G protein-coupled receptor kinases promote phosphorylation and beta-arrestin-mediated internalization of CCR5 homo- and hetero-oligomers

Expression levels of the chemokine receptor, CC chemokine receptor 5 (CCR5), at the cell surface determine cell susceptibility to HIV entry and infection. Cellular activation by CCR5 itself, but also by unrelated receptors leads to cross-phosphorylation and cross-internalization of CCR5. This study addresses the underlying molecular mechanisms of homologous and heterologous CCR5 regulation. As shown by bioluminescence resonance energy transfer experiments, CCR5 formed constitutive homo- as well as heterooligomeric complexes together with C5aR but not with the unrelated AT(1a)R in living cells. Stimulation with CCL5 of RBL cells, which co-expressed CCR5 together with an N-terminally truncated CCR5-DeltaNT mutant, resulted in both protein kinase C (PKC)- and G protein-coupled receptor (GPCR) kinase (GRK)-mediated cross-phosphorylation of the mutant unligated receptor, as determined by phosphosite-specific monoclonal antibody. Similarly, both PKC and GRK cross-phosphorylated CCR5 in a heterologous manner after C5a stimulation of RBL-CCR5/C5aR cells, whereas AT(1a)R stimulation resulted only in classical PKC-mediated CCR5 phosphorylation. Co-expression of CCR5-DeltaNT together with a phosphorylation-deficient CCR5 mutant that neither binds beta-arrestin nor undergoes internalization partially restored the CCL5-induced association of beta-arrestin with the homo-oligomeric receptor complex and augmented cellular uptake of (125)I-CCL5. Co-expression of C5aR, but not of AT(1a)R, promoted CCR5 co-internalization upon agonist stimulation by a mechanism independent of CCR5 phosphorylation. Co-internalization of phosphorylated CCR5 was also observed in C5a-stimulated macrophages. Finally, co-expression of a constitutively internalized C5aR-US28(CT) mutant led to intracellular accumulation of CCR5 in the absence of ligand stimulation. These results show that GRKs and beta-arrestin are involved in heterologous receptor regulation by cross-phosphorylating and co-internalizing unligated receptors within homo- or hetero-oligomeric protein complexes.     

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.     

Role of the human V1 vasopressin receptor COOH terminus in internalization and mitogenic signal transduction

We studied the role played by the intracellular COOH-terminal region of the human arginine vasopressin (AVP) V1-vascular receptor (V1R) in ligand binding, trafficking, and mitogenic signal transduction in Chinese hamster ovary cells stably transfected with the human AVP receptor cDNA clones that we had isolated previously. Truncations, mutations, or chimeric alterations of the V1R COOH terminus did not alter ligand binding, but agonist-induced V1R internalization and recycling were reduced in the absence of the proximal region of the V(1)R COOH terminus. Coupling to phospholipase C was altered as a function of the COOH-terminal length. Deletion of the proximal portion of the V1R COOH terminus or its replacement by the V2-renal receptor COOH terminus prevented AVP stimulation of DNA synthesis and progression through the cell cycle. Mutation of a kinase consensus motif in the proximal region of the V1R COOH terminus also abolished the mitogenic response. Thus the V1R cytoplasmic COOH terminus is not involved in ligand specificity but is instrumental in receptor trafficking and facilitates the interaction between the intracellular loops of the receptor, G protein, and phospholipase C. It is absolutely required for transmission of the mitogenic action of AVP, probably via a specific kinase phosphorylation site.     

Cytoplasmic tail of D1 dopaminergic receptor differentially regulates desensitization and phosphorylation by G protein-coupled receptor kinase 2 and 3

Herein, we investigate the differential D1 dopaminergic receptor (D1R) regulation by G protein-coupled receptor kinase (GRK) 2 and 3 using two truncated receptors lacking the distal (Δ425) and distal-central (Δ379) cytoplasmic tail (CT) regions. We first show the association between D1R and GRKs in co-transfected cells and rat striatum. Our studies further indicate that deletion of distal CT region of D1R does not alter the association between receptor and GRK2. Meanwhile, removal of both distal and central CT regions culminates in a drastic increase in the basal association between Δ379 and GRK2 relative to D1R and Δ425. Interestingly, CT truncations have no effect on the basal and DA-induced association of receptors with GRK3. Furthermore, we demonstrate that desensitization of D1R is considerably more robust in cells expressing GRK3. Notably, the robust GRK3-induced D1R desensitization is not attenuated by CT deletions. However, GRK2-induced Δ425 desensitization is not detectable whereas we unexpectedly find that Δ379 desensitization is similar to GRK2-induced D1R desensitization. GRK2 and GRK3-dependent desensitization of wild type D1R is not linked to differences in the extent of DA-induced receptor phosphorylation. Moreover, our studies show that GRK2-induced D1R phosphorylation is only modulated by deletion of distal CT region while distal and central CT regions control GRK3-induced D1R phosphorylation. Intriguingly, dopamine-induced Δ379 phosphorylation by GRK3 was significantly lower than receptor phosphorylation in cells harboring Δ379 alone or Δ379 and GRK2. Overall, our study suggests an intricate interplay between CT regions of D1R in differentially regulating receptor responsiveness by GRK2 and GRK3.     

The GRK4 subfamily of G protein-coupled receptor kinases. Alternative splicing, gene organization, and sequence conservation.

G protein-coupled receptor kinases (GRKs) desensitize G protein-coupled receptors by phosphorylating activated receptors. The six known GRKs have been classified into three subfamilies based on sequence and functional similarities. Examination of the mouse GRK4 subfamily (GRKs 4, 5, and 6) suggests that mouse GRK4 is not alternatively spliced in a manner analogous to human or rat GRK4, whereas GRK6 undergoes extensive alternative splicing to generate three variants with distinct carboxyl termini. Characterization of the mouse GRK 5 and 6 genes reveals that all members of the GRK4 subfamily share an identical gene structure, in which 15 introns interrupt the coding sequence at equivalent positions in all three genes. Surprisingly, none of the three GRK subgroups (GRK1, GRK2/3, and GRK4/5/6) shares even a single intron in common, indicating that these three subfamilies are distinct gene lineages that have been maintained since their divergence over 1 billion years ago. Comparison of the amino acid sequences of GRKs from various mammalian species indicates that GRK2, GRK5, and GRK6 exhibit a remarkably high degree of sequence conservation, whereas GRK1 and particularly GRK4 have accumulated amino acid changes at extremely rapid rates over the past 100 million years. The divergence of individual GRKs at vastly different rates reveals that strikingly different evolutionary pressures apply to the function of the individual GRKs.     

Multiple mechanisms involving protein phosphorylation are linked to desensitization of muscarinic receptors

Agonists induce phosphorylation of m2 muscarinic receptors (mAChR) in several cell types. This phosphorylation correlates with desensitization. The mechanisms underlying mAChR phosphorylation have been investigated using several in vitro approaches. Protein kinase C phosphorylated the purified and reconstituted m2 mAChR to a stoichiometry of approximately 5 mols P/mol receptor; this phosphorylation resulted in the decreased ability of receptors to activate G-proteins. Although the phosphorylation by PKC was not modulated by agonist binding to the mAChR, heterotrimeric G-proteins were able to completely block the PKC-mediated effects. If significant receptor/G-protein coupling occurs in vivo, agonists would be required to promote dissociation of the G-proteins from the receptors and reveal the phosphorylation sites for PKC. Members of the G-protein coupled receptor kinase (GRK) family also phosphorylated the purified and reconstituted m2 mAChR. In contrast to PKC, the GRKs phosphorylated the m2 mAChR strictly in an agonist-dependent manner. GRK mediated phosphorylation perturbed receptor/G-protein coupling. In addition, phosphorylation allowed for arrestin binding to the m2 mAChR which should further contribute to desensitization. Using a new strategy that does not require purification and reconstitution of receptors for GRK studies, the m3 mAChR were revealed as substrates for the GRKs. For both the m2 and m3 receptor subtypes, the most effective kinases were GRK 2 and 3. Phosphorylation of the receptors by these enzymes was stimulated by low concentrations of G-proteins and by membrane phospholipids. Thus, multiple mechanisms involving protein phosphorylation appear to contribute to the overall process of mAChR desensitization.     

Critical role of a subdomain of the N-terminus of the V1a vasopressin receptor for binding agonists but not antagonists; functional rescue by the oxytocin receptor N-terminus

A fundamental issue in molecular pharmacology is to define how agonist:receptor interaction differs from that of antagonist:receptor. The V(1a) receptor (V(1a)R) is a member of a family of related G-protein-coupled receptors that are activated by the neurohypophysial peptide hormone arginine-vasopressin (AVP). Here we define a short subdomain of the N-terminus of the V(1a)R from Glu(37) to Asn(47) that is an absolute requirement for binding AVP and other agonists. In marked contrast to the situation for agonists, deleting this segment has little or no effect on the binding of either peptide or non-peptide antagonists. In addition, we established that this subdomain was crucial for receptor activation and second messenger generation. The oxytocin receptor (OTR) also binds AVP with high affinity but exhibits a different pharmacological profile to the V(1a)R. Substitution of the N-terminus of the V(1a)R with the corresponding sequence from the OTR generated a chimeric receptor (OTR(N)-V(1a)R). The presence of the OTR N-terminus recovered high affinity agonist binding such that the OTR(N)-V(1a)R possessed almost wild-type V(1a)R pharmacology and signaling. Consequently, a domain within the N-terminus is required for agonist binding but it does not provide the molecular discriminator for subtype-selective agonist recognition. Cotransfection and peptide mimetic studies demonstrated that this N-terminal subdomain had to be contiguous with the receptor polypeptide to be functional. This study establishes that a segment of the V(1a)R N-terminus has a pivotal role in the mechanism of agonist binding and provides molecular insight into key differences between the interaction of agonists and antagonists with a peptide receptor family.     

G protein-coupled receptor kinase 5 regulates airway responses induced by muscarinic receptor activation

G protein-coupled receptors (GPCRs) transduce extracellular signals into intracellular events. The waning responsiveness of GPCRs in the face of persistent agonist stimulation, or desensitization, is a necessary event that ensures physiological homeostasis. GPCR kinases (GRKs) are important regulators of GPCR desensitization. GRK5, one member of the GRK family, desensitizes central M(2) muscarinic receptors in mice. We questioned whether GRK5 might also be an important regulator of peripheral muscarinic receptor responsiveness in the cardiopulmonary system. Specifically, we wanted to determine the role of GRK5 in regulating muscarinic receptor-mediated control of airway smooth muscle tone or regulation of cholinergic-induced bradycardia. Tracheal pressure, heart rate, and tracheal smooth muscle tension were measured in mice having a targeted deletion of the GRK5 gene (GRK5(-/-)) and littermate wild-type (WT) control mice. Both in vivo and in vitro results showed that the airway contractile response to a muscarinic receptor agonist was not different between GRK5(-/-) and WT mice. However, the relaxation component of bilateral vagal stimulation and the airway smooth muscle relaxation resulting from beta(2)-adrenergic receptor activation were diminished in GRK5(-/-) mice. These data suggest that M(2) muscarinic receptor-mediated opposition of airway smooth muscle relaxation is regulated by GRK5 and is, therefore, excessive in GRK5(-/-) mice. In addition, this study shows that GRK5 regulates pulmonary responses in a tissue- and receptor-specific manner but does not regulate peripheral cardiac muscarinic receptors. GRK5 regulation of airway responses may have implications in obstructive airway diseases such as asthma or chronic obstructive pulmonary disease.     

Molecular modeling of the oxytocin receptor/bioligand interactions

Oxytocin is a nonapeptide hormone (CYIQNCPLG-NH2, OT), controlling labor and lactation in mammalian females, via interactions with specific cellular membrane receptors (OTRs). The native hormone is cyclized via a 1-6 disulfide and its receptor belongs to the GTP-binding (G) protein-coupled receptor (GPCR) family, also known as heptahelical transmembrane (7TM) or serpentine receptors. Using a technique combining multiple sequence alignments with available experimental constraints, a reliable OTR model was built. Subsequently, the OTR complexes with a selective agonist [Thr4,Gly7]OT, a selective cyclohexapeptide antagonist L-366,948 and oxytocin itself were modeled and relaxed using a constrained simulated annealing (CSA) protocol. All three ligands seem to prefer similar modes of binding to the receptor, manifested by repeating receptor residues which directly interact with the ligands. Those involved in the three complexes are putative helices: TM3: R113, K116, Q119, M123; TM4: Q171, and TM5: I201 and T205. Most of them are the equivalent residues/positions to those found in our earlier studies, regarding related vasopressin V2 receptor/bioligand interactions.     

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.     

Human substance P receptor undergoes agonist-dependent phosphorylation by G protein-coupled receptor kinase 5 in vitro

G protein-coupled receptor kinases (GRKs) phosphorylate agonist-occupied G protein-coupled receptors, leading to receptor desensitization. Seven GRKs, designated GRK1 through 7, have been characterized. GRK5 is negatively regulated by protein kinase C. We investigated whether human substance P receptor (hSPR) is a substrate of GRK5. We report that membrane-bound hSPR is phosphorylated by purified GRK5, and that both the rate and extent of phosphorylation increase dramatically in the presence of substance P. The phosphorylation has a high stoichiometry (20+/-4 mol phosphate/mol hSPR) and a low K(m) (1.7+/-0.1 nM). These data provide the first evidence that hSPR is a substrate of GRK5.     

G protein-coupled receptor kinases and beta arrestins are relocalized and attenuate cyclic 3',5'-adenosine monophosphate response to follicle-stimulating hormone in rat primary Sertoli cells.

The FSH receptor (FSH-R) is a member of the rhodopsin-like subfamily of G protein-coupled receptors that undergoes homologous desensitization upon agonist stimulation. In immortalized cell lines overexpressing the FSH-R, G protein-coupled receptor kinases (GRKs) and beta-arrestins are involved in the phosphorylation, uncoupling, and internalization of this receptor. In an effort to appreciate the physiological relevance of GRK/beta-arrestin actions in natural FSH-R-bearing cells, we used primary rat Sertoli cells as a model. GRK2, -3, -5, -6a, and -6b and beta-arrestins 1 and 2 were expressed in primary rat Sertoli cells. Overexpression of these different GRKs and beta-arrestins in primary rat Sertoli cells significantly attenuated the FSH-induced cAMP response, and FSH rapidly triggered a relocalization of endogenously expressed GRK2, -3, -5, and -6 and beta-arrestins 1 and 2 from the cytosol to the membranes. These results highlight the relationship existing between the GRK/beta-arrestin regulatory system and the FSH-R signaling machinery in a physiological model.     

Kinase-inactive G-protein-coupled receptor kinases are able to attenuate follicle-stimulating hormone-induced signaling

Homologous desensitization of G-protein-coupled receptors (GPCR) is thought to occur in several steps: binding of G-protein-coupled receptor kinases (GRKs) to receptors, receptor phosphorylation, kinase dissociation, and finally binding of beta-arrestin to phosphorylated receptors and functional uncoupling of the associated Galpha protein. It has recently been reported that GRKs can inhibit Galphaq-mediated signaling in the absence of phosphorylation of some GPCRs. Whether or not comparable phosphorylation-independent effects are also possible with Galphas-coupled receptors remains unclear. In the present study, using the tightly Galphas-coupled FSR receptor (FSH-R) as a model, we observed inhibition of the cAMP-dependent signaling pathway using kinase-inactive mutants of GRK2, 5, and 6. These negative effects occur upstream of adenylyl cyclase activation and are likely independent of GRK interaction with G protein alpha or beta/gamma subunits. Moreover, we demonstrated that, when overexpressed in Cos 7 cells, mutated GRK2 associates with the FSH activated FSH-R. We hypothesize that phosphorylation-independent dampening of the FSH-R-associated signaling could be attributable to physical association between GRKs and the receptor, subsequently inhibiting G protein activation.     

G protein-coupled receptor kinase function is essential for chemosensation in C. elegans

G protein-coupled receptors (GPCRs) mediate diverse signaling processes, including olfaction. G protein-coupled receptor kinases (GRKs) are important regulators of G protein signal transduction that specifically phosphorylate activated GPCRs to terminate signaling. Despite previously described roles for GRKs in GPCR signal downregulation, animals lacking C. elegans G protein-coupled receptor kinase-2 (Ce-grk-2) function are not hypersensitive to odorants. Instead, decreased Ce-grk-2 function in adult sensory neurons profoundly disrupts chemosensation, based on both behavioral analysis and Ca(2+) imaging. Although mammalian arrestin proteins cooperate with GRKs in receptor desensitization, loss of C. elegans arrestin-1 (arr-1) does not disrupt chemosensation. Either overexpression of the C. elegans Galpha subunit odr-3 or loss of eat-16, which encodes a regulator of G protein signaling (RGS) protein, restores chemosensation in Ce-grk-2 mutants. These results demonstrate that loss of GRK function can lead to reduced GPCR signal transduction and suggest an important role for RGS proteins in the regulation of chemosensation.     

Effects of cholesterol manipulation on the signaling of the human oxytocin receptor

We have recently shown that oxytocin inhibits cell growth when the vast majority of oxytocin receptors (OTRs) are excluded from detergent-resistant membranes (DRMs; the biochemical counterpart of lipid rafts), but has a strong mitogenic effect when the receptors are targeted to these plasma membrane domains upon fusion with caveolin-2, a resident raft protein. The aim of this study was to investigate whether the manipulation of total cell cholesterol can influence OTR localization and signaling. Our data indicate that cholesterol depletion in HEK-293 cells does not affect the signaling events mediated by the OTRs located outside DRMs. When treated with 2 mM methyl-beta-cyclodextrin (MbetaCD), the receptors remained outside and continued to inhibit cell growth. On the contrary, the MbetaCD treatment of cells expressing receptors fused to caveolin-2 led to their redistribution outside DRMs, and converted the receptor-mediated proliferative effect into cell growth inhibition. These data indicate that 1) once released from DRMs, the receptors fused to caveolin-2 signal exactly as wild-type OTRs and 2) their DRM location is responsible for the specific OTR signaling leading to cell proliferation. Finally, we evaluated whether cholesterol loading could force the OTRs into lipid rafts and change their signaling, but, after cell treatment with an MbetaCD/cholesterol complex, receptor stimulation continued to lead to cell growth inhibition, thus indicating that increasing cell cholesterol levels is not sufficient per se to affect OTR signaling.     

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.     

Pathophysiological roles of G-protein-coupled receptor kinases

G-protein-coupled receptor kinases (GRKs) interact with the agonist-activated form of G-protein-coupled receptors (GPCRs) to effect receptor phosphorylation and to initiate profound impairment of receptor signalling, or desensitization. GPCRs form the largest family of cell surface receptors known and defects in GRK function have the potential consequence to affect GPCR-stimulated biological responses in many pathological situations. This review focuses on the physiological role of GRKs revealed by genetically modified animals but also develops the involvement of GRKs in human diseases as, Oguchi disease, heart failure, hypertension or rhumatoid arthritis. Furthermore, the regulation of GRK levels in opiate addiction, cancers, psychiatric diseases, cystic fibrosis and cardiac diseases is discussed. Both transgenic mice and human pathologies have demonstrated the importance of GRKs in the signalling pathways of rhodopsin, beta-adrenergic and dopamine-1 receptors. The modulation of GRK activity in animal models of cardiac diseases can be effective to restore cardiac function in heart failure and opens a novel therapeutic strategy in diseases with GPCR dysregulation.     

Real-time detection of interactions between the human oxytocin receptor and G protein-coupled receptor kinase-2

Although the oxytocin receptor (OTR) mediates many important functions including uterine contractions, milk ejection, and maternal behavior, the mechanisms controlling agonist-induced OTR desensitization have remained unclear, and attempts to demonstrate involvement of a G protein-coupled receptor kinase (GRK) have so far failed. Using the OTR as a model, we demonstrate here directly for the first time the dynamics of agonist-induced interactions of a GRK with a G protein-coupled receptor in real time, using time-resolved bioluminescence resonance energy transfer. GRK2/receptor interactions started within 4 sec, peaked at 10 sec, and decreased to less than 40% within 8 min. By contrast, beta-arrestin/OTR interactions initiated only at 10 sec, reached plateau levels at 120 sec, but remained stable with little decrease thereafter. Physical GRK2/OTR association was further demonstrated by coimmunoprecipitation of endogenous GRK2 with activated OTR. In COS-7 cells, which express low levels of GRK2 and beta-arrestin, overexpression of GRK2 and beta-arrestin increased receptor phosphorylation, desensitization, and internalization to the high levels observed in human embryonic kidney 293 cells. By contrast, specific inhibition of endogenous GRK2 by dominant-negative mutants robustly inhibited OTR phosphorylation and internalization as well as arrestin/OTR interactions. These data characterize the temporal and causal relationship of GRK-2/OTR and beta-arrestin/OTR interactions and establish GRK/OTR interaction as a prerequisite for beta-arrestin-mediated OTR desensitization.     

Amino acids in the COOH-terminal region of the oxytocin receptor third intracellular domain are important for receptor function

Previously, residue K6.30 in the COOH-terminal region of the third intracellular domain (3iC) of the oxytocin (OT) receptor (OTR) was identified as important for receptor function leading to phospholipase C activation in both OTR and the vasopressin V(2) receptor (V(2)R) chimera V(2)ROTR3iC. Substitution of either A6.28K or V6.30K in wild-type V(2)R did not recapitulate the increase in phosphatidylinositide (PI) turnover observed in V(2)ROTR3iC. Hence, the role of K6.30 may be context-specific. Deletion of two NH(2)-terminal OTR3iC segments in the V(2)ROTR3iC chimera did not diminish vasopressin-stimulated PI turnover, whereas deletion of RVSSVKL (residues 6.19-6.25) reduced receptor expression. Deletion of this sequence in wild-type OTR reduced expression by 50% without affecting affinity for [(3)H]OT. This OTR mutant was unable to activate PI turnover or extracellular signal-regulated kinase 1/2 phosphorylation. The effects of alanine substitution for individual residues in RVSSVKL indicated differential importance for OTR function. The R6.19A substitution lost high-affinity sites for [(3)H]OT and the ability to stimulate PI turnover. Affinity for [(3)H]OT and membrane expression was not affected by any other substitutions. OTR-V6.20A and OTR-K6.24A mutants functioned as well as wild-type OTR, whereas OTR S6.21A, S6.22A, and V6.23A mutants exhibited impaired abilities to activate PI turnover (20-40% of OTR), and the OTR-L6.25A mutant exhibited constitutive activity. In conclusion, specific amino acids in the RVSSVKL segment in the COOH-terminal region of the third intracellular domain of OTR influence the ability of OTR to activate G protein-mediated actions.     

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.