally important to the cardiovascular system remains to be determined.

Role for Additional Proteins in the Endocytosis of GPCRs

In addition to the well-recognized roles of the GRK and β-arrestin proteins in GPCR internalization, multiple other proteins have been demonstrated to be important in the endocytic process. A partial list and description of the role these proteins play in GPCR endocytosis is discussed below.

ADP-Ribosylation Factor 6

ADP-ribosylation factor 6 (ARF6) is 1 member of the ARF family of small GTP-binding proteins known to be key players in vesicular trafficking events. In addition to its role in binding AP-2 and clathrin, β-arrestin is also able to directly bind to ARF6 and modulate its activity. ARF6 activation requires the exchange of GTP for GDP, a reaction that is catalyzed by the ARF guanine nucleotide exchange factor (GEF) ARNO ( AR F N ucleotide-binding site O pener). Importantly, ARNO is constitutively associated with β-arrestin2. 92 As shown for the β2-AR, expression of mutant ARF6 proteins containing single amino acid substitutions rendering them deficient in their ability to either bind (T27N) or hydrolyze (Q67L) GTP inhibited agonist-induced β2-AR internalization. 92 Overexpression of ARNO alone, however, increases β2-AR internalization by stimulating GTP nucleotide exchange on ARF. Thus, agonist-promoted recruitment of β-arrestin to an activated receptor leads to the local regulation of endocytosis by β-arrestin attributable to its inherent ability to bind both ARNO and ARF6.

In addition to requiring a GEF protein, ARF proteins also require a GTPase-activating protein (GAP) to accelerate hydrolysis of bound GTP. Initially identified as GRK-interacting proteins (GITs), GIT1 and GIT2 are zinc finger–containing proteins that function as GAPs for ARF6. 93,94 GIT1 overexpression reduces the internalization of transmembrane receptors in CCPs and CCVs, including the β1- and β2-ARs, the adenosine 2B receptor, and the M1-muscarinic receptor. 95 Importantly, ARF-GAP activity of the GIT proteins is stimulated by phosphatidylinositol 3,4,5-trisphosphate, whereas other ARF-GAPs, such as ARF-GAP1, are stimulated by phosphatidylinositol 4,5-bisphosphate and diacylglycerol. 94 This raises the interesting possibility that GIT regulation of ARF6 activity may be integrated through activation of the phosphatidylinositol 3-kinase signaling pathway.

Phosphatidylinositol 3-Kinase

Phosphatidylinositol 3-kinases (PI3Ks) are a conserved family of kinases with both lipid and protein kinase activity which can be activated in response to GPCR stimulation. As a family, they have been shown to play important roles in an array of cellular functions as divergent as cell survival, cell motility, and receptor endocytosis. Within the cytosol, PI3K is constitutively complexed with GRK2. 96 As demonstrated for the β2-AR, agonist binding induces translocation of the GRK/P13K complex to the activated receptor, formation of phosphatidylinositol 3,4,5-trisphosphate, and subsequent recruitment of AP-2 and clathrin, leading to receptor endocytosis. Importantly, receptor internalization is blocked by overexpression of the portion of PI3K that mediates its interaction with GRK2, as well as by the specific 3,4,5 trisphosphate lipid phosphatase PTEN, demonstrating the importance of the lipid kinase activity of PI3K for the localized production of D-3 phosphoinositides in regulating ligand-induced endocytosis of the β2-AR. 97

As noted above, in addition to possessing lipid kinase activity, PI3K family members are also able to function as protein kinases, although the importance of this activity has remained largely obscure given the limited number of protein substrates recognized by PI3Ks. 98 Recently, however, the importance of PI3K protein kinase activity in the regulation of β2-AR endocytosis has been illuminated. In an elegant study, Naga Prasad et al identified the cytoskeletal protein nonmuscle tropomyosin (an actin filament binding protein) as a substrate for the γ isoform of PI3K and further demonstrated that PI3K can selectively phosphorylate a single site (S61) within tropomyosin. 99 Alteration of this site within tropomyosin to mimic constitutive phosphorylation (S61D) leads to complementation of a protein kinase defective PI3K, whereas change to a phospho-deficient residue (S61A) blocked agonist-induced β2-AR internalization. Thus, through both its lipid and kinase activity, PI3K plays a central role in the agonist-induced removal of the β2-AR, the prototypic model cardiovascular GPCR, from the cell surface. Whether this paradigm will extend to other members of the cardiovascular GPCR family is at present unknown but seems likely, given that endocytosis via AP-2 containing CCVs is the predominant mechanism of internalization for most ligand-activated GPCRs.

Intracellular Trafficking: Sorting/Recycling/Degradation

Once internalized from the cell surface, GPCRs can be sorted along multiple pathways (Figure, C). They may undergo dephosphorylation, resensitization, and be recycled back to the plasma membrane. Alternatively, GPCRs may be targeted for degradation via the endosomal/lysosomal pathway. Finally, multiple GPCRs have more recently been shown to initiate G protein–independent intracellular signaling pathways following endocytosis, as is discussed below. These multiple trafficking fates for internalized GPCRs are the subject of an excellent recent review. 100 We would like to highlight a few proteins important for these processes.

Na + /H + -Exchanger Regulatory Factor

Following adrenergic receptor stimulation, it has long been recognized that G protein–dependent changes in cellular metabolism, excitability, and growth occur. 101 Likewise, cellular changes apparently independent of G protein activation have been demonstrated, including alterations in cellular pH via regulation of the Na + /H + exchanger. Recent work has established that agonist stimulation of the β2-AR promotes direct association of the extreme carboxyl terminus of the receptor with the first PDZ domain within Na + /H + -exchanger regulatory factor-1 (NHERF1). 102 Similar associations have been shown to occur for other GPCRs containing sequences conforming to the consensus motif D-S/T-x-L, including the purinergic P2Y1 receptor and the CFTR, 103 whereas other GPCRs such as the parathyroid hormone receptor 104 have been shown to interact with both PDZ domains of both NHERF1 and NHERF2 via a slightly different (ETVM) PDZ consensus motif. For the β2-AR, disruption of this interaction markedly impairs agonist-induced changes in intracellular pH.

NHERF is also of critical importance for proper intracellular sorting of the β2-AR. In addition to its ability to bind the extreme carboxyl terminus of the β2-AR via its PDZ domain, NHERF is able via its ezrin–radixin–moesin (ERM) domain to bind to the actin cytoskeleton through association with ERM proteins. Importantly, mutations generated to disrupt the interaction of NHERF with either the β2-AR carboxyl terminus or with ERM proteins lead to significant agonist-induced lysosomal degradation of the β2-AR following endocytosis, rather than recycling. 105 As noted above, multiple additional GPCRs have been shown to interact with the NHERF proteins. Although NHERF has been shown to play a role in the recycling of other GPCRs such as the κ opioid receptor (reviewed by Liu-Chen 106 ), the generalizability of the role that NHERF plays in the recycling of other GPCRs, particularly in the cardiovascular system, remains to be determined.

N-Ethylmaleimide-Sensitive Fusion Protein

In a study to identify β-arrestin binding partners, β-arrestin1 was found to interact in both yeast 2-hybrid and in vitro assays with N-ethylmaleimide-sensitive fusion protein (NSF), an ATPase essential for many intracellular transport functions. 107 Furthermore, overexpression of NSF in HEK293 cells led to enhanced agonist-induced β2-AR internalization and could rescue the effects of a β-arrestin1 mutant (S412D) previously shown to function as a dominant negative for β2-AR internalization. Interestingly, the β2-AR is also able, via its extreme carboxyl terminus, to bind directly to NSF. 108 The β2-AR/NSF interaction is agonist dependent and requisite for efficient agonist-mediated β2-AR internalization. Importantly, whereas wild-type β2-ARs recycle to the cell surface following exposure to the antagonist propranolol, β2-ARs containing mutations in their distal carboxyl termini remain sequestered intracellularly, demonstrating the importance of the β2-AR/NSF interaction for proper β2-AR recycling. Thus, although it is clear that proteins that bind to the extreme carboxyl termini of GPCRs, such as NHERF and NSF, serve to regulate the intracellular sorting of the receptors such as the β2-AR, the extent to which the trafficking of other GPCRs is modulated by these or similar as yet unidentified proteins remains to be elucidated.

Sorting Nexin 1

Following internalization, GPCRs may be either recycled to the plasma membrane or undergo sorting to the endosomal/lysosomal pathway for degradation. Although fundamentally important as a mechanism for downregulation of certain GPCRs, such as the PAR1, which recognizes the coagulant protease thrombin, comparatively less is known about the molecular components that direct postendosomal sorting and recycling of the receptors.

Although sorting nexin 1 (SNX1) was originally identified as a membrane-associated protein that interacts with the receptor tyrosine kinase, epidermal growth factor receptor (EGFR) and functions in EGFR sorting to lysosomes, 109 SNX1 has also been demonstrated to interact with PAR1 in an agonist-dependent manner. 110 Overexpression of a SNX1 carboxyl terminal–deletion mutant did not significantly impair agonist-induced PAR1 internalization and accumulation in early endosomes but was able to impede endosome to lysosome sorting of PAR1 and thus markedly inhibit PAR1 degradation. 110 Additional studies, in which depletion of endogenous SNX1 in HeLa cells via small interfering RNA technology markedly impaired agonist-induced PAR1 degradation, buttress the suggestion that SNX1 plays a role in the intracellular trafficking of PAR1 from endosomes to lysosomes. 111 Whether SNX1 or other SNX proteins can mediate the endosomal to lysosomal sorting of GPCRs other than PAR1 remains to be determined but is certainly possible given the general conservation of trafficking themes shared across members of this large protein family.

Rab GTPases

A second class of proteins shown to regulate the movement of GPCRs both during and subsequent to endocytosis are the Rab proteins, a family of Ras-like small GTP binding proteins. Importantly, different Rab GTPase family members selectively associate with specific intracellular structures including both recycling and sorting endosomes, where they mediate multiple steps of vesicular membrane trafficking including vesicle budding, docking, and fusion (reviewed by Seachrist and Ferguson 112 ).

Within the cardiovascular system, Rab5 plays a central role in the agonist-dependent CCV-mediated internalization and early endosomal localization of some GPCRs, including the β2-AR. 113 Expression of a dominant negative Rab5 (S34N) blocked β2-AR internalization, whereas a constitutively active Rab5 (Q79L) did not significantly alter β2-AR internalization. Interestingly, whereas the β2-AR is not able to directly associate with Rab5, the AT1R does bind Rab5 directly via the carboxyl terminus of the receptor and localizes to Rab5-containing endosomal structures following endocytosis. 114 Furthermore, the interaction of Rab5 with the AT1R on endosomes appears to be necessary for preventing sorting of the internalized receptor to lysosomes. 115 Unlike internalization of the β2-AR, however, endocytosis of the AT1R does not appear to be dependent on Rab5, 114 demonstrating the intracellular diversity in GPCR trafficking roles played by even single members of the Rab protein family.

Whereas Rab5 plays a role in GPCR trafficking events associated with GPCR internalization and/or early endosomal formation, multiple additional Rab family members have been implicated in other GPCR-trafficking events, including Rab4 and Rab11 in receptor recycling, and Rab7 in endosomal to lysosomal sorting. 115 In a transgenic mouse model in which a dominant-negative Rab4 (S27N) was overexpressed in the heart, mice had impaired responsiveness to both endogenous and exogenous catecholamines. 116 Furthermore, when these mice were crossbred with mice also overexpressing a cardiac-specific human β2-AR, the resultant mice had an abnormal intracellular vesicular β2-AR accumulation, as well as significantly reduced cardiac contractility. Such results suggest that proper Rab4-mediated regulation of β2-AR receptor recycling is necessary for maintenance of normal catecholamine responsiveness and receptor resensitization. Whether Rab4 also plays a similar role in vivo in the recycling of other GPCRs within the cardiovascular system is currently unknown. For a more complete review of the diverse roles that members of the Rab protein family play throughout nearly all aspects of GPCR trafficking, refer to several recent excellent reviews. 100,112

Other Proteins With Roles in GPCR Endocytosis and Postendocytotic Trafficking

Many additional proteins have been identified and characterized that play fundamental roles in the endocytosis and postendocytic trafficking of GPCRs. These include, but are not limited to, proteins involved in CCV formation and membrane scission such as AP-2, clathrin, dynamin, Hrs, and GASP, 117–119 as well as multiple scaffolding proteins that have been demonstrated to interact with the β1-AR including postsynaptic density 95 (PSD95), 120 membrane-associated guanylate kinase inverted-2 (MAGI-2), 121 and the endophilins. 122 Because of the limited scope of this review, we have chosen to highlight only selected proteins as above.

Internalization/Trafficking and Signaling

Although internalization mechanisms leading to receptor sequestration away from the source of stimulus have been primarily considered as signal desensitization pathways, a large body of research now exists in support of continued signaling that is coupled with GPCR endocytosis. 123 Moreover, although it is generally believed that endocytic “addresses” determine the extent of signaling, continued signaling mechanisms during the trafficking of proteins may actually determine the exact endocytic route by regulating processes such as vesicle fusion and cargo transfer. As such, it is increasingly evident that the 2 processes, trafficking and signaling, are not only connected but likely inextricably intertwined (Figure, D).

In the case of GPCRs, β-arrestin has been clearly demonstrated to play an essential role in the desensitization of G protein–mediated second messenger signaling. More recently, however, the β-arrestin proteins have also been shown to facilitate both receptor internalization via CCVs, as well as to couple the receptors to and activate nonreceptor tyrosine kinases (eg, members of the c-Src family), thereby mediating a second wave of signaling. 124,125 In addition, on binding to activated GPCRs such as the AT1aR, β-arrestin has also been shown to be capable of sequestering and scaffolding signaling kinases, as well as targeting active signaling complexes into cytoplasmic endosomal microcompartments. 125,126 Importantly, chemical or molecular inhibition of receptor endocytosis (or, alternatively, expression of β-arrestin mutants that impair receptor internalization), can lead to a decline in such downstream signaling, demonstrating a coupling between endocytosis and signaling. 124,127

In addition to c-Src activation, β-arrestin has been shown in multiple studies to function as a signaling intermediate in GPCR signal transduction to mitogen-activated protein kinase (MAPK) pathways, under conditions where G protein coupling is virtually absent. 125,128,129 Although this β-arrestin–dependent signaling has been most well characterized for ERK activation, it may also apply to other signaling kinases and appears to play roles in a variety of cellular responses including chemotaxis and antiapoptosis. Although it has not yet been conclusively determined that receptor endocytosis is required for this β-arrestin pathway, recent data suggest that the trafficking patterns of β-arrestin/receptor complexes may play a significant role. 130,131 Interestingly, β-arrestin ubiquitination at specific acceptor sites (eg, Ang II–induced ubiquitination of lysines 11 and 12 in β-arrestin2) is crucial for stable β-arrestin/AT1R interaction as well as the formation and endosomal localization of AT1R-MAPK signalosomes. Whether this observation may be broadly applicable to other GPCRs relevant to cardiovascular biology is currently unknown and is an area of active investigation.

Conclusions

Within the cardiovascular system, regulation of GPCR trafficking is of fundamental importance both for physiological homeostasis and the molecular response to physiological perturbation. There is efficient and coordinated movement of GPCRs from synthesis to the cellular surface, where they can interact with components of the extracellular milieu to transduce signals to intracellular effectors and subsequent GPCR retrieval from the cell surface. These processes highlight the intricate cellular balance that has developed to exquisitely regulate hormonal responsiveness at the tissue level. Indeed, much has been learned about GPCR trafficking within the cardiovascular system over the past 2 decades, as evidenced by the ever-expanding ensemble of identified proteins crucial to this process. The recent recognition that GPCR signaling may occur within the cardiovascular system via G protein–independent/β-arrestin-dependent pathways raises the fascinating possibility that pharmacologically relevant cardiovascular ligands may be developed that allow for selective GPCR signaling via the activation of proteins initially believed only to have a role in limiting GPCR signaling and promoting GPCR removal from the cell surface.

Original received June 30, 2006 revision received August 10, 2006 accepted August 11, 2006.

We thank Donna Addison and Elizabeth Hall for excellent secretarial assistance.


If a cell has two different GPCRs, how does the cell differentiate between the phosphorylation cascade caused by each? - Biology

C2006/F2402 '14 OUTLINE OF LECTURE #14

(c) 2014 Dr. Deborah Mowshowitz, Columbia University, New York, NY . Last update 03/09/2014 12:35 PM .

Handouts: 14A -- Signaling -- GPCRs, G proteins, and cAMP
14B -- Homeostasis -- Seesaw view for Glucose and Temperature Regulation
14C
-- Lining of the GI Tract & Typical Circuit

We will be covering a lot about hormones A hypertext of endocrinology with some animations is at
http://arbl.cvmbs.colostate.edu/hbooks/pathphys/endocrine/index.html


I. Role of G proteins & 2nd Messengers in Signaling, cont.

A. What are the important properties of G proteins? See Becker fig. 14-5, Handout 14A.

  • G protein is activated by dumping GDP and picking up GTP in response to some signal.
  • It is NOT activated by phosphorylation of the bound GDP to GTP.
  • Activation is called GDP/GTP exchange -- more details below

b. Inactivation: G protein inactivates itself by catalyzing hydrolysis of GTP to GDP.

c. Why is it a switch? The G protein does not stay active for long. "Turns itself off."

B. Typical Pathway -- Role in signaling (see also handout 14A, middle panel)

ligand (1st messenger) binds outside cell activate receptor in membrane activate G protein in the membrane activate target enzyme in membrane generate small molecule (2nd messenger) inside cell

a. Terminology: Be careful not to confuse G proteins (activated by receptors) & GPCRs (the actual receptors in the membrane).

b. Location: The ligand (1st messenger) binds to the extracellular domain of its receptor. The remaining events are inside the cell.

c. Response: The action of the 2nd messenger (explained below) leads to a specific response in each responsive cell type. Typical responses:

(1) release of glucose from storage in liver in response to epinephrine.

(2) synthesis and release of TH (thyroid hormone) from the thyroid gland in response to TSH (thyroid stimulating hormone)

C. What are Second messengers? How do they fit in? See handout 14A or Sadava fig. 7.7 (7.8). More details, and examples, are covered below.

  • Active G protein (subunit) → binds to & activates enzyme in (or associated with) membrane.
  • Activated enzyme → generates second messenger, in cytoplasm and/or membrane.
  • Details of cAMP pathway are below. See Becker fig. 14-7 or Sadava figs. 7.12 (7.14).
  • We will get to IP3 pathway later. If you are curious, see Becker fig. 14-10 or Sadava fig. 7.13 (7.15).

II. How do G Proteins Work?

A. Activation & Inactivation of G Proteins in General

1 . Reactions of activation & inactivation

a. Activation Reaction (GTP/GDP exchange, NOT phosphorylation of GDP GTP replaces GDP):

Protein-GDP (inactive) + GTPProtein-GTP (active) + GDP

b. Inactivation Reaction (hydrolysis of bound GTP to GDP):

Protein-GTP (active)Protein-GDP (inactive) + phosphate.

c. Overall: GTP displaces GDP, activating the G protein GTP is then hydrolyzed (usually rapidly), returning the G protein to its inactive state.

(1). Net effect on GTP -- GTP hydrolysis: GTP ( + water)GDP + phosphate

(2). NetEffect on G protein -- none: Protein is temporarily activated, but then inactivated. However protein cycles from inactive to active and back to inactive -- acts as switch.

d. Terminology. Since the overall result is that GTP is hydrolyzed to GDP, G proteins are sometimes called "GTPases."

2. What triggers activation?

a. General Case: Binding of a protein called a GEF (guanine-nucleotide exchange factor) causes GDP to fall off, and GTP binds.

b. In signaling: Activated GPCR = GEF. Binding of activated receptor to G protein triggers activation of G protein (causes loss of GDP).

3. What triggers inactivation?

a. G protein itself has enzymatic activity -- catalyzes inactivation (hydrolysis).

b. No trigger required -- hydrolysis of GTP to GDP happens automatically.

c. Other proteins may increase speed of hydrolysis. They are called RGS proteins (Regulators of G protein Signaling) or GAP proteins (GTPase Activating Proteins).

4. How do G proteins compare to kinases? See table below.

B. Types of G proteins

1. Subunits -- Ordinary G proteins are trimeric = they have 3 subunits.

a. Inactive G prot = heterotrimer of alpha, beta, gamma

b. Separation occurs on activation: On activation, alpha subunit (with the GTP) separates from other 2 subunits.

c. Either part may be the effector that actually acts on target -- alpha, or beta + gamma, can act as activator or inhibitor of target protein.

d. Hydrolysis causes reassociation. Hydrolysis of GTP to GDP causes alpha to reassociate with other subunits → inactive heterotrimer

2. Small G proteins -- to be discussed further when we get to cell cycle & cancer. For reference:

a. Structure: Small G proteins have no subunits.

b. Example: the protein called ras -- important in growth control many cancer cells have over-active ras.

c. Role of GTP/GDP exchange: Are activated by GTP/GDP exchange, and inactivated by hydrolysis of GTP to GDP, as above.

d. Are not activated by GPCRs directly (other 'middle man' adapter proteins are involved)

3 . How many G proteins?

a. There are many different G proteins. G proteins are involved in a very large number of cellular processes, not just signaling. (We have ignored their importance until now. See Becker for details & many examples. )

b. Active G proteins can be inhibitory or stimulatory.

c. Method of action: Activated G proteins work by binding to and activating (or inhibiting) other target enzymes/proteins.

d. Terminology: The different trimeric G proteins are usually known as Gp, Gq Gi, Gs etc. (Books differ on details of naming.)

C. For reference: Comparison of Protein Kinases, Receptor Protein Kinases, & Trimeric G proteins

Protein Catalyzes What's added to Target Protein? Who gets the P or GTP? How Inactivated?
Protein Kinase Protein + ATP → ADP + protein-P Phosphate Usually a dif. protein Separate Phosphatase removes P
Trimeric
G Protein
Exchange & Hydrolysis as described above. GTP Itself Hydrolyzes GTP to GDP (by itself)
Receptor Protein Kinase ** Protein + ATP → ADP + protein-P Phosphate Usually separate subunit of self Separate Phosphatase removes P

**Receptor protein kinases have an extracellular ligand binding domain and an intracellular kinase (or kinase binding) domain. Ordinary kinases usually add phosphates to other proteins. Receptor kinases usually add phosphates to themselves. For an example, see Sadava fig. 7.6 (7.7)

Try problems 6-1 & 6-2.

III. An example of a second messenger -- cAMP & its target (PKA) See handout 14A or Becker fig. 14-7.

A. What is cAMP? The most famous second messenger. See handout 14A or Becker 14-6 for structure.

B. How is cAMP level regulated? What does it do?

1. How is cAMP made?

a. cAMP made from ATP by adenyl cyclase see handout and Becker fig. 14-6 or Sadava fig. 7.12 (7.14).

b. What activates the enzyme adenyl cyclase? G protein activates adenyl cyclase (also called adenylyl cyclase or AC)

2. What does cAMP do? See Becker table 14-1 & handout 13C.

a. cAMP binds to and activates protein kinase A = PKA . (Also called cyclic AMP dependent protein kinase = cAPK) See Becker fig. 14-8.

b. PKA adds phosphates to other proteins

(1). Effect of phosphorylation: Phosphorylation by PKA can activate or inhibit target protein (target = substrate of PKA)

(2). Amplification of PKA action. Active PKA can modify other kinases/phosphatases and start a cascade

(3). End result varies.Depends on which kinases and phosphatases in that tissue are targets (substrates) of PKA and/or the other kinases/phosphatases (at end of cascade). See example below.

3. How does signal system turn off when hormone leaves?

a. Existing cAMP is short lived -- it's hydrolyzed by phosphodiesterase (PDE) to ordinary5' AMP.

b. Synthesis of new cAMP stops (when signal removed). AC becomes inactive.

(1) G protein doesn't stay activated for long: Activated G protein hydrolyzes its own GTP → GDP ( → inactive G protein).

(2) Inactivated G protein no longer activates AC.

c. In absence of cAMP, action of kinases are stopped and/or reversed

(1) PKA becomes inactivated

(2) Phosphatases become active -- remove phosphates added by kinases, reversing PKA effect.

C . How do hormones work through cAMP? (see also handout 14A, middle panel)

1. Pathway from Ligand → PKA is the same*. Steps are as follows:

a. Ligand (hormone or other) binds to receptor, activating receptor.

b. Activated receptor activates G protein.

c. G protein activates adenyl cyclase. (But see note at *.)

d. Adenyl cyclase catalyzes production of cAMP

e. cAMP activates PKA

f. PKA phosphorylates target proteins.

g. Different tissues can respond differently to same hormone -- see below.

*Note: This applies to G proteins that activate adenyl cyclase. There are multiple G proteins -- some inhibit adenyl cyclase, and some affect different enzymes and cause (or inhibit) production of different second messengers.

2. Example #1: TSH -- effects thyroid gland.

a. Why does TSH affect only thyroid? Only thyroid gland has TSH receptors.

b. Generation of 2nd messenger In summary, TSH triggers synthesis of cAMP

TSH (1st messenger) binds activate GPCR in membrane activate G protein in the membrane activate enzyme in membrane (adenyl cyclase) generate small molecule (2nd messenger) inside cell = cAMP

c. Role of 2nd messenger (cAMP): cAMP activates PKA PKA phosphorylates (and activates) enzymes needed to make thyroid hormone.

cAMP activate protein kinase (PKA) in cytoplasm phosphorylate target enzymes stimulate multiple steps in synthesis and release of TH

d. Overall response: Thyroid stimulating hormone (TSH) -- promotes release of thyroid hormone (TH) from thyroid gland. Will be discussed in more detail when we do a summary of major hormones.

3. Why bother with all these steps?

a. Amplification. Many steps involve amplifications. For example, one molecule of active Adenyl cyclase can generate many molecules of cAMP and one molecule of PKA can phosphorylate many molecules of its target enzymes. For an example of the possibilities of a cascade of amplification, see Becker fig. 14-3 or Sadava fig. 7.18 (7.20). The two texts both agree on a high level of amplification, but don't agree on the exact numbers.

b. The classic example. The usual example for this type of modification cascade is the breakdown of glycogen, stimulated by the hormone epinephrine (adrenaline), which is the example in Becker fig. 14-3, and is explained in more detail below (example #2).

4. Example #2 -- Epinephrine -- effects on Regulation of Glycogen metabolism:

a. When is epinephrine secreted? In response to a crisis. It triggers the 'fight or flight' reaction.

b. History: This was the first case of regulation by cAMP to be discovered.

c. How does epinephrine work? Epinephrine binds to a receptor (GPCR) which activates AC and this triggers synthesis of cAMP and activation of PKA.

d. PKA phosphorylates many enzymes involved in glycogen metabolism. Effects of phosphorylation:

(1). Enzymes of glycogen breakdown to glucose-phosphate are stimulated.

(2). Enzymes of glycogen synthesis are inhibited.

(3). If you are interested in the details, see Sadava fig. 7.18 (7.20) or Becker figs.14-22 (14-25) & 6-17, or the handout at http://www.columbia.edu/cu/biology/courses/c2006/handouts12/glycogen09.pdf.

e. What is the net result? Glycogen is broken down to provide energy to cope with a stressful situation.

Try problems 6-8 & 6-9.

  • The receptor acts in in combination with other proteins, to bind to different EREs -- different genes affected

  • Effect of binding to ERE not necessarily the same (inhibition vs activation).

  • In muscle, lactate can be released to blood.

  • In liver, glucose can be released into blood.

  • Why is the result different? Liver contains phosphatase for G-6-P and muscle does not. (Additional enzymes also differ.)

(4). General principle -- final effect depends on all proteins present in the cell, not just the receptors or immediate targets.

(5). Mechanism of Hormone Action -- The process that is regulated may be different-- in example #1, transcription is affected in example #2 enzyme activity is affected.

b. Different hormones can trigger the same signaling pathway in the same cell

(1). How? Epi.(epinephrine) & glucagon bind to different receptors, but both receptors activate the same G protein and trigger the same series of events → cAMP → etc. so can get same response to both hormones in same tissue (if both receptors are present).

(2). Why? Two hormones control same process (glycogen metabolism) for different purposes -- Epi to respond to stress glucagon to respond to low blood sugar (maintain homeostasis -- details below or next time).

Question: Since both hormones trigger the same pathway, will any cell that responds to one hormone respond to the other hormone?

Try problem 6-11.

IV. Introduction to Physiology & Multicellular organisms -- What is signaling good for? Why do you need epinephrine, glucagon, TSH, etc?

A. Single cell Life Style vs. Multicellular

1. Single celled organisms

a. Surrounded by external environment -- Can't change or regulate it

b. Have one basic function -- grow and multiply

c. Respond to external conditions (since can't change them) to maintain optimal intracellular state

(1). Pick up and/or dump what is necessary for metabolism

(2). Keep intracellular conditions(pH, level of amino acids, oxygen, etc.) as constant as possibleand expend minimal energy by adjusting rates of transcription, enzyme activity, etc.

d. Note no specialization: each cell does all possible functions

2. Multicellular organisms & Homeostasis

  • plasma = liquid part of blood = fluid between blood cells

  • interstitial fluid (IF) = fluid between all other cells

c. Each cell has two basic functions

(1). Grow or maintain itself as above

(2). Specialized role in maintaining homeostasis of whole organism

d. Cells are Specialized. Maintenance of homeostasis requires co-operation of many different cell types, not just circuits within a single cell.

Unicellular Organisms Multicellular Organisms
What surrounds cell? External environment Internal environment of organism
Can organism regulate what surrounds each cell? No Yes
How many functions of each cell? 1 2 or more
Is cell specialized? No Yes

B. Organization -- How are cells set up to co-operate in a multicellular organism?

1. Cells, Tissues & the 4 major tissue types (5, if you count the blood separately) -- see lecture #4, & Sadava figs. 40.3 to 40.6.

2. Organs

a. Consist of more than one kind of tissue.

b. Example: Lining of GI tract. (Demo now or next time.) Lining has layers of different tissues -- epithelial, connective, muscle, and nervous. These tissues serve primarily for absorption (of material from lumen), support, contraction, and regulation respectively. (See Sadava fig. 40.7) The blood (a type of connective) doesn't really fit in this classification -- serves for transport of materials in and out.

3. Systems -- Group of Organs → body or organ system. Work together to maintain homeostasis for some component. Number of systems depends on who's counting. Usual # is 8-12.

a. Immune system -- responds primarily to internal changes caused by presence of foreign organisms (or their macromolecules) -- responds to viruses, bacteria, cancer cells, etc. (Graft rejection, allergy, etc. are side effects of this.)

b. Other systems -- respond to changes in internal mileu caused by other factors.

C. Consequence of Multicellularity -- How does co-operation of parts (tissues, organs, systems, etc.) occur? Need signaling to maintain homeostasis.

IV . How is a component of the internal milieu regulated?

A. General Principle -- Homeostasis is maintained by Negative Feedback

  • In physiology, negative and positive feedback are defined as above. For negative fb, it doesn't matter whether the corrections are achieved by inhibition (turning off the heater stopping glucose production) or acceleration (turning on the air conditioner, increasing glucose uptake from blood).

  • In biochemistry, negative feedback usually means inhibition of an earlier step (& positive fb usually means activation).

  • In normal English speech, positive and negative feedback are used in a third way. Positive fb means complimentary, positive comments while negative fb means critical, negative comments. Positive fb means the listener liked it negative fb. means s/he didn't.

  • 'Effector' is also used differently in physio and biochem see below.

B. Example #1 -- Regulation of blood glucose levels. The see-saw view. See handout 14B or Sadava fig. 51.17 (51.18). The fig. in the 9th edition is not a see saw, but the point is the same.

1. Have a regulated variable -- glucose level in blood.

2. Need a sensor (or receptor) -- to measure levels of "regulated variable" (glucose). Here, sensor is in pancreas.

3. Need effector(s) -- to control levels of regulated variable (glucose) -- usually have one or more effectors that respond in opposing ways. In this case, effectors for uptake of glucose are liver, adipose tissue, and skeletal muscle effector for release of glucose is liver.

Note on terminology: In physiology, "effector" usually means "a tissue or organ (like muscle or liver) that carries out an action and thus produces an effect." In this example, the effectors = organs that act to raise or lower the blood glucose. In molecular biology, the term "effector" is usually used to mean "a modulator of protein function." A modulator = a small molecule (like an inducer, enzyme activator etc.) that binds to a protein, alters the shape and/or function of the protein, and thus triggers an effect.

4. Have a set point -- the level the regulated variable (blood glucose) should be. Set point is also sometimes used to mean the level at which corrections (to raise or lower the value) kick in.

In most cases, there is no significant difference between these two definitions of set point. In some cases, the desired value (first definition) and the value at which corrections occur (second definition) may be different. For example, there may be two cut-off points -- upper and lower, that bracket the desired level of a regulated variable. At levels above or below the respective cut-off points, messages are sent to the appropriate effectors to take corrective action. The term "critical values" is sometimes used instead of "set points" to describe the cut-off point(s).

5. Signaling -- need some signal system to connect the sensor(s) and the effector(s). Can be nervous &/or hormonal. In this case, primary (but not only) signal is hormonal & primary hormones (signals) are insulin & glucagon.

6. Operation of Negative Feedback -- the system responds to negate deviations from the set point. Important features:

a. Works to stabilize levels of blood glucose (the regulated variable)

b. System is self-correcting -- Deviations in either direction (if blood glucose is either too high or too low) are corrected.

c. There are two opposing actions by effectors, not just one.

(1). If [G] gets too high, effectors take G up from blood. (top half of seesaw diagram)

(2). If blood [G] gets too low, effector releases G to blood. (bottom half of seesaw diagram)

d. Important: Negative feedback is not always inhibition. In negative feedback, deviations from the set point can be corrected either by speeding up a process (such as glucose uptake) or slowing down a process (such as glycogen breakdown to glucose). Therefore deviation from the set point in either direction may be fixed by accelerating, not inhibiting, a process. For example:

(1). If blood [G] goes up, the uptake from blood increases. In this case, an increase in glucose uptake is used to help decrease the deviation from the set point. (Note this problem can also be fixed by decreasing glycogen breakdown.)

(2). If blood [G] falls,release into blood increases. In this case, an increase in glucose release is used to help decrease the deviation from the set point. (Note this problem can also be fixed by decreasing glycogen synthesis.)

(3). In both cases, whether the deviation is positive or negative (above or below the set point) the deviation can be reduced by increasing a process or by inhibiting another.

7. Net Result -- regulated variable ([G] in blood) is not constant, but stays close to set point.

See problem 5-1 & 5-2 a & b.

C. Example #2 -- Regulation of body temperature (in humans) -- the see-saw view (handout 14B)

1. Note many features are same as in glucose case. (Can you list them??)

2. Features not found in glucose case:

a. Multiple sensors in different places (for core and skin temp.) How to integrate multiple inputs?

b. Nature of Signal -- Signals are neuronal, not hormonal

c. Integrative center (IC) = thermostat in Sadava fig. 40.2 (10th ed)

(1). Role of IC: Compares set-point to actual value, sends appropriate message to effectors.

(2). Type of IC

(a). Sensor/IC function may be combined, as in Glucose example.

(b). Separate IC needed if there are multiple sensors, as in this case. IC co-ordinates incoming information from multiple sensors

(3). In this example, IC = hypothalamus (HT)


Apoptosis and Cancer

One primary function of apoptosis is to destroy cells that are dangerous to the rest of the organism. A common reason for apoptosis is when a cell recognizes that its DNA has been badly damaged. In these cases, the DNA damage triggers apoptosis pathways, ensuring that the cell cannot become a malignant cancer.

However, clearly this process sometimes fails. All instances of cancer are presumably instances where a damaged cell did not commit apoptosis, but instead went on to make more of itself.

Apoptosis may be unable to occur if essential genes required for it are among those that are damaged. However, some doctors and scientists have been studying apoptosis intensely in hopes that they may be able to learn to trigger it specifically in cancer cells using new medications or other therapies.

As with all drugs designed to kill cancer cells, the challenge with drugs designed to induce apoptosis is to ensure that these drugs only effect cancer cells. A medication that causes healthy cells as well as cancerous ones to commit programmed cell death could be very dangerous.

The picture may also not be quite as simple as “cancer occurs when apoptosis fails.” Research has suggested that some cancers may arise in cell populations where apoptosis occurs more easily than it should possibly these cells have been forced to “learn” to ignore over-enthusiastic apoptosis signals, and subsequently don’t commit apoptosis even when they have sustained severe damage.

Other research has revealed that cancer cells which die due to the effects of medication often die by apoptosis – suggesting that cancers that are especially apoptosis-resistant may also be especially treatment-resistant.

Much more research is needed on the subject of cancer treatment, and understanding apoptosis pathways is one extremely promising avenue for making new breakthroughs!


G protein–coupled receptors (GPCRs) play an integral role in the signal transduction of an enormous array of biological phenomena, thereby serving to modulate at a molecular level almost all components of human biology. This role is nowhere more evident than in cardiovascular biology, where GPCRs regulate such core measures of cardiovascular function as heart rate, contractility, and vascular tone. GPCR/ligand interaction initiates signal transduction cascades, and requires the presence of the receptor at the plasma membrane. Plasma membrane localization is in turn a function of the delivery of a receptor to and removal from the cell surface, a concept defined most broadly as receptor trafficking. This review illuminates our current view of GPCR trafficking, particularly within the cardiovascular system, as well as highlights the recent and provocative finding that components of the GPCR trafficking machinery can facilitate GPCR signaling independent of G protein activation.

G protein–coupled receptors (GPCRs) are central mediators of nearly all aspects of cardiovascular biology. GPCRs were originally identified as receptors capable of coupling to specific guanine nucleotide-binding proteins (G proteins), thereby transducing an extracellular signal to an intracellular effector, although more recently, several GPCRs have been demonstrated to signal via G protein–independent mechanisms both in vitro and in vivo. 1 As a family of proteins, GPCRs share common structural features, including seven membrane-spanning domains, and thus are alternatively referred to as 7-transmembrane receptors. GPCRs are the largest superfamily of cell-surface receptors, accounting for approximately 2% of the human genome. 2 Further, ligands directed at GPCRs (primarily agonists and antagonists) represent the largest family of pharmacological agents, accounting for nearly 30% of current clinical pharmaceutical agents available. 3 Both hormones and neurotransmitters exert their effects on the cardiovascular system via GPCRs. Examples of GPCRs with well-ascribed roles in cardiovascular biology include the β1- and β2-adrenergic receptors (ARs), the α1- and α2-ARs, the M2- and M3-muscarinic acetylcholine receptors, the angiotensin II (Ang II) receptors, the endothelin receptors, the adenosine receptor, the thrombin receptor, and the vasopressin receptor.

Over the past nearly 3 decades, a wealth of information has revealed much about the signaling properties of this family of seven membrane-spanning receptors. Much work has focused on revealing the ways in which the GPCRs regulate discrete effector molecules including adenylyl cyclase, phospholipases, and ion channels. Still further work has shed light on mechanisms by which GPCR signaling is regulated and has led to the discovery of additional proteins including the GPCR kinases (GRKs) 4,5 and β-arrestin proteins, 6,7 which respectively phosphorylate agonist-activated GPCRs and bind phosphorylated GPCRs to physically disrupt the receptor/G protein interaction, thereby leading to desensitization of receptor-mediated G protein activation. In addition to its role in GPCR desensitization, β-arrestin binding also promotes the cytosol to cell surface translocation of components of the endocytic machinery, namely adaptor protein-2 (AP-2) 8 and clathrin, 9 thereby facilitating receptor removal from the plasma membrane.

Although still substantial, comparatively less work has focused on GPCR trafficking, much of it related to mechanisms regulating endocytosis of GPCRs from the cellular surface, including the role of β-arrestin in facilitating GPCR endocytosis as above. Indeed, the appropriate delivery of GPCRs to the cell surface to permit receptor/ligand interactions, and their subsequent retrieval from the plasma membrane, are of fundamental importance for the regulation of GPCR activity. This review highlights our current understanding of GPCR movement from synthesis onward, with special emphasis on studies of GPCRs from the cardiovascular system. Lastly, we discuss the importance of the newly recognized role that GPCR trafficking itself can have on cellular signaling, including the recently recognized and expanding role for β-arrestins in GPCR signaling independent of G proteins.

GPCR Trafficking: Posttranslation

Both during and subsequent to synthesis, membrane proteins including GPCRs undergo a continual process of maturation before reaching residence at the plasma membrane. They must be properly inserted into the membrane (a process believed to occur cotranslationally for most membrane proteins), achieve proper folding while still resident in the endoplasmic reticulum, traverse from the cis- to the trans-Golgi while undergoing modification, and finally be targeted to the plasma membrane where they attain residence as mature proteins. This section will discuss various aspects of this maturational process that have been determined for GPCRs (Figure, A).

General model of GPCR trafficking. A, Following synthesis, GPCRs initially reside in the ER, where they undergo processing and folding guided by chaperone and quality-control proteins. Within the ER, many GPCRs likely form either homo- or heterodimeric structures. Following ER exit, GPCRs transit through the Golgi apparatus, where they may undergo additional modifications such as oligosaccharide processing. On the distal edge of the Golgi, GPCRs are packaged in exocytic transport vesicles and enter the endosomal system, where they are subsequently targeted to the plasma membrane. Multiple proteins, as listed, have been identified that affect GPCR stability at the cell surface. ERAD indicates ER-associated degradation. B, Although variations have been described, GPCR endocytosis from the plasma membrane most commonly occurs in a GRK- and β-arrestin–dependent manner. Ligand binding promotes GRK-mediated phosphorylation of the cytoplasmic surface of GPCR and subsequent β-arrestin translocation and binding to the receptor. β-Arrestin binding, in turn, facilitates the subsequent recruitment of AP-2 and clathrin and GPCR inclusion in CCPs before endocytosis via CCVs. C, Following endocytosis, GPCRs may be either recycled to the plasma membrane or sorted for lysosomal degradation. The Rab family of small GTPases is integral in determining the fate of a GPCR, whereas SNX1 has more recently been shown to play a role in endosomal to lysosomal GPCR sorting. Receptor ubiquitination also plays a role in receptor degradation via lysosomes. D, More recently, several GPCRs have been shown to be capable of signaling via β-arrestin–dependent pathways. Well-characterized β-arrestin–dependent signaling cascades that have been described include agonist-dependent nonreceptor tyrosine kinase activation as well as activation of the MAPK signaling pathway.

Folding and Chaperones

Strict quality-control mechanisms within cells ensure that improperly or incompletely folded proteins are targeted for degradation, usually via the proteasome pathway. For most nascent proteins studied, folding into a proper/functional conformation requires the presence of endogenous accessory chaperone proteins. 10 GPCRs are no exception to this quality-control process. As example, the human DnaJ protein HSJ1b, a member of the heat shock protein (HSP) family of cytoplasmic cochaperones, regulates trafficking of rhodopsin from the endoplasmic reticulum (ER) to the cell surface. 11 Alternatively, single-membrane–spanning chaperone proteins can facilitate GPCR exit from the ER, as recently demonstrated for the calcitonin receptor-like receptor, which must form a heterodimeric complex with the receptor activity modifying protein (RAMP) before ER egress (reviewed by Tan et al 12 ). Whether these or similar endogenous chaperone proteins regulate the folding of GPCRs important to some aspects of cardiovascular biology is currently unknown, but certainly possible given the structural conservation across this large protein family.

Despite such checks to ensure proper protein folding errors do, however, occur. As such, a variety of human diseases have been identified in which naturally occurring mutations result in the misfolding and/or mistargeting of a mutant protein. Such “protein conformational” diseases are thus considered to result from mutations that do not affect the functional domain(s) of the mutant protein, but rather interfere with the normal cellular trafficking of the protein (reviewed by Bernier et al 13 ). Such misfolded proteins typically either form aggregates that are deleterious to the cell or are recognized as improperly folded and therefore targeted for degradation by the cellular quality-control mechanisms noted above (reviewed by Sitia and Braakman 14 ). Interestingly, recent work has demonstrated that in some instances, chemical or pharmacological manipulation can rescue misfolded proteins and lead to their proper translocation to the plasma membrane where the proteins are functionally active.

Nephrogenic diabetes insipidus (NDI) is an X-linked disorder 15 with an incidence in the population of approximately 1 per 250 000. NDI is characterized by renal resistance to the posterior pituitary-derived antidiuretic hormone (also called arginine vasopressin), an octapeptide which normally acts at the vasopressin 2 receptor (V2R) present on renal epithelial cells to allow for normal urinary concentration. 15 In patients with NDI and the complete absence of renal epithelial V2R cell surface expression, daily urinary volume can exceed 15 L and lead to rapid death. More than 150 different mutations in the V2R have been described, the majority of which (≈70%) impair V2R cell surface trafficking (reviewed by Bernier et al 13 ). The addition of a cell-permeable V2R antagonist to a subset of mutant V2Rs previously shown to accumulate in the ER resulted in the proper folding, ER exit, correct targeting to the cell surface, and functional rescue of receptor activity of the mutant proteins. 16 This is presumably attributable to the ability of the small molecule ligand to stabilize the native state of the protein, thereby facilitating the proper trafficking of the receptor. Additional mutant 7-transmembrane receptors known to have altered trafficking properties that result in human disease, and for which molecular chaperones have been identified, include rhodopsin, the sulfonylurea receptor 1 (SUR1), smoothened, and the gonadotropin-releasing hormone receptor (reviewed by Bernier et al 13 ). It remains to be seen, however, whether pharmacological treatment of the mistrafficked receptor targets identified above in cellular systems will translate to amelioration of human disease.

Oligomerization

Early studies using rhodopsin, 17,18 muscarinic, 19 and β-adrenergic 20 receptors as model GPCRs suggested that GPCRs exist primarily as monomers, although modification of the detergent extraction systems used for protein purification led early investigators to suggest that a varying fraction of GPCRs may also be present in oligomeric form. 19,20 Much recent work using coimmunoprecipitation and resonance energy-transfer techniques have convincingly demonstrated that dimeric GPCR structures are present at the plasma membrane and are the topic of several recent reviews. 21–24 Indeed, it has been postulated that homo- and heterodimeric GPCRs may represent the basic functional unit necessary for most, if not all, GPCR signaling (reviewed by Park et al 25 ). Whether higher-order oligomeric complexes can form at the plasma membrane has not, however, been clearly demonstrated, although the M2-muscarinic receptor has been suggested to be capable of forming a trimer. 26

Not well appreciated nor understood, however, is the likely important role that oligomerization of GPCRs plays in the biosynthesis and trafficking of nascent GPCRs to the cellular surface. Indeed, multiple GPCRs including the β2-AR 27 and vasopressin receptors 28 undergo constitutive homodimerization early in the biosynthetic pathway, likely occurring in the ER. Expression of mutant β2-ARs constructed to either lack an ER-export motif or to contain a heterologous ER-retention signal led to entrapment of wild-type β2-AR in the ER, likely because of receptor dimerization. 27 Importantly, addition of a peptide corresponding to the putative glycophorin-like dimerization motif in the sixth-transmembrane domain of the β2-AR inhibited both receptor dimerization and transit to the cell surface. 29 Similar results in which mutants of the V2R 30 or the D2 dopamine receptor 31 act as dominant negatives for plasma membrane expression of their respective wild-type receptors suggest that receptor oligomerization before cell surface delivery may be a general mechanism by which multiple members of the GPCR family are regulated.

In addition to homodimerization, early heterodimerization is also like to play an important role in the proper targeting of some GPCRs, as recently demonstrated for the α1D-AR, which required heterodimerization with the closely related α1B-AR for cell surface expression. 32 Immunoprecipitation studies in which epitope-tagged β2-AR and Ang II type 1 (AT1) receptors (AT1Rs) were coexpressed suggested that the receptors are able to form oligomers before their localization on the plasma membrane, as the amount of immunoprecipitated receptor complex was unaffected by exposure to either agonist or antagonist. 33 Further studies have demonstrated the unanticipated finding that heterodimerization between the β2-AR and either an olfactory receptor 34 or the α1D-AR 35 facilitates receptor ER export and cell surface expression. Finally, expression of the β2-AR along with the δ and κ opioid receptors in cultured cells leads to heterooligomerization of the β2-AR with either the δ or κ opioid receptor at the plasma membrane. 36 Interestingly, this association did not affect ligand binding or functional properties of the receptors but did alter the trafficking properties. In the δ-β2 cells, δ receptors underwent β2-AR agonist-stimulated internalization and β2-AR underwent opioid-mediated endocytosis, whereas in κ-β2-AR cells, the β2-AR did not internalize in response to either β2-AR agonist or opioid. 36 Although this is a single example, such results suggest that GPCR heterooligomerization may be an important way of modulating GPCR trafficking and signaling. It is important to note, however, that in each oligomerization study described above, overexpression of the receptor(s) of interest was performed and that such alterations in cellular receptor content may modify the endogenous molecular interactions that occur in the absence of receptor overexpression.

Thus, in addition to the important role that endogenous molecular chaperones such as the HSP and RAMP family proteins play in protein folding and ER export of GPCRs, homo- and heteroreceptor oligomerization also likely play a critical step in the pathway used by at least some GPCRs for cellular trafficking, although it is at present unclear whether oligomerization following protein synthesis is a general pathway used by all GPCRs.

Cell Surface Stability

In order for a GPCR to transduce an extracellular signal, it must both traffic correctly to and be retained at the cellular surface to allow for receptor/ligand interaction. Multiple proteins not directly involved in the signal transduction cascade have been identified which stabilize receptor surface expression. These include spinophilin, Homer, actin-binding protein 280/filamin A, protein 4.1N, muskelin, and postsynaptic density-95 (PSD-95) (reviewed by Tan et al 12 ). Of these, PSD-95, a multiple PDZ domain–containing scaffolding protein, has been most conclusively shown to specifically interact with a GPCR fundamental to cardiovascular biology, namely the β1-AR. This interaction occurs via the third PDZ domain of PSD-95, which interacts with the carboxyl terminus of the β1-AR. Interestingly, overexpression of PSD-95 decreased β1-AR internalization but did not affect agonist-stimulated cAMP production or receptor desensitization, suggesting a role for PSD-95 in maintaining the β1-AR at the cellular surface. 37

GPCR Trafficking: Endocytosis

Much work over the past several decades has illuminated our current understanding of the molecular mechanisms underlying GPCR removal from the cell surface. Fundamental to this process are 2 families of proteins, the GPCR kinases (GRKs) and the β-arrestins, both of which were initially identified in studies of GPCR desensitization and which are involved in removal of ligand-activated GPCRs from the plasma membrane. Additional work has identified an ensemble of accessory proteins, which interact with the GRK and β-arrestin classes of proteins, and much recent effort has been devoted to delineating the details of these multiple interactions that are inherent to the process of GPCR endocytosis. Furthermore, as will be described below, the agonist-induced posttranslational ubiquitination of both receptor and β-arrestin play definitive and discrete roles in regulating the life cycle of GPCRs (Figure, B).

Role of the Lipid Microenvironment in GPCR Trafficking

Understanding of the importance of the membrane lipid microenvironment for GPCR signaling and trafficking is rapidly evolving. As example, it has recently been demonstrated that following translation, the AT1R requires caveolin as an intracellular molecular chaperone for trafficking to the plasma membrane. 38 Moreover, once at the cell surface, it is clear that some subsets of GPCRs are preferentially segregated to discrete regions of the membrane defined as lipid rafts. 39–41 GPCRs of fundamental importance to cardiovascular biology that have been localized to lipid rafts and/or caveolae include the adenosine A1, α1-AR, β1-AR, β2-AR, AT1R, the endothelin (ETA-A and ET-B) receptors, and the M2-muscarinic receptors. 42

Caveolae, a specific type of lipid microdomain, represent for some GPCRs the preferred microenvironment for certain events such as signaling. However, a receptor’s maintenance within a specific microenvironment may be subject to dynamic regulation. Indeed, reversible GPCR modifications have been described, including both covalent attachment of a lipid to the GPCR or GPCR phosphorylation, which can shift the GPCR between different membrane milieus. For example, a reversible lipid modification (eg, palmitoylation/depalmitoylation of cysteine residues) has been demonstrated to target GPCRs such as the 5-HT1a receptor to lipid rafts. 43 Interestingly, agonist-induced endocytosis of the β1-AR via clathrin-coated pits (CCPs) in human embryonic kidney (HEK) 293 cells requires GRK phosphorylation of the receptor, whereas endocytosis of the β1-AR in lipid rafts/caveolae is dependent on the receptor undergoing protein kinase A phosphorylation. 44 Further evidence supporting the importance of GPCR membrane microdomain restriction is that confinement of the β2-AR to caveolae has been reported to be of critical importance for regulation of the intrinsic contraction rate in neonatal cardiac myocyte membrane preparations. 45 The importance of the lipid microenvironment for the assemblage of signaling scaffolds beneath the GPCR/membrane interface is also an area of active investigation and may play a role in multiple aspects of GPCR trafficking but is beyond the scope of this review. For a more complete review of the role of the lipid microenvironment on both GPCR signaling and trafficking, refer to several recent excellent reviews. 39,42

Agonist-Dependent Versus Agonist-Independent GPCR Internalization

Receptor internalization following agonist exposure is a well-documented response for a wide variety of GPCRs important for cardiovascular biology. As example, the prototypic GPCR, the β2-AR, was initially shown to internalize following exposure to agonist, as demonstrated by loss of surface binding of a nonpermeable membrane ligand. 46 Use of a membrane permeant ligand, however, demonstrated that the β2-AR was still ligand accessible, suggesting that the receptor was sequestered in an intracellular compartment following agonist treatment. 46 Alternatively, whereas exposure of the AT1R to Ang II leads to receptor internalization and endosomal sequestration, the Ang II type 2 receptor (AT2R) does not undergo endocytosis with Ang II addition, demonstrating that subtype-specific receptor sorting and internalization can occur within the cardiovascular GPCR system. 47

Internalization for most GPCRs occurs on the order of minutes and correlates with receptor phosphorylation by the GRKs and subsequent β-arrestin translocation, as will be discussed below. Indeed, agonist-induced β2-AR receptor internalization can be inhibited either by mutations of the β2-AR, which inhibit agonist-induced GRK phosphorylation 48 or by mutations in the β-arrestin proteins. 49 Following internalization, receptors may be either recycled to the cell surface or targeted for lysosomal degradation (reviewed by Bohm et al 50 ).

Internalization of GPCRs in the absence of agonist has also been examined. Although mean rates of internalization vary between receptors assayed, rates are in general substantially slowed in the absence relative to the presence of the cognate ligand of a GPCR. The β2-AR, as example, undergoes sequestration from the cell surface with a half-life of approximately 10 minutes in the presence of agonist but remains on the cell surface for greater than 1 hour in the absence of agonist. 51

The Role of Accessory Proteins in the Endocytosis of GPCRs

Endocytosis of GPCRs can occur via caveolae, clathrin-coated vesicles, or uncoated vesicles. 52 Although short linear amino acid stretches in the cytoplasmic domains of GPCRs likely play a role in their endocytosis, the majority of work to date has demonstrated that much of GPCR endocytosis is primarily regulated by GRK and β-arrestin–dependent processes involving clathrin-coated pits.

GPCR Kinases

As shown for multiple GPCRs, the serine/threonine-specific GPCR kinases (GRKs) are recruited following agonist binding to the cytoplasmic surface of the activated receptor, leading to receptor phosphorylation. The phosphorylated surface of the GPCR is then competent to serve as a platform for the cytosol to membrane translocation of the β-arrestin proteins (reviewed by Shenoy and Lefkowitz 53 ).

The GRK family of kinases is composed of 7 members that share significant amino acid and structural homology (reviewed previously 54,55 ). Within this family, 4 kinases (GRKs 2, 3, 5, and 6) are expressed broadly and are believed to play a role in GPCR phosphorylation within the cardiovascular system. GRK2 and GRK3 reside in the cytosol in the absence of agonist and translocate to the membrane following GPCR stimulation. GRK2/3 translocation and membrane localization are mediated in part by their binding to heterotrimeric G protein βγ subunits. 56 GRK5 and GRK6, on the other hand, are constitutively localized to the plasma membrane. Whereas GRK6 palmitoylation is essential for membrane association, 57 localization of GRK5 to the plasma membrane is believed to be attributable to an electrostatic interaction between the highly basic carboxyl terminus of GRK5 and phospholipids at the plasma membrane. 58,59

Although GRK-specific phosphorylation of the cytoplasmic surface of agonist-occupied GPCRs mediates β-arrestin recruitment, the structural features common to activated receptors that are recognized by the GRKs remain largely unknown. Indeed, it is a question of fundamental importance as to how members of this limited group of broadly expressed GRKs are able to phosphorylate such a diverse array of activated GPCRs and thereby lead to β-arrestin recruitment.

Functionally, 2 classes of GPCRs, denoted “class A” and “class B,” can be defined, based on the relative stability of the GPCR/β-arrestin interaction. For the β2-AR (a class A receptor) and the vasopressin receptor (a class B receptor), these determinants appear to be present within the carboxyl termini of the receptors, as the stability of their interaction with β-arrestin, as well as their ability to be dephosphorylated, recycled, and resensitized was completely reversed in mutant receptors in which their carboxyl-terminal tails were switched. 60

Interestingly, whereas in vitro studies have localized GRK2- and GRK5-mediated phosphorylation sites of the β2-AR to distal portion of the cytoplasmic tail of the receptor, 61 more recent studies in intact cells have suggested that agonist induced β2-AR phosphorylation occurs in the proximal portion of the carboxyl terminus of the receptor. 62 Although the observed β2-AR proximal tail phosphorylation was believed to be mediated by GRK rather than protein kinase A, this was not confirmed. Thus although GRK-mediated phosphorylation of agonist-stimulated GPCRs underlies β-arrestin recruitment and thereby initiates GPCR endocytosis in CCPs, many of the molecular details remain to be determined. Notably, a recent study using high-throughput RNA interference implicated GRKs as playing a more general role in the process of clathrin-mediated endocytosis itself. 63

Β-Arrestin As an Endocytic Adaptor Protein

Within humans, there exist 2 isoforms of the nonvisual β-arrestin proteins, namely β-arrestin1 and β-arrestin2, both of which show ubiquitous tissue distribution. In addition to their well-described role in limiting receptor-G protein interaction, the β-arrestin proteins also serve to both recruit and physically bridge the receptor to the endocytic machinery. Experimentally, receptor mutations that impair agonist-induced GPCR phosphorylation limit β-arrestin recruitment and lead to poor receptor internalization, as demonstrated for a β2-AR in which all of the GRK phosphorylation sites had been altered. 48 Further, expression of “dominant-negative” mutant β-arrestin proteins (such as β-arrestin1 V53D or β-arrestin2 V54D) inhibit β2-AR internalization. 64 In addition, the β-arrestin proteins themselves are able to interact directly with the essential components of the clathrin-coated vesicle (CCV) coat machinery, namely the heterotetrameric AP-2 complex, 8 as well as clathrin, 9 and these interactions are critical both for recruitment of the β2-AR into clathrin-coated pits as well as for receptor internalization. Studies with other GPCRs, including the α2-AR 65 and the A2B adenosine receptor 66 have also shown important roles for the β-arrestins in receptor endocytosis.

Interestingly, although the 2 β-arrestin isoforms exhibit nearly 80% amino acid identity, 6 they do not appear to perform redundant biologic roles and, indeed, exhibit differences in their regulation. Whereas β-arrestin1 is phosphorylated by extracellular signal regulated kinase (ERK) enzymes, 67 β-arrestin2 is phosphorylated by casein kinase II. 68 For both β-arrestin proteins, however, the phosphorylation/dephosphorylation status appears to regulate the ability of the β-arrestin protein to promote internalization of the β2-AR via clathrin-coated vesicles.

As noted above, analysis of agonist-stimulated β-arrestin translocation for a variety of GPCRs suggests there exist 2 largely distinct classes receptors with which the β-arrestins associate, denoted class A and class B GPCRs. Following agonist exposure, class A receptors including the β2-AR, endothelin A receptor, and α1b-AR preferentially recruit β-arrestin2. 69 In contrast, class B receptors, including the AT1aR and V2 receptor, are able to bind both β-arrestin isoforms with nearly equal affinity. 70 Whereas the β-arrestin1/2–class A receptor interactions occur solely at the plasma membrane and are lost following GPCR internalization, β-arrestin1/2 interactions with class B receptors are much more stable and can be detected on endosomal structures following receptor endocytosis (reviewed by Pierce and Lefkowitz 71 ). Further, class A receptors generally recycle to the plasma membrane rapidly, whereas class B GPCRs recycle more slowly. The role of ubiquitination in modulating GPCR/arrestin interaction is likely important and is discussed below. Mouse embryonic fibroblasts generated to lack both β-arrestin isoforms showed a marked impairment of agonist-stimulated internalization of either the class A β2-AR or the class B AT1aR, whereas only β2-AR internalization was affected by the single deletion of the β-arrestin2 isoform. 69 Studies using RNA interference technology to selectively ablate the β-arrestin proteins have shown similar results with respect to internalization of the β2-AR and AT1-AR as model class A and B receptors, respectively. 72

Agonist-Induced GPCR Ubiquitination and Sorting

Posttranslational modification of substrate proteins by the covalent attachment of ubiquitin (ubiquitination), originally discovered in the context of cellular protein degradation, has recently been shown to play a noncanonical role in regulating the postendocytic sorting of several membrane proteins including GPCRs. 73,74 Protein ubiquitination is mediated by the concerted action of 3 enzymes. The first 2 enzymes (E1 and E2) are responsible, respectively, for activating ubiquitin and escorting the activated ubiquitin. The third enzyme, E3 ubiquitin ligase (E3), recognizes and modifies the substrate in a timely fashion. 75 For the β2-AR, both GRK-mediated phosphorylation and β-arrestin binding are essential for receptor ubiquitination to occur. 76 Importantly, this agonist-stimulated β2-AR ubiquitination modification is necessary for the receptor to undergo degradation in lysosomes. Further, ubiquitin-dependent lysosomal degradation is applicable to other GPCRs such as V2R and the protease-activated receptor2 (PAR2). 77,78 For both the β2-AR and V2R, receptor ubiquitination requires the β-arrestin proteins. Although the nature of this requirement is not entirely clear, 1 supposition is that the β-arrestins may serve as adaptors to bring as yet unidentified E3 ligase(s) to the receptors in a stimulus dependent fashion. In the case of the PAR2 and the CXCR4 receptors, ubiquitination is mediated by the E3 ligases c-Cbl 78 and AIP4, 79 respectively, but no involvement of the β-arrestins has as yet been demonstrated.

For the above mammalian GPCRs, as well as for others such as the chemokine receptor CXCR4, 80 receptor ubiquitination is not required for internalization per se but is crucial for the sorting of ubiquitinated receptors to lysosomes. A recent study of the β1-AR in a heterologous cellular system reported the resistance of the receptor protein to ubiquitination as well as agonist-mediated degradation, suggesting a strong relationship between this receptor modification and downregulation pathways. 81

Whereas the degradation of GPCRs and other membrane proteins is known to occur in lysosomes, that of some membrane receptors such as the single-membrane spanning erythropoietin receptor involve both lysosomes and 26S proteasomes, the megaprotease complexes that degrade most cellular proteins. 82 Interestingly, however, a recent report suggests that ubiquitination and proteasomal degradation of newly synthesized intracellular A2A adenosine receptors serves as a method of ER quality control (Figure, A). Importantly, this degradation could be overcome by the coexpression of USP4, a deubiquitinating enzyme belonging to a family of enzymes that catalyze removal of ubiquitin from the modified substrates. 83 USP4 expression led to more robust functional expression of the A2A receptor at the plasma membrane, suggesting that deubiquitination can facilitate cell-surface targeting of membrane proteins (Figure, A).

On the other hand, GPCR internalization can be regulated by the agonist-dependent ubiquitination of β-arrestin by the E3 ligase Mdm2, as demonstrated for the β2-AR. 76 Moreover, the stability of β-arrestin/GPCR binding that defines GPCRs as class A or B (as described above) also correlates with the ubiquitination status of the β-arrestin proteins. The separation of β-arrestin from class A GPCRs results from rapid β-arrestin deubiquitination, whereas the more stable β-arrestin interaction with class B receptors is caused by the sustained ubiquitination of β-arrestin. 84 As will be described below, ubiquitination of β-arrestin appears to not only be capable of regulating GPCR trafficking properties but also likely plays an important role in directing downstream signaling events.

Sorting Signals Used for GPCR Intracellular Trafficking and Endocytosis

The identification of short, linear amino acid signals present in the intracellular domains of transmembrane proteins responsible for mediating the intracellular sorting and endocytosis of a transmembrane protein from the plasma membrane has been the focus of much work over the past 2 decades. Such sequences are believed to act as recognition sites for components of the cellular adaptor protein machinery necessary for intracellular protein trafficking. The importance of such signals contained within the intracellular domains of GPCRs, however, is less well described than that for other transmembrane proteins. Albeit limited in number, exceptions to the general paradigm of GRK/β-arrestin–mediated endocytosis have been delineated for 7-membrane-spanning receptors important to the cardiovascular system. Interestingly, the best-described motifs are analogous to those used by non-GPCR transmembrane receptors and include di-leucine–based (LL or LXL) and tyrosine-based (NPXY or NPXXY, or YXXO) motifs.

As a prototypic GPCR, the β2-AR contains a di-leucine motif within its carboxyl terminus, alteration of which does not affect the ability of the receptor to traffic correctly to the cell surface, bind agonist, or to activate adenylyl cyclase. Agonist addition, however, does not lead to internalization of this mutant β2-AR, 85 demonstrating a role for di-leucine motif of the β2-AR in agonist-induced receptor endocytosis. Similarly, mutations introduced into the di-leucine motif in the cytoplasmic tail of the vasopressin 1a receptor (V1aR) significantly impaired agonist-induced receptor internalization. 86 Interestingly, however, mutation of the analogous di-leucine motif in the V2R resulted in a receptor that was unable to escape from the ER, suggesting a role for this motif in V2R maturation. 87

Tyrosine-based sorting signals have also been shown to be necessary in the trafficking of the β2-AR. Mutation of Y326 in the human β2-AR, located at the proposed junction of the seventh-transmembrane domain and the proximal portion of the carboxyl terminus and conserved in position across many members of the large superfamily of GPCRs, does not affect the ability of the receptor to traffic correctly to the cell surface, bind agonist, or to activate adenylyl cyclase when the receptor is overexpressed. 88 The mutation, however, did completely abolish β2-AR agonist-induced internalization. Complicating this interpretation, however, was the finding that lower expression levels of mutant β2-AR resulted in the loss of ligand binding and adenylyl cyclase coupling, likely because of intracellular retention of the mutant receptor. 89 More recently, a highly conserved tyrosine-based motif (YXXO) in the cytoplasmic tail of the protease-activated receptor-1 (PAR1), a GPCR for thrombin, which has previously been shown to undergo β-arrestin–independent internalization, was shown to be necessary for agonist-mediated but not constitutive internalization. 90 Finally, more recent studies have shown a direct interaction between a stretch of 8 arginines contained in the carboxyl terminus of the α1b-AR and the AP-2 μ subunit. 91 Whether the alternative tyrosine-based motif found in PAR1 or the nonclassical arginine motif identified in the α1b-AR plays a role in the endocytosis and intracellular trafficking of other GPCRs classically identified as fundamentally important to the cardiovascular system remains to be determined.

Role for Additional Proteins in the Endocytosis of GPCRs

In addition to the well-recognized roles of the GRK and β-arrestin proteins in GPCR internalization, multiple other proteins have been demonstrated to be important in the endocytic process. A partial list and description of the role these proteins play in GPCR endocytosis is discussed below.

ADP-Ribosylation Factor 6

ADP-ribosylation factor 6 (ARF6) is 1 member of the ARF family of small GTP-binding proteins known to be key players in vesicular trafficking events. In addition to its role in binding AP-2 and clathrin, β-arrestin is also able to directly bind to ARF6 and modulate its activity. ARF6 activation requires the exchange of GTP for GDP, a reaction that is catalyzed by the ARF guanine nucleotide exchange factor (GEF) ARNO ( AR F N ucleotide-binding site O pener). Importantly, ARNO is constitutively associated with β-arrestin2. 92 As shown for the β2-AR, expression of mutant ARF6 proteins containing single amino acid substitutions rendering them deficient in their ability to either bind (T27N) or hydrolyze (Q67L) GTP inhibited agonist-induced β2-AR internalization. 92 Overexpression of ARNO alone, however, increases β2-AR internalization by stimulating GTP nucleotide exchange on ARF. Thus, agonist-promoted recruitment of β-arrestin to an activated receptor leads to the local regulation of endocytosis by β-arrestin attributable to its inherent ability to bind both ARNO and ARF6.

In addition to requiring a GEF protein, ARF proteins also require a GTPase-activating protein (GAP) to accelerate hydrolysis of bound GTP. Initially identified as GRK-interacting proteins (GITs), GIT1 and GIT2 are zinc finger–containing proteins that function as GAPs for ARF6. 93,94 GIT1 overexpression reduces the internalization of transmembrane receptors in CCPs and CCVs, including the β1- and β2-ARs, the adenosine 2B receptor, and the M1-muscarinic receptor. 95 Importantly, ARF-GAP activity of the GIT proteins is stimulated by phosphatidylinositol 3,4,5-trisphosphate, whereas other ARF-GAPs, such as ARF-GAP1, are stimulated by phosphatidylinositol 4,5-bisphosphate and diacylglycerol. 94 This raises the interesting possibility that GIT regulation of ARF6 activity may be integrated through activation of the phosphatidylinositol 3-kinase signaling pathway.

Phosphatidylinositol 3-Kinase

Phosphatidylinositol 3-kinases (PI3Ks) are a conserved family of kinases with both lipid and protein kinase activity which can be activated in response to GPCR stimulation. As a family, they have been shown to play important roles in an array of cellular functions as divergent as cell survival, cell motility, and receptor endocytosis. Within the cytosol, PI3K is constitutively complexed with GRK2. 96 As demonstrated for the β2-AR, agonist binding induces translocation of the GRK/P13K complex to the activated receptor, formation of phosphatidylinositol 3,4,5-trisphosphate, and subsequent recruitment of AP-2 and clathrin, leading to receptor endocytosis. Importantly, receptor internalization is blocked by overexpression of the portion of PI3K that mediates its interaction with GRK2, as well as by the specific 3,4,5 trisphosphate lipid phosphatase PTEN, demonstrating the importance of the lipid kinase activity of PI3K for the localized production of D-3 phosphoinositides in regulating ligand-induced endocytosis of the β2-AR. 97

As noted above, in addition to possessing lipid kinase activity, PI3K family members are also able to function as protein kinases, although the importance of this activity has remained largely obscure given the limited number of protein substrates recognized by PI3Ks. 98 Recently, however, the importance of PI3K protein kinase activity in the regulation of β2-AR endocytosis has been illuminated. In an elegant study, Naga Prasad et al identified the cytoskeletal protein nonmuscle tropomyosin (an actin filament binding protein) as a substrate for the γ isoform of PI3K and further demonstrated that PI3K can selectively phosphorylate a single site (S61) within tropomyosin. 99 Alteration of this site within tropomyosin to mimic constitutive phosphorylation (S61D) leads to complementation of a protein kinase defective PI3K, whereas change to a phospho-deficient residue (S61A) blocked agonist-induced β2-AR internalization. Thus, through both its lipid and kinase activity, PI3K plays a central role in the agonist-induced removal of the β2-AR, the prototypic model cardiovascular GPCR, from the cell surface. Whether this paradigm will extend to other members of the cardiovascular GPCR family is at present unknown but seems likely, given that endocytosis via AP-2 containing CCVs is the predominant mechanism of internalization for most ligand-activated GPCRs.

Intracellular Trafficking: Sorting/Recycling/Degradation

Once internalized from the cell surface, GPCRs can be sorted along multiple pathways (Figure, C). They may undergo dephosphorylation, resensitization, and be recycled back to the plasma membrane. Alternatively, GPCRs may be targeted for degradation via the endosomal/lysosomal pathway. Finally, multiple GPCRs have more recently been shown to initiate G protein–independent intracellular signaling pathways following endocytosis, as is discussed below. These multiple trafficking fates for internalized GPCRs are the subject of an excellent recent review. 100 We would like to highlight a few proteins important for these processes.

Na + /H + -Exchanger Regulatory Factor

Following adrenergic receptor stimulation, it has long been recognized that G protein–dependent changes in cellular metabolism, excitability, and growth occur. 101 Likewise, cellular changes apparently independent of G protein activation have been demonstrated, including alterations in cellular pH via regulation of the Na + /H + exchanger. Recent work has established that agonist stimulation of the β2-AR promotes direct association of the extreme carboxyl terminus of the receptor with the first PDZ domain within Na + /H + -exchanger regulatory factor-1 (NHERF1). 102 Similar associations have been shown to occur for other GPCRs containing sequences conforming to the consensus motif D-S/T-x-L, including the purinergic P2Y1 receptor and the CFTR, 103 whereas other GPCRs such as the parathyroid hormone receptor 104 have been shown to interact with both PDZ domains of both NHERF1 and NHERF2 via a slightly different (ETVM) PDZ consensus motif. For the β2-AR, disruption of this interaction markedly impairs agonist-induced changes in intracellular pH.

NHERF is also of critical importance for proper intracellular sorting of the β2-AR. In addition to its ability to bind the extreme carboxyl terminus of the β2-AR via its PDZ domain, NHERF is able via its ezrin–radixin–moesin (ERM) domain to bind to the actin cytoskeleton through association with ERM proteins. Importantly, mutations generated to disrupt the interaction of NHERF with either the β2-AR carboxyl terminus or with ERM proteins lead to significant agonist-induced lysosomal degradation of the β2-AR following endocytosis, rather than recycling. 105 As noted above, multiple additional GPCRs have been shown to interact with the NHERF proteins. Although NHERF has been shown to play a role in the recycling of other GPCRs such as the κ opioid receptor (reviewed by Liu-Chen 106 ), the generalizability of the role that NHERF plays in the recycling of other GPCRs, particularly in the cardiovascular system, remains to be determined.

N-Ethylmaleimide-Sensitive Fusion Protein

In a study to identify β-arrestin binding partners, β-arrestin1 was found to interact in both yeast 2-hybrid and in vitro assays with N-ethylmaleimide-sensitive fusion protein (NSF), an ATPase essential for many intracellular transport functions. 107 Furthermore, overexpression of NSF in HEK293 cells led to enhanced agonist-induced β2-AR internalization and could rescue the effects of a β-arrestin1 mutant (S412D) previously shown to function as a dominant negative for β2-AR internalization. Interestingly, the β2-AR is also able, via its extreme carboxyl terminus, to bind directly to NSF. 108 The β2-AR/NSF interaction is agonist dependent and requisite for efficient agonist-mediated β2-AR internalization. Importantly, whereas wild-type β2-ARs recycle to the cell surface following exposure to the antagonist propranolol, β2-ARs containing mutations in their distal carboxyl termini remain sequestered intracellularly, demonstrating the importance of the β2-AR/NSF interaction for proper β2-AR recycling. Thus, although it is clear that proteins that bind to the extreme carboxyl termini of GPCRs, such as NHERF and NSF, serve to regulate the intracellular sorting of the receptors such as the β2-AR, the extent to which the trafficking of other GPCRs is modulated by these or similar as yet unidentified proteins remains to be elucidated.

Sorting Nexin 1

Following internalization, GPCRs may be either recycled to the plasma membrane or undergo sorting to the endosomal/lysosomal pathway for degradation. Although fundamentally important as a mechanism for downregulation of certain GPCRs, such as the PAR1, which recognizes the coagulant protease thrombin, comparatively less is known about the molecular components that direct postendosomal sorting and recycling of the receptors.

Although sorting nexin 1 (SNX1) was originally identified as a membrane-associated protein that interacts with the receptor tyrosine kinase, epidermal growth factor receptor (EGFR) and functions in EGFR sorting to lysosomes, 109 SNX1 has also been demonstrated to interact with PAR1 in an agonist-dependent manner. 110 Overexpression of a SNX1 carboxyl terminal–deletion mutant did not significantly impair agonist-induced PAR1 internalization and accumulation in early endosomes but was able to impede endosome to lysosome sorting of PAR1 and thus markedly inhibit PAR1 degradation. 110 Additional studies, in which depletion of endogenous SNX1 in HeLa cells via small interfering RNA technology markedly impaired agonist-induced PAR1 degradation, buttress the suggestion that SNX1 plays a role in the intracellular trafficking of PAR1 from endosomes to lysosomes. 111 Whether SNX1 or other SNX proteins can mediate the endosomal to lysosomal sorting of GPCRs other than PAR1 remains to be determined but is certainly possible given the general conservation of trafficking themes shared across members of this large protein family.

Rab GTPases

A second class of proteins shown to regulate the movement of GPCRs both during and subsequent to endocytosis are the Rab proteins, a family of Ras-like small GTP binding proteins. Importantly, different Rab GTPase family members selectively associate with specific intracellular structures including both recycling and sorting endosomes, where they mediate multiple steps of vesicular membrane trafficking including vesicle budding, docking, and fusion (reviewed by Seachrist and Ferguson 112 ).

Within the cardiovascular system, Rab5 plays a central role in the agonist-dependent CCV-mediated internalization and early endosomal localization of some GPCRs, including the β2-AR. 113 Expression of a dominant negative Rab5 (S34N) blocked β2-AR internalization, whereas a constitutively active Rab5 (Q79L) did not significantly alter β2-AR internalization. Interestingly, whereas the β2-AR is not able to directly associate with Rab5, the AT1R does bind Rab5 directly via the carboxyl terminus of the receptor and localizes to Rab5-containing endosomal structures following endocytosis. 114 Furthermore, the interaction of Rab5 with the AT1R on endosomes appears to be necessary for preventing sorting of the internalized receptor to lysosomes. 115 Unlike internalization of the β2-AR, however, endocytosis of the AT1R does not appear to be dependent on Rab5, 114 demonstrating the intracellular diversity in GPCR trafficking roles played by even single members of the Rab protein family.

Whereas Rab5 plays a role in GPCR trafficking events associated with GPCR internalization and/or early endosomal formation, multiple additional Rab family members have been implicated in other GPCR-trafficking events, including Rab4 and Rab11 in receptor recycling, and Rab7 in endosomal to lysosomal sorting. 115 In a transgenic mouse model in which a dominant-negative Rab4 (S27N) was overexpressed in the heart, mice had impaired responsiveness to both endogenous and exogenous catecholamines. 116 Furthermore, when these mice were crossbred with mice also overexpressing a cardiac-specific human β2-AR, the resultant mice had an abnormal intracellular vesicular β2-AR accumulation, as well as significantly reduced cardiac contractility. Such results suggest that proper Rab4-mediated regulation of β2-AR receptor recycling is necessary for maintenance of normal catecholamine responsiveness and receptor resensitization. Whether Rab4 also plays a similar role in vivo in the recycling of other GPCRs within the cardiovascular system is currently unknown. For a more complete review of the diverse roles that members of the Rab protein family play throughout nearly all aspects of GPCR trafficking, refer to several recent excellent reviews. 100,112

Other Proteins With Roles in GPCR Endocytosis and Postendocytotic Trafficking

Many additional proteins have been identified and characterized that play fundamental roles in the endocytosis and postendocytic trafficking of GPCRs. These include, but are not limited to, proteins involved in CCV formation and membrane scission such as AP-2, clathrin, dynamin, Hrs, and GASP, 117–119 as well as multiple scaffolding proteins that have been demonstrated to interact with the β1-AR including postsynaptic density 95 (PSD95), 120 membrane-associated guanylate kinase inverted-2 (MAGI-2), 121 and the endophilins. 122 Because of the limited scope of this review, we have chosen to highlight only selected proteins as above.

Internalization/Trafficking and Signaling

Although internalization mechanisms leading to receptor sequestration away from the source of stimulus have been primarily considered as signal desensitization pathways, a large body of research now exists in support of continued signaling that is coupled with GPCR endocytosis. 123 Moreover, although it is generally believed that endocytic “addresses” determine the extent of signaling, continued signaling mechanisms during the trafficking of proteins may actually determine the exact endocytic route by regulating processes such as vesicle fusion and cargo transfer. As such, it is increasingly evident that the 2 processes, trafficking and signaling, are not only connected but likely inextricably intertwined (Figure, D).

In the case of GPCRs, β-arrestin has been clearly demonstrated to play an essential role in the desensitization of G protein–mediated second messenger signaling. More recently, however, the β-arrestin proteins have also been shown to facilitate both receptor internalization via CCVs, as well as to couple the receptors to and activate nonreceptor tyrosine kinases (eg, members of the c-Src family), thereby mediating a second wave of signaling. 124,125 In addition, on binding to activated GPCRs such as the AT1aR, β-arrestin has also been shown to be capable of sequestering and scaffolding signaling kinases, as well as targeting active signaling complexes into cytoplasmic endosomal microcompartments. 125,126 Importantly, chemical or molecular inhibition of receptor endocytosis (or, alternatively, expression of β-arrestin mutants that impair receptor internalization), can lead to a decline in such downstream signaling, demonstrating a coupling between endocytosis and signaling. 124,127

In addition to c-Src activation, β-arrestin has been shown in multiple studies to function as a signaling intermediate in GPCR signal transduction to mitogen-activated protein kinase (MAPK) pathways, under conditions where G protein coupling is virtually absent. 125,128,129 Although this β-arrestin–dependent signaling has been most well characterized for ERK activation, it may also apply to other signaling kinases and appears to play roles in a variety of cellular responses including chemotaxis and antiapoptosis. Although it has not yet been conclusively determined that receptor endocytosis is required for this β-arrestin pathway, recent data suggest that the trafficking patterns of β-arrestin/receptor complexes may play a significant role. 130,131 Interestingly, β-arrestin ubiquitination at specific acceptor sites (eg, Ang II–induced ubiquitination of lysines 11 and 12 in β-arrestin2) is crucial for stable β-arrestin/AT1R interaction as well as the formation and endosomal localization of AT1R-MAPK signalosomes. Whether this observation may be broadly applicable to other GPCRs relevant to cardiovascular biology is currently unknown and is an area of active investigation.

Conclusions

Within the cardiovascular system, regulation of GPCR trafficking is of fundamental importance both for physiological homeostasis and the molecular response to physiological perturbation. There is efficient and coordinated movement of GPCRs from synthesis to the cellular surface, where they can interact with components of the extracellular milieu to transduce signals to intracellular effectors and subsequent GPCR retrieval from the cell surface. These processes highlight the intricate cellular balance that has developed to exquisitely regulate hormonal responsiveness at the tissue level. Indeed, much has been learned about GPCR trafficking within the cardiovascular system over the past 2 decades, as evidenced by the ever-expanding ensemble of identified proteins crucial to this process. The recent recognition that GPCR signaling may occur within the cardiovascular system via G protein–independent/β-arrestin-dependent pathways raises the fascinating possibility that pharmacologically relevant cardiovascular ligands may be developed that allow for selective GPCR signaling via the activation of proteins initially believed only to have a role in limiting GPCR signaling and promoting GPCR removal from the cell surface.

Original received June 30, 2006 revision received August 10, 2006 accepted August 11, 2006.

We thank Donna Addison and Elizabeth Hall for excellent secretarial assistance.


If a cell has two different GPCRs, how does the cell differentiate between the phosphorylation cascade caused by each? - Biology

C2006/F2402 '11 OUTLINE OF LECTURE #15

(c) 2011 Dr. Deborah Mowshowitz, Columbia University, New York, NY . Last update 03/24/2011 02:16 PM .
Handouts: 14A -- Overview of Signaling -- the Biological Big Bang Theory
14B -- Structure of G proteins and GPCR's cAMP pathway
15A -- Lining of the GI Tract & Typical Circuit
15B -- Homeostasis)-- Seesaw view for Glucose and Temperature Regulation

I . How do Intracellular Receptors Work? (cont). See Sadava fig. 7.9 (15.8)

A-E. See last lecture.

F. Example -- Estrogen (A steroid)

1. Basic Mechanism . E → binds to estrogen receptors → complex complex binds to estrogen response elements (EREs) in regulatory regions of (multiple) target genes. Binding → increased transcription of some genes (genes activated) decreased transcription of others (genes repressed).

2. Example of some proteins controlled by E -- controls production of receptors for other hormones. For example, during pregnancy controls production of receptors for oxytocin (in uterus) and prolactin (in breast). Oxytocin controls birth contractions prolactin controls milk production.

a. In uterus: estrogen binding → activates transcription of gene for oxytocin receptors → production of new receptors for oxytocin = up regulation of oxytocin receptors. Receptors needed to allow response to contraction signal (oxytocin) → contractions → birth.

b. In breast: estrogen binding → inhibits transcription of gene for prolactin receptors → down regulation of prolactin receptors at birth, estrogen level falls and inhibition stops → transcription of gene for prolactin receptors → synthesis of prolactin receptors → response to lactation signal (prolactin).

  • All cells (except immune system) have the same cis acting regulatory sites -- the same HRE's, enhancers, etc.
  • It's the trans acting factors such as hormone receptors that vary, not the cis acting regulatory sites.
  • All cells have the same genes for the trans acting factors, receptors etc., but different genes are used to make regulatory proteins in different cells.

Other examples of how hormones can give different results in different tissues will be discussed later. In general, what any hormone does depends on combination of proteins (enzymes, TF's, etc.) already in target cell.

Try problem 6-19. By now you should be able to do 6-12 to 6-15.

II . Types of Cell Surface (Transmembrane) Receptors (See bottom of 14A)

A. Channels. Some receptors are themselves (parts of) channels. See AcCh receptor in previous lecture and Sadava fig. 7.6 (15.5) Other receptors are not channels, but work by opening or closing separate channels. Channels will be discussed next week by Dr. Firestein.

B. Types of Cell Surface receptors that are not channels

1. Type 1: Linked to G proteins.

a. Terminology: Called G protein linked receptors, or G Protein Coupled Receptors (GPCRs). For a generalized case, see Sadava fig. 7.8 (15.7).

b. Structure: All are 7 pass transmembrane proteins with same basic structure all belong to same protein/gene family. (See Becker 14-4 or top of handout 14B.)

c. What are they receptors for? Many hormones such as TSH & epinephrine use GPCRs.

d. How do they work? Activate a G protein, which acts as a switch to trigger amplification (details below). G proteins usually:

(1). Activate enzymes that generate second messengers (see Sadava fig. 7.8 (15.7)), or

(2). Open/close ion channels.

2. Type 2: Not linked to G proteins. To be discussed in more detail later when we get to cell cycle and cancer. For reference:

a. Many are protein kinases. In addition to extracellular, ligand binding domain, have an intracellular kinase domain, or interact with an intracellular kinase (when activated).

b. Most well known type: Receptor Tyrosine Kinases (RTKs) -- also called TK linked receptors.

c. Structure: Usually are single pass proteins that aggregate into dimers when activated.

d. What are they receptors for? Many Growth Factors use TK linked receptors or related receptors. (See Becker table 14-3 if you are curious).

e. How do they work? These usually generate cascades of modifications, but do not always use 2nd messengers. If you want to see an example, see Sadava figs. 7.6 & 7.12 (15.6 & 15.10). We won't get to details of how these work for a while.


III. G proteins -- How do they Fit In? How do they work?

A. What are the important properties of G proteins? (See Becker fig. 14-5 & Handout 14B)

1. Have active and inactive forms

a. Active form is bound to GTP

b. Inactive form is bound to GDP.

2. G-proteins act as switches in many processes (not just signaling)

a. Activation: G protein is activated by dumping GDP and picking up GTP in response to some signal.

b. Inactivation: G protein inactivates itself by catalyzing hydrolysis of GTP to GDP.

c. Why is it a switch? The G protein does not stay active for long. "Turns itself off."

B. Typical Pathway -- Role in signaling (see also handout 14B, middle panel)

ligand (1st messenger) binds outside cell activate receptor in membrane activate G protein in the membrane activate target enzyme in membrane generate small molecule (2nd messenger) inside cell

Note that the ligand (1st messenger) binds to the extracellular domain of its receptor. The remaining events are inside the cell. More on 2nd messengers below.

C. Activation & Inactivation of G Proteins

1 . GTP exchange: Mechanism of activation & inactivation

a. Activation Reaction (GTP/GDP exchange, NOT phosphorylation of GDP GTP replaces GDP):

Protein-GDP (inactive) + GTP → Protein-GTP (active) + GDP

b. Inactivation Reaction (hydrolysis of bound GTP to GDP):

Protein-GTP (active) → Protein-GDP (inactive) + phosphate.

c. Overall: GTP displaces GDP, activating the G protein GTP is then hydrolyzed (usually rapidly), returning the G protein to its inactive state.

(1). Net effect on GTP -- GTP hydrolysis: GTP ( + water) → GDP + phosphate

(2). NetEffect on G protein -- none: Protein is temporarily activated, but then inactivated. However protein cycles from inactive to active and back to inactive -- acts as switch.

d. Terminology. Since the overall result is that GTP is hydrolyzed to GDP, G proteins are sometimes called "GTPases."

2. What triggers activation?

a. General Case: Binding of a protein called a GEF (guanine-nucleotide exchange factor) causes GDP to fall off, and GTP binds.

b. In signaling: Activated GPCR = GEF. Binding of activated receptor to G protein triggers activation of G protein (causes loss of GDP).

3. What triggers inactivation?

a. G protein itself has enzymatic activity -- catalyzes inactivation (hydrolysis).

b. No trigger required -- hydrolysis of GTP to GDP happens automatically.

c. Other proteins may increase speed of hydrolysis. They are called RGS proteins (Regulators of G protein Signaling) or GAP proteins (GTPase Activating Proteins).

D. Types of G proteins

1. Subunits -- Ordinary G proteins are trimeric = they have 3 subunits.

a. Inactive G prot = heterotrimer of alpha, beta, gamma

b. Separation occurs on activation: On activation, alpha subunit (with the GTP) separates from other 2 subunits.

c. Either part may be the effector that actually acts on target -- alpha, or beta + gamma, can act as activator or inhibitor of target protein.

d. Hydrolysis causes reassociation. Hydrolysis of GTP to GDP causes alpha to reassociate with other subunits → inactive heterotrimer

2. Small G proteins -- to be discussed further when we get to cell cycle & cancer. For reference:

a. Structure: Small G proteins have no subunits.

b. Example: the protein called ras -- important in growth control many cancer cells have over-active ras.

c. Role of GTP/GDP exchange: Are activated by GTP/GDP exchange, and inactivated by hydrolysis of GTP to GDP, as above.

d. Are not activated by GPCRs directly (other 'middle man' adapter proteins are involved)

3 . How many G proteins?

a. There are many different G proteins. G proteins are involved in a very large number of cellular processes, not just signaling. (We have ignored their importance until now. See Becker for details & many examples. )

b. Active G proteins can be inhibitory or stimulatory.

c. Method of action: Activated G proteins work by binding to and activating (or inhibiting) other target enzymes/proteins.

d. Terminology: The different trimeric G proteins are usually known as Gp, Gq Gi, Gs etc. (Books differ on details of naming.)

E. For reference: Comparison of Protein Kinases, Receptor Protein Kinases, & Trimeric G proteins

Protein Catalyzes What's added to Target Protein? Who gets the P or GTP? How Inactivated?
Protein Kinase Protein + ATP → ADP + protein-P Phosphate Usually a dif. protein Separate Phosphatase removes P
Receptor Protein Kinase ** Protein + ATP → ADP + protein-P Phosphate Usually separate subunit of self Separate Phosphatase removes P
Trimeric
G Protein
Exchange & Hydrolysis as described above. GTP Itself Hydrolyzes GTP to GDP (by itself)

**Receptor protein kinases have an extracellular ligand binding domain and an intracellular kinase (or kinase binding) domain. Ordinary kinases usually add phosphates to other proteins. Receptor kinases usually add phosphates to themselves. (For an example, see Sadava fig. 7.7 (15.6)

Try problems 6-1 & 6-2.

III. 2nd Messengers -- How do they fit in? How do they work?

A. Typical Pathway -- where does 2nd messenger fit in? (see also handout 14B, middle panel)

ligand (1st messenger) binds outside cell activate receptor in membrane activate G protein in the membrane activate target enzyme in membrane generate small molecule (2nd messenger) inside cell

B. What are Second messengers? -- See handout 14B or Sadava fig. 7.8 (15.7)

1. What are they? Small molecules or ions that move through the cell and bind to their target proteins.

2 . The usual second messengers -- see handout 14B for structure of cAMP and mode of action

2nd Messenger Where does it come from? How is it made?
cAMP ATP by action of adenyl cyclase
DAG & IP3 membrane lipid by action of phospholipase C
Ca 2+ stored Ca 2+ in ER (or extracellular) by opening channels (in ER/plasma memb.)

3. What do 2nd messengers do? Bind to and thereby activate (or inactivate) target proteins.

4. How they are made : Active G protein (subunit) → binds to & activates enzyme in (or associated with) membrane → generates second messenger in cytoplasm. See Becker fig. 14-7 or Sadava figs. 7.8 & 7.14 (15.7 & 15.12) for cAMP pathway. We will get to IP3 pathway later. If you are curious, see Becker fig. 14-10 or Sadava fig. 7.15 (15.13).

5. A Specific Example: Thyroid stimulating hormone (TSH) -- promotes release of thyroid hormone (TH).

a. Generation of 2nd messenger (cAMP)

TSH (1st messenger) binds activate GPCR in membrane activate G protein in the membrane activate enzyme in membrane (adenyl cyclase) generate small molecule (2nd messenger) inside cell = cAMP

b. Action of 2nd messenger

cAMP activate protein kinase (PKA) in cytoplasm phosphorylate target enzymes stimulate multiple steps in synthesis and release of TH

6. Why bother with all these steps?

a. Amplification. Many steps involve amplifications. For example, one molecule of active Adenyl cyclase can generate many molecules of cAMP and one molecule of PKA can phosphorylate many molecules of its target enzymes. For an example of the possibilities of a cascade of amplification, see Becker fig. 14-3 or Sadava fig. 7.20 (15.18). (They don't agree on the exact numbers.)

b. Examples. The usual example for this type of modification cascade is the breakdown of glycogen, stimulated by the hormone epinephrine (adrenaline), which is the example in Becker fig. 14-3. This example was the first to be discovered, but is more complex than the TSH case.

If you are interested in the details or want to read ahead, see Sadava fig. 7.20 (15.18) or Becker figs. 14-25 (14-24) & 6-17 (6-18 ), or the handout at http://www.columbia.edu/cu/biology/courses/c2006/handouts/glycogen09.pdf.


IV. An example of a second messenger -- cAMP & its target (PKA) See handout 14B
or Becker fig. 14-7.

A. How is cAMP level regulated? What does it do?

1. How is cAMP made?

a. G protein activates adenyl cyclase (also called adenylyl cyclase or AC)

b. cAMP made from ATP by adenyl cyclase for structure of cAMP see handout and Becker fig. 14-6 or Sadava fig. 7.14 (15.12).

2. What does cAMP do? See Becker table 14-1 (14-5).

a. cAMP binds to and activates protein kinase A = PKA . (Also called cyclic AMP dependent protein kinase = cAPK) See Becker fig. 14-8.

b. PKA adds phosphates to other proteins

(1). Phosphorylation by PKA can activate or inhibit target protein (target = substrate of PKA)

(2). PKA action can modify other kinases/phosphatases and start a cascade

(3). End result varies. Depends on which kinases and phosphatases in that tissue are targets (substrates) of PKA and/or the other kinases/phosphatases (at end of cascade). See example below.

3. How does signal system turn off when hormone leaves?

a. G protein doesn't stay activated for long: Activated G protein hydrolyzes its own GTP → GDP ( → inactive G protein).

b. cAMP is short lived -- it's hydrolyzed by phosphodiesterase (PDE)

c. In absence of cAMP, action of kinases are stopped and/or reversed

(1) PKA becomes inactivated

(2) Phosphatases become active -- remove phosphates added by kinases

B. How do hormones work through cAMP?

1. TSH -- see above. PKA phosphorylates (and activates) enzymes needed to make thyroid hormone.

2 . Glycogen metabolism: This case is very complex and will be discussed later. See above for references.


V. Introduction to Physiology & Multicellular organisms -- What is signaling good for?

A. Single cell Life Style vs. Multicellular

1. Single celled organisms

a. Surrounded by external environment -- Can't change or regulate it

b. Have one basic function -- grow and multiply

c. Respond to external conditions (since can't change them) to maintain optimal intracellular state

(1). Pick up and/or dump what is necessary for metabolism

(2). Keep intracellular conditions (pH, level of amino acids, oxygen, etc.) as constant as possible and expend minimal energy by adjusting rates of transcription, enzyme activity, etc.

d. Note no specialization: each cell does all possible functions

2. Multicellular organisms & Homeostasis

  • plasma = liquid part of blood = fluid between blood cells

  • interstitial fluid (IF) = fluid between all other cells

c. Each cell has two basic functions

(1). Grow or maintain itself as above

(2). Specialized role in maintaining homeostasis of whole organism

d. Cells are Specialized. Maintenance of homeostasis requires co-operation of many different cell types, not just circuits within a single cell.

Unicellular Organisms Multicellular Organisms
What surrounds cell? External environment Internal environment of organism
Can organism regulate what surrounds each cell? No Yes
How many functions of each cell? 1 2 or more
Is cell specialized? No Yes

B. Organization -- How are cells set up to co-operate in a multicellular organism? See 15A.

1. Cells, Tissues & the 4 major tissue types (5, if you count the blood separately) -- see lecture #4, & Sadava fig. 40.7

2. Organs

a. Made of (different kinds of) tissues.

b. Example: lining of GI tract. Has layers of different tissues -- epithelial, connective, muscle, and nervous these serve primarily for absorption (of material from lumen), support, contraction, and regulation respectively. (See handout 15A or Sadava fig. 40.7) The blood (a type of connective) doesn't really fit in this classification -- serves for transport of materials in and out.

3. Systems -- Group of Organs → body or organ system. Work together to maintain homeostasis for some component. See Sadava 40.1 (8th ed). Number of systems depends on who's counting. Usual # is 8-12.

a. Immune system -- responds primarily to internal changes caused by presence of foreign organisms (or their macromolecules) -- responds to viruses, bacteria, cancer cells, etc. (Graft rejection, allergy, etc. are side effects of this.)

b. Other systems -- respond to changes in internal mileu caused by other factors.


VI. How is a component of the internal milieu regulated?

A. General Principle -- Homeostasis is maintained by Negative Feedback

  • In physiology, negative and positive feedback are defined as above. For negative fb, it doesn't matter whether the corrections are achieved by inhibition (turning off the heater stopping glucose production) or acceleration (turning on the air conditioner, increasing glucose uptake from blood).

  • In biochemistry, negative feedback usually means inhibition of an earlier step (& positive fb usually means activation).

  • 'Effector' is also used differently in physio and biochem see below.

B. Example #1 -- Regulation of blood glucose levels. The see-saw view. See handout 15B or Sadava fig. 51.18 (50.19). The fig. in the 9th ed. is not a see-saw, but the point is the same.

1. Have a regulated variable -- glucose level in blood.

2. Need a sensor (or receptor) -- to measure levels of "regulated variable" (glucose). Here, sensor is in pancreas.

3. Need effector(s) -- to control levels of regulated variable (glucose) -- usually have one or more effectors that respond in opposing ways. In this case, effectors for uptake of glucose are liver, adipose tissue, and skeletal muscle effector for release of glucose is liver.

Note on terminology: In physiology, "effector" usually means "a tissue or organ (like muscle or liver) that carries out an action and thus produces an effect." In this example, the effectors = organs that act to raise or lower the blood glucose. In molecular biology, the term "effector" is usually used to mean "a modulator of protein function." A modulator = a small molecule (like an inducer, enzyme activator etc.) that binds to a protein, alters the shape and/or function of the protein, and thus triggers an effect.

4. Have a set point -- the level the regulated variable (blood glucose) should be. Set point is also sometimes used to mean the level at which corrections (to raise or lower the value) kick in.

In most cases, there is no significant difference between these two definitions of set point. In some cases, the desired value (first definition) and the value at which corrections occur (second definition) may be different. For example, there may be two cut-off points-- upper and lower, that bracket the desired level of a regulated variable. At levels above or below the respective cut-off points, messages are sent to the appropriate effectors to take corrective action. The term "critical values" is sometimes used instead of "set points" to describe the cut-off point(s).

5. Signaling -- need some signal system to connect the sensor(s) and the effector(s). Can be nervous &/or hormonal. In this case, primary (but not only) signal is hormonal & primary hormones (signals) are insulin & glucagon.

6. Operation of Negative Feedback -- the system responds to negate deviations from the set point. Important features:

a. Works to stabilize levels of blood glucose (the regulated variable)

b. System is self-correcting -- Deviations in either direction (if blood glucose is either too high or too low) are corrected.

c. There are two opposing actions by effectors, not just one.

(1). If [G] gets too high, effectors take G up from blood. (top half of seesaw diagram)

(2). If blood [G] gets too low, effector releases G to blood. (bottom half of seesaw diagram)

d. Negative feedback is not always inhibition. The deviation from the set point may be fixed by accelerating, not inhibiting, a process. In negative feedback, deviations from the set point can be corrected either by speeding up a process (such as glucose uptake) or slowing down a process (such as glycogen breakdown to glucose). For example:

(1). If blood [G] goes up, uptake from blood increases and glycogen breakdown decreases. In this case, an increase in glucose uptake is used to help decrease the deviation from the set point.

(2). If blood [G] falls, release into blood increases, and glycogen synthesis decreases. In this case, an increase in glucose release is used to help decrease the deviation from the set point.

(3). In both cases, one process is increased and another is inhibited to help decrease the deviation from the set point.

7. Net Result -- regulated variable ([G] in blood) is not constant, but stays close to set point.

See problem 5-1 & 5-2 a & b.

C. Example #2 -- Regulation of body temperature (in humans) -- the see-saw view (handout 15B)

1. Note many features are same as in glucose case. (Can you list them??)

2. Features not found in glucose case:

a. Multiple sensors in different places (for core and skin temp.). How to integrate multiple inputs?

b. Nature of Signal -- Signals are neuronal, not hormonal

c. Integrative center (IC)

(1). Role of IC: Compares set-point to actual value, sends appropriate message to effectors.

(2). Type of IC

(a). Sensor/IC function may be combined, as in Glucose example.

(b). Separate IC needed if there are multiple sensors, as in this case. IC co-ordinates incoming information from multiple sensors

(3). In this example, IC = hypothalamus (HT)