Beta-arrestin mediates desensitization and internalization but does not affect dephosphorylation of the thyrotropin-releasing hormone receptor

The G protein-coupled thyrotropin-releasing hormone (TRH) receptor is phosphorylated and binds to beta-arrestin after agonist exposure. To define the importance of receptor phosphorylation and beta-arrestin binding in desensitization, and to determine whether beta-arrestin binding and receptor endocytosis are required for receptor dephosphorylation, we expressed TRH receptors in fibroblasts from mice lacking beta-arrestin-1 and/or beta-arrestin-2. Apparent affinity for [(3)H]MeTRH was increased 8-fold in cells expressing beta-arrestins, including a beta-arrestin mutant that did not permit receptor internalization. TRH caused extensive receptor endocytosis in the presence of beta-arrestins, but receptors remained primarily on the plasma membrane without beta-arrestin. beta-Arrestins strongly inhibited inositol 1,4,5-trisphosphate production within 10 s. At 30 min, endogenous beta-arrestins reduced TRH-stimulated inositol phosphate production by 48% (beta-arrestin-1), 71% (beta-arrestin-2), and 84% (beta-arrestins-1 and -2). In contrast, receptor phosphorylation, detected by the mobility shift of deglycosylated receptor, was unaffected by beta-arrestins. Receptors were fully phosphorylated within 15 s of TRH addition. Receptor dephosphorylation was identical with or without beta-arrestins and almost complete 20 min after TRH withdrawal. Blocking endocytosis with hypertonic sucrose did not alter the rate of receptor phosphorylation or dephosphorylation. Expressing receptors in cells lacking Galpha(q) and Galpha(11) or inhibiting protein kinase C pharmacologically did not prevent receptor phosphorylation or dephosphorylation. Overexpression of dominant negative G protein-coupled receptor kinase-2 (GRK2), however, retarded receptor phosphorylation. Receptor activation caused translocation of endogenous GRK2 to the plasma membrane. The results show conclusively that receptor dephosphorylation can take place on the plasma membrane and that beta-arrestin binding is critical for desensitization and internalization.     

Post-endocytic fates of delta-opioid receptor are regulated by GRK2-mediated receptor phosphorylation and distinct beta-arrestin isoforms

Once internalized, some G protein-coupled receptors (GPCRs) can recycle back to the cell surface, while some of them are delivered to lysosomes for degradation. Because recycling and degradation represent two opposing receptor fates, understanding the mechanisms that determine post-endocytic fate of GPCRs is of great importance. Our recent work has verified that agonist-induced internalization of delta-opioid receptor (DOR) employs both phosphorylation-dependent and -independent mechanisms in HEK293 cells. To investigate whether these two internalization mechanisms work differently in receptor regulation, we monitored receptor post-endocytic fates using flow cytometry, surface receptor biotinylation and radioligand binding assays. Results showed that the internalized wild type DOR could either recycle to the cell surface or be degraded. Mutant DOR M4/5/6, which lacks all three G protein-coupled receptor kinase 2 (GRK2) phosphorylation sites, could also internalize upon agonist challenge although in a reduced level as compared with the wild type counterpart. However, the internalized mutant DOR could not recycle back to the cell surface and all mutant DOR was degraded after internalization. Inhibition of GRK2 expression by GRK2 RNAi also strongly attenuated recycling of DOR. Furthermore, overexpression of GRK2, which significantly increased receptor phosphorylation and internalization, also targeted more internalized receptors to the recycling pathway. These data suggest that GRK2-catalyzed receptor phosphorylation is critically involved in DOR internalization and recycling, and the phosphorylation-independent internalization leads to receptor degradation. Data obtained from beta-arrestin1 and beta-arrestin2 RNAi experiments indicated that both beta-arrestin1 and beta-arrestin2 participate in phosphorylation-dependent internalization and the subsequent recycling of DOR. However, phosphorylation-independent internalization and degradation of DOR were strongly blocked by beta-arrestin2 RNAi, but not beta-arrestin1 RNAi. Taken together, these data demonstrate for the first time that GRK2 phosphorylation-dependent internalization mediated by both beta-arrestin1 and beta-arrestin2 leads DOR to recycle, whereas GRK2-independent internalization mediated by beta-arrestin2 alone leads to receptor degradation. Thus, the post-endocytic fate of internalized DOR can be regulated by GRK2-catalyzed receptor phosphorylation as well as distinct beta-arrestin isoforms.     

Role of the G protein-coupled receptor kinase site serine cluster in beta2-adrenergic receptor internalization, desensitization, and beta-arrestin translocation

There is considerable evidence for the role of carboxyl-terminal serines 355, 356, and 364 in G protein-coupled receptor kinase (GRK)-mediated phosphorylation and desensitization of beta(2)-adrenergic receptors (beta(2)ARs). In this study we used receptors in which these serines were changed to alanines (SA3) or to aspartic acids (SD3) to determine the role of these sites in beta-arrestin-dependent beta(2)AR internalization and desensitization. Coupling efficiencies for epinephrine activation of adenylyl cyclase were similar in wild-type and mutant receptors, demonstrating that the SD3 mutant did not drive constitutive GRK desensitization. Treatment of wild-type and mutant receptors with 0.3 nm isoproterenol for 5 min induced approximately 2-fold increases in the EC(50) for agonist activation of adenylyl cyclase, consistent with protein kinase A (PKA) site-mediated desensitization. When exposed to 1 mum isoproterenol to trigger GRK site-mediated desensitization, only wild-type receptors showed significant further desensitization. Using a phospho site-specific antibody, we determined that there is no requirement for these GRK sites in PKA-mediated phosphorylation at high agonist concentration. The rates of agonist-induced internalization of the SD3 and SA3 mutants were 44 and 13%, respectively, relative to that of wild-type receptors, but the SD3 mutant recruited enhanced green fluorescent protein (EGFP)-beta-arrestin 2 to the plasma membrane, whereas the SA3 mutant did not. EGFP-beta-Arrestin2 overexpression triggered a significant increase in the extent of SD3 mutant desensitization but had no effect on the desensitization of wild-type receptors or the SA3 mutant. Expression of a phosphorylation-independent beta-arrestin 1 mutant (R169E) significantly rescued the internalization defect of the SA3 mutant but inhibited the phosphorylation of serines 355 and 356 in wild-type receptors. Our data demonstrate that (i) the lack of GRK sites does not impair PKA site phosphorylation, (ii) the SD3 mutation inhibits GRK-mediated desensitization although it supports some agonist-induced beta-arrestin binding and receptor internalization, and (iii) serines 355, 356, and 364 play a pivotal role in the GRK-mediated desensitization, beta-arrestin binding, and internalization of beta(2)ARs.     

Beta-arrestin1 and beta-arrestin2 are differentially required for phosphorylation-dependent and -independent internalization of delta-opioid receptors

Beta-arrestins are key negative regulators and scaffolds of G protein-coupled receptor (GPCR) signalling. Beta-arrestin1 and beta-arrestin2 preferentially bind to the phosphorylated GPCRs in response to agonist stimulation, resulting in receptor internalization and desensitization. The critical roles of GPCR kinases (GRKs)-catalyzed receptor phosphorylation and interaction of beta-arrestins with the phosphorylated receptor in receptor internalization are well established. However, emerging evidence suggests that an agonist-stimulated internalization mechanism that is independent of receptor phosphorylation may also be employed in some cases, although the molecular mechanism for the phosphorylation-independent GPCR internalization is not clear. The current study investigated the role of receptor phosphorylation and the involvement of different beta-arrestin subtypes in agonist-induced delta-opioid receptor (DOR) internalization in HEK293 cells. Results from flow cytometry, fluorescence microscopy, and surface biotin labelling experiments showed that elimination of agonist-induced DOR phosphorylation by mutation GRK binding or phosphorylation sites only partially blocked agonist-induced receptor internalization, indicating the presence of an agonist-induced, GRK-independent mechanism for DOR internalization. Fluorescence and co-immunoprecipitation studies indicated that both the wild-type DOR and the phosphorylation-deficient mutant receptor could bind and recruit beta-arrestin1 and beta-arrestin2 to the plasma membrane in an agonist-stimulated manner. Furthermore, internalization of both the wild-type and phosphorylation-deficient receptors was increased by overexpression of either type of beta-arrestins and blocked by dominant-negative mutants of beta-arrestin-mediated internalization, demonstrating that both phosphorylation-dependent and -independent internalization require beta-arrestin. Moreover, double-stranded RNA-mediated interference experiments showed that either beta-arrestin1 or beta-arrestin2 subtype-specific RNAi only partially inhibited agonist-induced internalization of the wild-type DOR. However, agonist-induced internalization of the phosphorylation-deficient DOR was not affected by beta-arrestin1-specific RNAi but was blocked by RNAi against beta-arrestin2 subtype. These data indicate that endogenous beta-arrestin1 functions exclusively in the phosphorylation-dependent receptor internalization, whereas endogenous beta-arrestin2, but not beta-arrestin1, is required for the phosphorylation-independent receptor internalization. These results thus provide the first evidence of different requirement for beta-arrestin isoforms in the agonist induced phosphorylation-dependent and -independent GPCR internalization.     

Vasoactive intestinal polypeptide type-1 receptor regulation. Desensitization, phosphorylation, and sequestration

The vasoactive intestinal polypeptide type-1 (VPAC(1)) receptor is a class II G protein-coupled receptor, distinct from the adrenergic receptor superfamily. The mechanisms involved in the regulation of the VPAC(1) receptor are largely unknown. We examined agonist-dependent VPAC(1) receptor signaling, phosphorylation, desensitization, and sequestration in human embryonic kidney 293 cells. Agonist stimulation of cells overexpressing this receptor led to a dose-dependent increase in cAMP that peaked within 5-10 min and was completely desensitized after 20 min. Cells cotransfected with the VPAC(1) receptor (VPAC(1)R) and G protein-coupled receptor kinases (GRKs) 2, 3, 5, and 6 exhibited enhanced desensitization that was not evident with GRK 4. Immunoprecipitation of the epitope-tagged VPAC(1) receptor revealed dose-dependent phosphorylation that was increased with cotransfection of any GRK. Agonist-stimulated internalization of the VPAC(1)R peaked in 10 min, and neither overexpressed beta-arrestin nor its dominant-negative mutant altered internalization. However, a dynamin-dominant negative mutant did inhibit VPAC(1) receptor internalization. Interestingly, VPAC(1)R specificity in desensitization was not evident by study of the overexpressed receptor; however, we determined that human embryonic kidney 293 cells express an endogenous VPAC(1)R that did demonstrate dose-dependent GRK specificity. Therefore, VPAC(1) receptor regulation involves agonist-stimulated, GRK-mediated phosphorylation, beta-arrestin translocation, and dynamin-dependent receptor internalization. Moreover, study of endogenously expressed receptors may provide information not evident in overexpressed systems.     

Homologous desensitization of signalling by the beta (beta) isoform of the human thromboxane A2 receptor

Thromboxane (TX) A(2) is a potent stimulator of platelet activation/aggregation and smooth muscle contraction and contributes to a variety of pathologies within the vasculature. In this study, we investigated the mechanism whereby the cellular responses to TXA(2) mediated through the TPbeta isoform of the human TXA(2) receptor (TP) are dynamically regulated by examining the mechanism of agonist-induced desensitization of intracellular signalling and second messenger generation by TPbeta. It was established that TPbeta is subject to profound agonist-induced homologous desensitization of signalling (intracellular calcium mobilization and inositol 1,3,5 trisphosphate generation) in response to stimulation with the TXA(2) mimetic U46619 and this occurs through two key mechanisms: TPbeta undergoes partial agonist-induced desensitization that occurs through a GF 109203X-sensitive, protein kinase (PK)C mechanism whereby Ser(145) within intracellular domain (IC)(2) has been identified as the key phospho-target. In addition, TPbeta also undergoes more profound and sustained agonist-induced desensitization involving G protein-coupled receptor kinase (GRK)2/3-phosphorylation of both Ser(239) and Ser(357) within its IC(3) and carboxyl-terminal C-tail domains, respectively. Inhibition of phosphorylation of either Ser(239) or Ser(357), through site directed mutagenesis, impaired desensitization while mutation of both Ser(239) and Ser(357) almost completely abolished desensitization of signalling, GRK phosphorylation and beta-arrestin association, thereby blocking TPbeta internalization. These data suggest a model whereby agonist-induced PKC phosphorylation of Ser(145) partially impairs. TPbeta signalling while GRK2/3 phosphorylation at both Ser(239) and Ser(357) within its IC(3) and C-tail domains, respectively, sterically inhibits G-protein coupling, profoundly desensitizing signalling, and promotes beta-arrestin association and, in turn, facilitates TPbeta internalization. Thromboxane (TX) A(2) is a potent stimulator of platelet aggregation and smooth muscle contraction and contributes to a variety of vascular pathologies. Herein the mechanism whereby the cellular responses to TXA(2) mediated through the TPbeta isoform of the human TXA(2) receptor (TP) are dynamically regulated was investigated by examining the mechanism of its agonist-induced desensitization of intracellular signalling and second messenger generation. TPbeta is subject to profound agonist-induced homologous desensitization of signalling (intracellular calcium mobilization and inositol 1,3,5 trisphosphate generation) in response to stimulation with the TXA(2) mimetic U46619 and this occurs through two key mechanisms: TPbeta undergoes partial agonist-induced desensitization that occurs through a GF 109203X-sensitive, protein kinase (PK)C mechanism whereby Ser(145) within intracellular domain (IC)(2) was identified as the key phospho-target. In addition, TPbeta also undergoes more profound and sustained agonist-induced desensitization involving G protein-coupled receptor kinase (GRK)2/3-phosphorylation of both Ser(239) and Ser(357) within its IC(3) and carboxyl-terminal C-tail domains, respectively. Inhibition of phosphorylation of either Ser(239) or Ser(357), through site directed mutagenesis, impaired desensitization while mutation of both Ser(239) and Ser(357) almost completely abolished desensitization of signalling, GRK phosphorylation and beta-arrestin association, thereby blocking TPbeta internalization. These data suggest a model whereby agonist-induced PKC phosphorylation of Ser(145) partially impairs TPbeta signalling while GRK2/3 phosphorylation at both Ser(239) and Ser(357) within its IC(3) and C-tail domains, respectively, sterically inhibits G-protein coupling, profoundly desensitizing signalling, and promotes beta-arrestin association and, in turn, facilitates TPbeta internalization.     

Regulation of EGF-induced ERK/MAPK activation and EGFR internalization by G protein-coupled receptor kinase 2

G protein-coupled receptor kinases （GRKs） mediate agonist-induced phosphorylation and desensitization of various G protein-coupled receptors （GPCRs）. We investigate the role of GRK2 on epidermal growth factor （EGF） receptor signaling, including EGF-induced extracellular signal-regulated kinase and mitogen-activated protein kinase （ERK/MAPK） activation and EGFR internalization. Immunoprecipitation and immunofluorescence experiments show that EGF stimulates GRK2 binding to EGFR complex and GRK2 translocating from cytoplasm to the plasma membrane in human embryonic kidney 293 cells. Western blotting assay shows that EGF-induced ERK/MAPK phosphorylation increases 1.9-fold, 1.1-fold and 1.5fold （P〈0.05） at time point 30, 60 and 120 min, respectively when the cells were transfected with GRK2,suggesting the regulatory role of GRK2 on EGF-induced ERK/MAPK activation. Flow cytometry experiments show that GRK2 overexpression has no effect on EGF-induced EGFR internalization, however, it increases agonist-induced G protein-coupled δ5 opioid receptor internalization by approximately 40% （P〈0.01）. Overall,these data suggest that GRK2 has a regulatory role in EGF-induced ERK/MAPK activation, and that the mechanisms underlying the modulatory role of GRK2 in EGFR and GPCR signaling pathways are somewhat different at least in receptor internalization.     

Protein kinase A and G protein-coupled receptor kinase phosphorylation mediates beta-1 adrenergic receptor endocytosis through different pathways

Agonist-induced phosphorylation of beta-adrenergic receptors (beta ARs) by G protein-coupled receptor kinases (GRKs) results in their desensitization followed by internalization. Whether protein kinase A (PKA)-mediated phosphorylation of beta ARs, particularly the beta 1AR subtype, can also trigger internalization is currently not known. To test this, we cloned the mouse wild type beta 1AR (WT beta 1AR) and created 3 mutants lacking, respectively: the putative PKA phosphorylation sites (PKA-beta 1AR), the putative GRK phosphorylation sites (GRK-beta 1AR), and both sets of phosphorylation sites (PKA-/GRK-beta 1AR). Following agonist stimulation, both PKA-beta 1AR and GRK-beta 1AR mutants showed comparable increases in phosphorylation and desensitization. Saturating concentrations of agonist induced only 50% internalization of either mutant compared with wild type, suggesting that both PKA and GRK phosphorylation of the receptor contributed to receptor sequestration in an additive manner. Moreover, in contrast to the WT beta 1AR and PKA-beta 1AR, sequestration of the GRK-beta 1AR and PKA-/GRK-beta 1AR was independent of beta-arrestin recruitment. Importantly, clathrin inhibitors abolished agonist-dependent internalization for both the WT beta 1AR and PKA-beta 1AR, whereas caveolae inhibitors prevented internalization only of the GRK-beta 1AR mutant. Taken together, these data demonstrate that: 1) PKA-mediated phosphorylation can trigger agonist-induced internalization of the beta 1AR and 2) the pathway selected for beta 1AR internalization is primarily determined by the kinase that phosphorylates the receptor, i.e. PKA-mediated phosphorylation directs internalization via a caveolae pathway, whereas GRK-mediated phosphorylation directs it through clathrin-coated pits.     

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.     

Agonist-dependent desensitization of the kappa opioid receptor by G protein receptor kinase and beta-arrestin.

We used the Xenopus oocyte expression system to examine the regulation of rat kappa opioid receptor (rKOR) function by G protein receptor kinases (GRKs). kappa agonists increased the conductance of G protein-activated inwardly rectifying potassium channels in oocytes co-expressing KOR with Kir3.1 and Kir3.4. In the absence of added GRK and beta-arrestin 2, desensitization of the kappa agonist-induced potassium current was modest. Co-expression of either GRK3 or GRK5 along with beta-arrestin 2 significantly increased the rate of desensitization, whereas addition of either beta-arrestin 2, GRK3, or GRK5 alone had no effect on the KOR desensitization rate. The desensitization was homologous as co-expressed delta opioid receptor-evoked responses were not affected by KOR desensitization. The rate of GRK3/beta-arrestin 2-dependent desensitization was reduced by truncation of the C-terminal 26 amino acids, KOR(Q355Delta). In contrast, substitution of Ala for Ser within the third intracellular loop [KOR(S255A,S260A, S262A)] did not reduce the desensitization rate. Within the C-terminal region, KOR(S369A) substitution significantly attenuated desensitization, whereas the KOR(T363A) and KOR(S356A,T357A) point mutations did not. These results suggest that co-expression of GRK3 or GRK5 and beta-arrestin 2 produced homologous, agonist-induced desensitization of the kappa opioid receptor by a mechanism requiring the phosphorylation of the serine 369 of rKOR.     

Beta-arrestin- and G protein receptor kinase-mediated calcium-sensing receptor desensitization

Extracellular calcium rapidly controls PTH secretion through binding to the G protein-coupled calcium-sensing receptor (CASR) expressed in parathyroid glands. Very little is known about the regulatory proteins involved in desensitization of CASR. G protein receptor kinases (GRK) and beta-arrestins are important regulators of agonist-dependent desensitization of G protein-coupled receptors. In the present study, we investigated their role in mediating agonist-dependent desensitization of CASR. In heterologous cell culture models, we found that the transfection of GRK4 inhibits CASR signaling by enhancing receptor phosphorylation and beta-arrestin translocation to the CASR. In contrast, we found that overexpression of GRK2 desensitizes CASR by classical mechanisms as well as through phosphorylation-independent mechanisms involving disruption of Galphaq signaling. In addition, we observed lower circulating PTH levels and an attenuated increase in serum PTH after hypocalcemic stimulation in beta-arrestin2 null mice, suggesting a functional role of beta-arrestin2-dependent desensitization pathways in regulating CASR function in vivo. We conclude that GRKs and beta-arrestins play key roles in regulating CASR responsiveness in parathyroid glands.     

Agonist-specific regulation of delta-opioid receptor trafficking by G protein-coupled receptor kinase and beta-arrestin

Opioid receptors mediate multiple biological functions through their interaction with endogenous opioid peptides as well as opioid alkaloids including morphine and etorphine. Previously we have reported that the ability of distinct opioid agonists to differentially regulate mu-opioid receptor (mu OR) responsiveness is related to their ability to promote G protein-coupled receptor kinase (GRK)-dependent phosphorylation of the receptor (1). In the present study, we further examined the role of GRK and beta-arrestin in agonist-specific regulation of the delta-opioid receptor (delta OR). While both etorphine and morphine effectively activate the delta OR, only etorphine triggers robust delta OR phosphorylation followed by plasma membrane translocation of beta-arrestin and receptor internalization. In contrast, morphine is unable to either elicit delta OR phosphorylation or stimulate beta-arrestin translocation, correlating with its inability to cause delta OR internalization. Unlike for the mu OR, overexpression of GRK2 results in neither the enhancement of delta OR sequestration nor the rescue of delta OR-mediated beta-arrestin translocation. Therefore, our findings not only point to the existence of marked differences in the ability of different opioid agonists to promote delta OR phosphorylation by GRK and binding to beta-arrestin, but also demonstrate differences in the regulation of two opioid receptor subtypes. These observations may have important implications for our understanding of the distinct ability of various opioids in inducing opioid tolerance and addiction.     

Targeted construction of phosphorylation-independent beta-arrestin mutants with constitutive activity in cells

Arrestin proteins play a key role in the desensitization of G protein-coupled receptors (GPCRs). Recently we proposed a molecular mechanism whereby arrestin preferentially binds to the activated and phosphorylated form of its cognate GPCR. To test the model, we introduced two different types of mutations into beta-arrestin that were expected to disrupt two crucial elements that make beta-arrestin binding to receptors phosphorylation-dependent. We found that two beta-arrestin mutants (Arg169 --> Glu and Asp383 --> Ter) (Ter, stop codon) are indeed "constitutively active." In vitro these mutants bind to the agonist-activated beta2-adrenergic receptor (beta2AR) regardless of its phosphorylation status. When expressed in Xenopus oocytes these beta-arrestin mutants effectively desensitize beta2AR in a phosphorylation-independent manner. Constitutively active beta-arrestin mutants also effectively desensitize delta opioid receptor (DOR) and restore the agonist-induced desensitization of a truncated DOR lacking the critical G protein-coupled receptor kinase (GRK) phosphorylation sites. The kinetics of the desensitization induced by phosphorylation-independent mutants in the absence of receptor phosphorylation appears identical to that induced by wild type beta-arrestin + GRK3. Either of the mutations could have occurred naturally and made receptor kinases redundant, raising the question of why a more complex two-step mechanism (receptor phosphorylation followed by arrestin binding) is universally used.     

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.     

Heterologous activation of protein kinase C stimulates phosphorylation of delta-opioid receptor at serine 344, resulting in beta-arrestin- and clathrin-mediated receptor internalization

The purpose of the current study is to investigate the effect of opioid-independent, heterologous activation of protein kinase C (PKC) on the responsiveness of opioid receptor and the underlying molecular mechanisms. Our result showed that removing the C terminus of delta opioid receptor (DOR) containing six Ser/Thr residues abolished both DPDPE- and phorbol 12-myristate 13-acetate (PMA)-induced DOR phosphorylation. The phosphorylation levels of DOR mutants T352A, T353A, and T358A/T361A/S363S were comparable to that of the wild-type DOR, whereas S344G substitution blocked PMA-induced receptor phosphorylation, indicating that PKC-mediated phosphorylation occurs at Ser-344. PKC-mediated Ser-344 phosphorylation was also induced by activation of G(q)-coupled alpha(1A)-adrenergic receptor or increase in intracellular Ca(2+) concentration. Activation of PKC by PMA, alpha(1A)-adrenergic receptor agonist, and ionomycin resulted in DOR internalization that required phosphorylation of Ser-344. Expression of dominant negative beta-arrestin and hypertonic sucrose treatment blocked PMA-induced DOR internalization, suggesting that PKC mediates DOR internalization via a beta-arrestin- and clathrin-dependent mechanism. Further study demonstrated that agonist-dependent G protein-coupled receptor kinase (GRK) phosphorylation sites in DOR are not targets of PKC. Agonist-dependent, GRK-mediated receptor phosphorylation and agonist-independent, PKC-mediated DOR phosphorylation were additive, but agonist-induced receptor phosphorylation could inhibit PKC-catalyzed heterologous DOR phosphorylation and subsequent internalization. These data demonstrate that the responsiveness of opioid receptor is regulated by both PKC and GRK through agonist-dependent and agonist-independent mechanisms and PKC-mediated receptor phosphorylation is an important molecular mechanism of heterologous regulation of opioid receptor functions.     

Agonist-induced signaling, desensitization, and internalization of a phosphorylation-deficient AT1A angiotensin receptor

An analysis of the functional role of a diacidic motif (Asp236-Asp237) in the third intracellular loop of the AT1A angiotensin II (Ang II) receptor (AT1-R) revealed that substitution of both amino acids with alanine (DD-AA) or asparagine (DD-NN) residues diminished Ang II-induced receptor phosphorylation in COS-7 cells. However, Ang II-stimulated inositol phosphate production, mitogen-activated protein kinase, and AT1 receptor desensitization and internalization were not significantly impaired. Overexpression of dominant negative G protein-coupled receptor kinase 2 (GRK2)K220M decreased agonist-induced receptor phosphorylation by approximately 40%, but did not further reduce the impaired phosphorylation of DD-AA and DD-NN receptors. Inhibition of protein kinase C by bisindolylmaleimide reduced the phosphorylation of both the wild-type and the DD mutant receptors by approximately 30%. The inhibitory effects of GRK2K220M expression and protein kinase C inhibition by bisindolylmaleimide on agonist-induced phosphorylation were additive for the wild-type AT1-R, but not for the DD mutant receptor. Agonist-induced internalization of the wild-type and DD mutant receptors was similar and was unaltered by coexpression of GRK2K220M. These findings demonstrate that an acidic motif at position 236/237 in the third intracellular loop of the AT1-R is required for optimal Ang II-induced phosphorylation of its carboxyl-terminal tail by GRKs. Furthermore, the properties of the DD mutant receptor suggest that not only Ang II-induced signaling, but also receptor desensitization and internalization, are independent of agonist-induced GRK-mediated phosphorylation of the AT1 receptor.     

Beta-arrestin binding to the beta2-adrenergic receptor requires both receptor phosphorylation and receptor activation

Homologous desensitization of beta2-adrenergic receptors has been shown to be mediated by phosphorylation of the agonist-stimulated receptor by G-protein-coupled receptor kinase 2 (GRK2) followed by binding of beta-arrestins to the phosphorylated receptor. Binding of beta-arrestin to the receptor is a prerequisite for subsequent receptor desensitization, internalization via clathrin-coated pits, and the initiation of alternative signaling pathways. In this study we have investigated the interactions between receptors and beta-arrestin2 in living cells using fluorescence resonance energy transfer. We show that (a) the initial kinetics of beta-arrestin2 binding to the receptor is limited by the kinetics of GRK2-mediated receptor phosphorylation; (b) repeated stimulation leads to the accumulation of GRK2-phosphorylated receptor, which can bind beta-arrestin2 very rapidly; and (c) the interaction of beta-arrestin2 with the receptor depends on the activation of the receptor by agonist because agonist withdrawal leads to swift dissociation of the receptor-beta-arrestin2 complex. This fast agonist-controlled association and dissociation of beta-arrestins from prephosphorylated receptors should permit rapid control of receptor sensitivity in repeatedly stimulated cells such as neurons.     

Clathrin box in G protein-coupled receptor kinase 2

beta(1)-Adrenergic receptor (beta(1)AR) shows the resistance to agonist-induced internalization. However, beta(1)AR can internalize as G protein-coupled receptor kinase 2 (GRK2) is fused to its carboxyl terminus. Internalization of the beta(1)AR and GRK2 fusion protein (beta(1)AR/GRK2) is dependent on dynamin but independent of beta-arrestin and phosphorylation. The beta(1)AR/GRK2 fusion protein internalizes via clathrin-coated pits and is found to co-localize with the endosome that contains transferrin. The fusion proteins consisting of beta(1)AR and various portions of GRK2 reveal that the residues 498-502 in the carboxyl-terminal domain of GRK2 are critical to promote internalization of the fusion proteins. This domain contains a consensus sequence of a clathrin-binding motif defined as a clathrin box. In vitro binding assays show that the residues 498-502 of GRK2 bind the amino-terminal domain of clathrin heavy chain to almost the same extent as beta-arrestin1. The mutation of the clathrin box in the carboxyl-terminal domain of GRK2 results in the loss of the ability to promote internalization of the fusion protein. GRK2 activity increases and then decreases as the concentration of clathrin heavy chain increases. Taken together, these results imply that GRK2 contains a functional clathrin box and directly interacts with clathrin to modulate its function.     

Internalization and desensitization of the oxytocin receptor is inhibited by Dynamin and clathrin mutants in human embryonic kidney 293 cells

Oxytocin (OT) has long been used as an uterotonic during labor management in women, and yet responses to OT infusion remain variable and unpredictable among patients. The investigation of oxytocin receptor (OTR) regulation will benefit labor management, because the clinical practice of continuous iv infusion of OT is not optimal. As with other G protein-coupled receptors, it is likely that the OTR internalizes and/or desensitizes upon continuous agonist exposure. The mechanisms by which this might occur, however, are unclear. Here we explore OTR internalization and desensitization in human embryonic kidney cells by utilizing inhibitors of heterologous second messenger systems and recently available mutant cDNA constructs. We report rapid and extensive internalization and desensitization of the OTR upon agonist exposure. Internalization was unaffected by inhibitors of protein kinase C or Ca(2+) calmodulin-dependant kinase II but was significantly reduced after transfection with dominant-negative mutant cDNAs of G protein-coupled receptor kinase 2, beta-Arrestin2, Dynamin, and Eps15 (a component of clathrin-coated pits). Moreover, desensitization of the OTR, measured by a calcium mobilization assay, was also inhibited by the aforementioned cDNA constructs. Thus, our data demonstrate, for the first time, the importance of the classical clathrin-mediated pathway during agonist-induced OTR internalization and desensitization.     

Exposure to TARC alters beta2-adrenergic receptor signaling in human peripheral blood T lymphocytes

The beta(2)-adrenergic receptor (beta(2)-AR) negatively regulates T cell activity through the activation of the G(s)/adenylyl cyclase/cAMP pathway. beta(2)-AR desensitization, which can be induced by its phosphorylation, may have important consequences for the regulation of T cell function in asthma. In the present study we demonstrate that the C-C chemokine thymus and activation-regulated chemokine (TARC) impairs the ability of beta(2)-agonist fenoterol to activate the cAMP downstream effector cAMP-responsive element binding protein (CREB) in freshly isolated human T cells. The TARC-induced activation of Src kinases resulted in membrane translocation of both G protein-coupled receptor kinase (GRK) 2 and beta-arrestin. Moreover, TARC was able to induce Src-dependent serine phosphorylation of the beta(2)-AR as well as its association with GRK2 and beta-arrestin. Finally, in contrast to CREB, phosphorylation of Src and extracellular signal-regulated kinase was enhanced by fenoterol upon TARC pretreatment. In summary, we show for the first time that TARC exposure impairs beta(2)-AR function in T cells. Our data suggest that this is mediated by Src-dependent activation of GRK2, resulting in receptor phosphorylation, binding to beta-arrestin, and a switch from cAMP-dependent signaling to activation of the MAPK pathway. We propose that aberrant T cell control in the presence of endogenous beta-agonists promotes T cell-mediated inflammation in asthma.