Relationship between the G protein signaling and homologous desensitization of G protein-coupled receptors

Signaling and desensitization of G protein-coupled receptor are intimately related, and measuring them separately requires certain parameters that represent desensitization independently of signaling. In this study, we tested whether desensitization requires signaling in three different receptors, beta2-adrenergic receptor (beta2AR) in S49 lymphoma cells, alpha-factor pheromone receptor (Ste2p) in Saccharomyces cerevisiae LM102 cells, and dopamine D3 receptor (D3R) in HEK-293 cells. Agonist-induced beta-arrestin translocation to the plasma membrane or receptor sequestration was measured to estimate homologous desensitization. To separate the signaling and desensitization of beta2AR, which mediates stimulation of adenylyl cyclase, S49 lymphoma cys- cells that lack the alpha subunit of Gs were used. Stimulation of beta2AR in these cells failed to increase intracellular cAMP, but beta-arrestin translocation still occurred, suggesting that feedback from beta2AR signaling is not required for homologous desensitization to occur. Agonist-induced sequestration of the yeast Ste2p-L236R, which showed reduced signaling through G protein, was not different from that of wildtype Ste2p, suggesting that the receptor signaling and sequestration are not directly linked cellular events. Both G protein coupling and D3R signaling, measured as inhibition of cAMP production, were greatly enhanced by co-expression of exogenous alpha subunit of Go (Goalpha) or adenylyl cyclase type 5 (AC5), respectively. However, agonist-induced beta-arrestin translocation, receptor phosphorylation, and sequestration were not affected by co-expression of Galphao and AC5, suggesting that the extent of signaling does not determine desensitization intensity. Taken together, our results consistently suggest that G protein signaling and homologous desensitization are independent cellular processes.     

Involvement of G protein-coupled receptor kinases and arrestins in desensitization to follicle-stimulating hormone action.

FSH rapidly desensitizes the FSH-receptor (FSH-R) upon binding. Very little information is available concerning the regulatory proteins involved in this process. In the present study, we investigated whether G protein-coupled receptor kinases (GRKs) and arrestins have a role in FSH-R desensitization, using a mouse Ltk 7/12 cell line stably overexpressing the rat FSH-R as a model. We found that these cells, which express GRK2, GRK3, GRK5, and GRK6 as well as beta-arrestins 1 and 2 as detected by RT-PCR and by Western blotting, were rapidly desensitized in the presence of FSH. Overexpression of GRKs and/or beta-arrestins in Ltk 7/12 cells allowed us to demonstrate 1) that GRK2, -3, -5, -6a, and -6b inhibit the FSH-R-mediated signaling (from 71% to 96% of maximal inhibition depending on the kinase, P < 0.001); 2) that beta-arrestins 1 or 2 also decrease the FSH action when overexpressed (80% of maximal inhibition, P < 0.01) whereas dominant negative beta-arrestin 2 [319-418] potentiates it 8-fold (P < 0.001); 3) that beta-arrestins and GRKs (except GRK6a) exert additive inhibition on FSH-induced response; and 4) that FSH-R desensitization depends upon the endogenous expression of GRKs, since there is potentiation of the FSH response (2- to 3-fold, P < 0.05) with antisenses cDNAs for GRK2, -5, and -6, but not GRK3. Our results show that the desensitization of the FSH-induced response involves the GRK/arrestin system.     

Multiple functions of G protein-coupled receptor kinases

Desensitization is a physiological feedback mechanism that blocks detrimental effects of persistent stimulation. G protein-coupled receptor kinase 2 (GRK2) was originally identified as the kinase that mediates G protein-coupled receptor (GPCR) desensitization. Subsequent studies revealed that GRK is a family composed of seven isoforms (GRK1–GRK7). Each GRK shows a differential expression pattern. GRK1, GRK4, and GRK7 are expressed in limited tissues. In contrast, GRK2, GRK3, GRK5, and GRK6 are ubiquitously expressed throughout the body. The roles of GRKs in GPCR desensitization are well established. When GPCRs are activated by their agonists, GRKs phosphorylate serine/threonine residues in the intracellular loops and the carboxyl-termini of GPCRs. Phosphorylation promotes translocation of β-arrestins to the receptors and inhibits further G protein activation by interrupting receptor-G protein coupling. The binding of β-arrestins to the receptors also helps to promote receptor internalization by clathrin-coated pits. Thus, the GRK-catalyzed phosphorylation and subsequent binding of β-arrestin to GPCRs are believed to be the common mechanism of GPCR desensitization and internalization. Recent studies have revealed that GRKs are also involved in the β-arrestin-mediated signaling pathway. The GRK-mediated phosphorylation of the receptors plays opposite roles in conventional G protein- and β-arrestin-mediated signaling. The GRK-catalyzed phosphorylation of the receptors results in decreased G protein-mediated signaling, but it is necessary for β-arrestin-mediated signaling. Agonists that selectively activate GRK/β-arrestin-dependent signaling without affecting G protein signaling are known as β-arrestin-biased agonists. Biased agonists are expected to have potential therapeutic benefits for various diseases due to their selective activation of favorable physiological responses or avoidance of the side effects of drugs. Furthermore, GRKs are recognized as signaling mediators that are independent of either G protein- or β-arrestin-mediated pathways. GRKs can phosphorylate non-GPCR substrates, and this is found to be involved in various physiological responses, such as cell motility, development, and inflammation. In addition to these effects, our group revealed that GRK6 expressed in macrophages mediates the removal of apoptotic cells (engulfment) in a kinase activity-dependent manner. These studies revealed that GRKs block excess stimulus and also induce cellular responses. Here, we summarized the involvement of GRKs in β-arrestin-mediated and G protein-independent signaling pathways.     

Role of G protein-coupled receptor kinases on the agonist-induced phosphorylation and internalization of the follitropin receptor.

The experiments presented herein were designed to identify members of the G protein-coupled receptor kinase (GRK) family that participate in the agonist-induced phosphorylation and internalization of the rat FSH receptor (rFSHR). Western blots of human kidney 293 cells (the cell line used in transfection experiments) and MSC-1 cells (a cell line derived from Sertoli cells that displays many of the differentiated functions of their normal counterparts) reveal the presence of GRK2 and GRK6 in both cell lines as well as GRK4 in MSC-1 cells. Cotransfection of 293 cells with the rFSHR and GRK2, GRK4alpha, or GRK6 resulted in an increase in the agonist-induced phosphorylation of the rFSHR. Cotransfections of the rFSHR with GRKs or arrestin-3 enhanced the agonist-induced internalization of the rFHSR, and combinations of GRKs and arrestin-3 were more effective than the individual components. To characterize the involvement of endogenous GRKs on phosphorylation and internalization, we inhibited endogenous GRK2 by overexpression of a kinase-deficient mutant of GRK2 or G alpha t, a scavenger of G betagamma. We also inhibited endogenous GRK6 by overexpression of a kinase-deficient mutant of GKR6. All three constructs were effective inhibitors of phosphorylation, but only the kinase-deficient mutant of GRK2 and G alpha t inhibited internalization. The inhibition of internalization induced by these two constructs was less pronounced than that induced by a dominant-negative mutant of the nonvisual arrrestins, however. The finding that inhibitors of GRK2 and GRK6 impair phosphorylation, but only the inhibitors of GRK2 impair internalization, suggests that different GRKs have differential effects on receptor internalization.     

Regulation of G protein-coupled receptor kinases by caveolin

G protein-coupled receptor kinases (GRKs) have been principally characterized by their ability to phosphorylate and desensitize G protein-coupled receptors. However, recent studies suggest that GRKs may have more diverse protein/protein interactions in cells. Based on the identification of a consensus caveolin binding motif within the pleckstrin homology domain of GRK2, we tested the direct binding of purified full-length GRK2 to various glutathione S-transferase-caveolin-1 fusion proteins, and we discovered a specific interaction of GRK2 with the caveolin scaffolding domain. Interestingly, analysis of GRK1 and GRK5, which lack a pleckstrin homology domain, revealed in vitro binding properties similar to those of GRK2. Maltose-binding protein caveolin and glutathione S-transferase-GRK fusion proteins were used to map overlapping regions in the N termini of both GRK2 and GRK5 that appear to mediate conserved GRK/caveolin interactions. In vivo association of GRK2 and caveolin was suggested by co-fractionation of GRK2 with caveolin in A431 and NIH-3T3 cells and was further supported by co-immunoprecipitation of GRK2 and caveolin in COS-1 cells. Functional significance for the GRK/caveolin interaction was demonstrated by the potent inhibition of GRK-mediated phosphorylation of both receptor and peptide substrates by caveolin-1 and -3 scaffolding domain peptides. These data reveal a novel mode for the regulation of GRKs that is likely to play an important role in their cellular function.     

The origin and evolution of G protein-coupled receptor kinases

G protein-coupled receptor (GPCR) kinases (GRKs) play key role in homologous desensitization of GPCRs. GRKs phosphorylate activated receptors, promoting high affinity binding of arrestins, which precludes G protein coupling. Direct binding to active GPCRs activates GRKs, so that they selectively phosphorylate only the activated form of the receptor regardless of the accessibility of the substrate peptides within it and their Ser/Thr-containing sequence. Mammalian GRKs were classified into three main lineages, but earlier GRK evolution has not been studied. Here we show that GRKs emerged at the early stages of eukaryotic evolution via an insertion of a kinase similar to ribosomal protein S6 kinase into a loop in RGS domain. GRKs in Metazoa fall into two clades, one including GRK2 and GRK3, and the other consisting of all remaining GRKs, split into GRK1-GRK7 lineage and GRK4-GRK5-GRK6 lineage in vertebrates. One representative of each of the two ancient clades is found as early as placozoan Trichoplax adhaerens. Several protists, two oomycetes and unicellular brown algae have one GRK-like protein, suggesting that the insertion of a kinase domain into the RGS domain preceded the origin of Metazoa. The two GRK families acquired distinct structural units in the N- and C-termini responsible for membrane recruitment and receptor association. Thus, GRKs apparently emerged before animals and rapidly expanded in true Metazoa, most likely due to the need for rapid signalling adjustments in fast-moving animals.     

Comprehensive repertoire and phylogenetic analysis of the G protein-coupled receptors in human and mouse

Understanding differences in the repertoire of orthologous gene pairs is vital for interpretation of pharmacological and physiological experiments if conclusions are conveyed between species. Here we present a comprehensive dataset for G protein-coupled receptors (GPCRs) in both human and mouse with a phylogenetic road map. We performed systematic searches applying several search tools such as BLAST, BLAT, and Hidden Markov models and searches in literature data. We aimed to gather a full-length version of each human or mouse GPCR in only one copy referring to a single chromosomal position. Moreover, we performed detailed phylogenetic analysis of the transmembrane regions of the receptors to establish accurate orthologous pairs. The results show the identity of 495 mouse and 400 human functional nonolfactory GPCRs. Overall, 329 of the receptors are found in one-to-one orthologous pairs, while 119 mouse and 31 human receptors originate from species-specific expansions or deletions. The average percentage similarity of the orthologue pairs is 85%, while it varies between the main GRAFS families from an average of 59 to 94%. The orthologous pairs for the lipid-binding GPCRs had the lowest levels of conservation, while the biogenic amines had highest levels of conservation. Moreover, we searched for expressed sequence tags (ESTs) and identified more than 17,000 ESTs matching GPCRs in mouse and human, providing information about their expression patterns. On the whole, this is the most comprehensive study of the gene repertoire that codes for human and mouse GPCRs. The datasets are available for downloading.     

Role of G protein-coupled receptor kinase 4 and beta-arrestin 1 in agonist-stimulated metabotropic glutamate receptor 1 internalization and activation of mitogen-activated protein kinases

The metabotropic glutamate 1 (mGlu(1)) receptor in cerebellar Purkinje cells plays a key role in motor learning and motor coordination. Here we show that the G protein-coupled receptor kinases (GRK) 2 and 4, which are expressed in these cells, regulate the mGlu(1) receptor by at least in part different mechanisms. Using kinase-dead mutants in HEK293 cells, we found that GRK4, but not GRK2, needs the intact kinase activity to desensitize the mGlu(1) receptor, whereas GRK2, but not GRK4, can interact with and regulate directly the activated Galpha(q). In cells transfected with GRK4 and exposed to agonist, beta-arrestin was first recruited to plasma membranes, where it was co-localized with the mGlu(1) receptor, and then internalized in vesicles. The receptor was also internalized but in different vesicles. The expression of beta-arrestin V53D dominant negative mutant, which did not affect the mGlu(1) receptor internalization, reduced by 70-80% the stimulation of mitogen-activated protein (MAP) kinase activation by the mGlu(1) receptor. The agonist-stimulated differential sorting of the mGlu(1) receptor and beta-arrestin as well as the activation of MAP kinases by mGlu(1) agonist was confirmed in cultured cerebellar Purkinje cells. A major involvement of GRK4 and of beta-arrestin in agonist-dependent receptor internalization and MAP kinase activation, respectively, was documented in cerebellar Purkinje cells using an antisense treatment to knock down GRK4 and expressing beta-arrestin V53D dominant negative mutant by an adenovirus vector. We conclude that GRK2 and GRK4 regulate the mGlu(1) receptor by different mechanisms and that beta-arrestin is directly involved in glutamate-stimulated MAP kinase activation by acting as a signaling molecule.     

G protein-coupled receptor kinase-mediated desensitization of metabotropic glutamate receptor 1A protects against cell death

Metabotropic glutamate receptors (mGluRs) constitute a unique subclass of G protein-coupled receptors (GPCRs) that bear little sequence homology to other members of the GPCR superfamily. The mGluR subtypes that are coupled to the hydrolysis of phosphoinositide contribute to both synaptic plasticity and glutamate-mediated excitotoxicity in neurons. In the present study, the expression of mGluR1a in HEK 293 cells led to agonist-independent cell death. Since G protein-coupled receptor kinases (GRKs) desensitize a diverse variety of GPCRs, we explored whether GRKs contributed to the regulation of both constitutive and agonist-stimulated mGluR1a activity and thereby may prevent mGluR1a-mediated excitotoxicity associated with mGluR1a overactivation. We find that the co-expression of mGluR1a with GRK2 and GRK5, but not GRK4 and GRK6, reduced both constitutive and agonist-stimulated mGluR1a activity. Agonist-stimulated mGluR1a phosphorylation was enhanced by the co-expression of GRK2 and was blocked by two different GRK2 dominant-negative mutants. Furthermore, GRK2-dependent mGluR1a desensitization protected against mGluR1a-mediated cell death, at least in part by blocking mGluR1a-stimulated apoptosis. Our data indicate that as with other members of the GPCR superfamily, a member of the structurally distinct mGluR family (mGluR1a) serves as a substrate for GRK-mediated phosphorylation and that GRK-dependent "feedback" modulation of mGluR1a responsiveness protects against pathophysiological mGluR1a signaling.     

Molecular basis for activation of G protein-coupled receptor kinases

G protein‐coupled receptor (GPCR) kinases (GRKs) selectively recognize and are allosterically regulated by activated GPCRs, but the molecular basis for this interaction is not understood. Herein, we report crystal structures of GRK6 in which regions known to be critical for receptor phosphorylation have coalesced to stabilize the kinase domain in a closed state and to form a likely receptor docking site. The crux of this docking site is an extended N‐terminal helix that bridges the large and small lobes of the kinase domain and lies adjacent to a basic surface of the protein proposed to bind anionic phospholipids. Mutation of exposed, hydrophobic residues in the N‐terminal helix selectively inhibits receptor, but not peptide phosphorylation, suggesting that these residues interact directly with GPCRs. Our structural and biochemical results thus provide an explanation for how receptor recognition, phospholipid binding, and kinase activation are intimately coupled in GRKs.     

Pleiotropic coupling of G protein-coupled receptors to the mitogen-activated protein kinase cascade. Role of focal adhesions and receptor tyrosine kinases.

G protein-coupled receptors (GPCRs) initiate Ras-dependent activation of the Erk 1/2 mitogen-activated protein kinase cascade by stimulating recruitment of Ras guanine nucleotide exchange factors to the plasma membrane. Both integrin-based focal adhesion complexes and receptor tyrosine kinases have been proposed as scaffolds upon which the GPCR-induced Ras activation complex may assemble. Using specific inhibitors of focal adhesion complex assembly and receptor tyrosine kinase activation, we have determined the relative contribution of each to activation of the Erk 1/2 cascade following stimulation of endogenous GPCRs in three different cell types. The tetrapeptide RGDS, which inhibits integrin dimerization, and cytochalasin D, which depolymerizes the actin cytoskeleton, disrupt the assembly of focal adhesions. In PC12 rat pheochromocytoma cells, both agents block lysophosphatidic acid (LPA)- and bradykinin-stimulated Erk 1/2 phosphorylation, suggesting that intact focal adhesion complexes are required for GPCR-induced mitogen-activated protein kinase activation in these cells. In Rat 1 fibroblasts, Erk 1/2 activation via LPA and thrombin receptors is completely insensitive to both agents. Conversely, the epidermal growth factor receptor-specific tyrphostin AG1478 inhibits GPCR-mediated Erk 1/2 activation in Rat 1 cells but has no effect in PC12 cells. In HEK-293 human embryonic kidney cells, LPA and thrombin receptor-mediated Erk 1/2 activation is partially sensitive to both the RGDS peptide and tyrphostin AG1478, suggesting that both focal adhesion and receptor tyrosine kinase scaffolds are employed in these cells. The dependence of GPCR-mediated Erk 1/2 activation on intact focal adhesions correlates with expression of the calcium-regulated focal adhesion kinase, Pyk2. In all three cell types, GPCR-stimulated Erk 1/2 activation is significantly inhibited by the Src kinase inhibitors, herbimycin A and 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo-D-3,4-pyrimidine (PP1), suggesting that Src family nonreceptor tyrosine kinases represent a point of convergence for signals originating from either scaffold.     

Phosphorylation of G protein-coupled receptors: GPCR kinases in heart disease

In the heart, beta -adrenergic receptors (beta ARs), members of the superfamily of G protein-coupled receptors (GPCRs), modulate cardiac responses to catecholamines. beta AR signaling, which is compromised in many cardiac diseases (e.g., congestive heart failure), is regulated by GPCR kinases (GRKs). Levels of the most abundant cardiac GRK, known as GRK2 or beta AR kinase 1 (beta ARK1), are increased in both animal and human heart failure. Transgenic mouse models have demonstrated that beta ARK1 plays a vital role in cardiac function and development, as well as in the regulation of myocardial signaling, and pharmacological studies have further implicated GRKs in the impairment of cardiac GPCR signaling. Gene therapy, along with the development of small-molecule modulators of GRK activity, has indicated in multiple animal models that the manipulation of GRK activity may elicit therapeutic benefits in many forms of cardiac disease.     

Real-time visualization of the cellular redistribution of G protein-coupled receptor kinase 2 and beta-arrestin 2 during homologous desensitization of the substance P receptor

The substance P receptor (SPR) is a G protein-coupled receptor (GPCR) that plays a key role in pain regulation. The SPR desensitizes in the continued presence of agonist, presumably via mechanisms that implicate G protein-coupled receptor kinases (GRKs) and beta-arrestins. The temporal relationship of these proposed biochemical events has never been established for any GPCR other than rhodopsin beyond the resolution provided by biochemical assays. We investigate the real-time activation and desensitization of the human SPR in live HEK293 cells using green fluorescent protein conjugates of protein kinase C, GRK2, and beta-arrestin 2. The translocation of protein kinase C betaII-green fluorescent protein to and from the plasma membrane in response to substance P indicates that the human SPR becomes activated within seconds of agonist exposure, and the response desensitizes within 30 s. This desensitization process coincides with a redistribution of GRK2 from the cytosol to the plasma membrane, followed by a robust redistribution of beta-arrestin 2 and a profound change in cell morphology that occurs after 1 min of SPR stimulation. These data establish a role for GRKs and beta-arrestins in homologous desensitization of the SPR and provide the first visual and temporal resolution of the sequence of events underlying homologous desensitization of a GPCR in living cells.     

G protein-coupled receptor kinases: Past,present and future

This review is provided in recognition of the extensive contributions of Dr. Robert J. Lefkowitz to the G protein-coupled receptor (GPCR) field and to celebrate his 75th birthday. Since one of the authors trained with Bob in the 80s, we provide a history of work done in the Lefkowitz lab during the 80s that focused on dissecting the mechanisms that regulate GPCR signaling, with a particular emphasis on the GPCR kinases (GRKs). In addition, we highlight structure/function characteristics of GRK interaction with GPCRs as well as a review of two recent reports that provide a molecular model for GRK-GPCR interaction. Finally, we offer our perspective on some future studies that we believe will drive this field.     

Differential regulation of the dopamine D2 and D3 receptors by G protein-coupled receptor kinases and beta-arrestins

The D(2) and D(3) receptors (D(2)R and D(3)R), which are potential targets for antipsychotic drugs, have a similar structural architecture and signaling pathway. Furthermore, in some brain regions they are expressed in the same cells, suggesting that differences between the two receptors might lie in other properties such as their regulation. In this study we investigated, using COS-7 and HEK-293 cells, the mechanism underlying the intracellular trafficking of the D(2)R and D(3)R. Activation of D(2)R caused G protein-coupled receptor kinase-dependent receptor phosphorylation, a robust translocation of beta-arrestin to the cell membrane, and profound receptor internalization. The internalization of the D(2)R was dynamin-dependent, suggesting that a clathrin-coated endocytic pathway is involved. In addition, the D(2)R, upon agonist-mediated internalization, localized to intracellular compartments distinct from those utilized by the beta(2)-adrenergic receptor. However, in the case of the D(3)R, only subtle agonist-mediated receptor phosphorylation, beta-arrestin translocation to the plasma membrane, and receptor internalization were observed. Interchange of the second and third intracellular loops of the D(2)R and D(3)R reversed their phenotypes, implicating these regions in the regulatory properties of the two receptors. Our studies thus indicate that functional distinctions between the D(2)R and D(3)R may be found in their desensitization and cellular trafficking properties. The differences in their regulatory properties suggest that they have distinct physiological roles in the brain.     

Muscarinic supersensitivity and impaired receptor desensitization in G protein-coupled receptor kinase 5-deficient mice

G protein-coupled receptor kinase 5 (GRK5) is a member of a family of enzymes that phosphorylate activated G protein-coupled receptors (GPCR). To address the physiological importance of GRK5-mediated regulation of GPCRs, mice bearing targeted deletion of the GRK5 gene (GRK5-KO) were generated. GRK5-KO mice exhibited mild spontaneous hypothermia as well as pronounced behavioral supersensitivity upon challenge with the nonselective muscarinic agonist oxotremorine. Classical cholinergic responses such as hypothermia, hypoactivity, tremor, and salivation were enhanced in GRK5-KO animals. The antinociceptive effect of oxotremorine was also potentiated and prolonged. Muscarinic receptors in brains from GRK5-KO mice resisted oxotremorine-induced desensitization, as assessed by oxotremorine-stimulated [5S]GTPgammaS binding. These data demonstrate that elimination of GRK5 results in cholinergic supersensitivity and impaired muscarinic receptor desensitization and suggest that a deficit of GPCR desensitization may be an underlying cause of behavioral supersensitivity.     

Integration of signals from receptor tyrosine kinases and g protein-coupled receptors

Activation of G protein-coupled receptors (GPCRs) leads to stimulation of classical G protein signaling pathways. In addition, GPCRs can activate the mitogen-activated protein kinases (MAPKs) such as the extracellular signal-regulated kinases, c-Jun NH(2)-terminal kinases (JNKs), and p38 MAPKs, and thereby influence cell proliferation, cell differentiation and mitogenesis. Cross talk between GPCRs and receptor tyrosine kinases (RTKs) is an incredibly complex process, and the exact signaling molecules involved are largely dependent on the cell type and the type of receptor that is activated. In this review we investigate recent advances that have been made in understanding the mechanisms of cross talk between GPCRs and RTKs, with a focus on GPCR-mediated activation of the Ras/MAPK pathway, GPCR-induced transactivation of RTKs, GPCR-mediated activation of JNK, and p38 MAPK, integration of signals by RhoGTPases, and activation of G protein signaling pathways by RTKs.     

Changes in G protein-coupled receptor sorting protein affinity regulate postendocytic targeting of G protein-coupled receptors

After activation, most G protein-coupled receptors (GPCRs) are regulated by a cascade of events involving desensitization and endocytosis. Internalized receptors can then be recycled to the plasma membrane, retained in an endosomal compartment, or targeted for degradation. The GPCR-associated sorting protein, GASP, has been shown to preferentially sort a number of native GPCRs to the lysosome for degradation after endocytosis. Here we show that a mutant beta(2) adrenergic receptor and a mutant mu opioid receptor that have previously been described as lacking "recycling signals" due to mutations in their C termini in fact bind to GASP and are targeted for degradation. We also show that a mutant dopamine D1 receptor, which has likewise been described as lacking a recycling signal, does not bind to GASP and is therefore not targeted for degradation. Together, these results indicate that alteration of receptors in their C termini can expose determinants with affinity for GASP binding and consequently target receptors for degradation.     

Desensitization of the mouse thromboxane A2 receptor (TP) by G protein-coupled receptor kinases (Grks)

GRKs play a key role in regulating G protein-coupled receptor (GPCR) responsiveness. To investigate the role of GRKs in desensitization of TP, we replaced threonines with favorable phosphorylation motifs for GRKs (positions 226 and 230) with alanine. Mutant and wild-type receptors were expressed in cell culture models and clones expressing similar numbers of receptors were studied. We found that: (1) affinity and specificity of thromboxane A2 (TxA2) binding to mutant TP were identical to the wild-type, (2) replacement of threonines 226 and 230 with alanines delayed the onset of agonist-induced desensitization, and (3) inhibition of endogenous GRK activity with a dominant-negative construct inhibited agonist-induced phosphorylation and enhanced responsiveness of wild-type TP but had little effect on responsiveness of the receptor mutant. These data are consistent with the notion that GRKs contribute to desensitization of TP.