Cross-regulation of VPAC(2) receptor desensitization by M(3) receptors via PKC-mediated phosphorylation of RKIP and inhibition of GRK2

In gastrointestinal smooth muscle cells, VPAC(2) receptor desensitization is exclusively mediated by G protein-coupled receptor kinase 2 (GRK2). The present study examined the mechanisms by which acetylcholine (ACh) acting via M(3) receptors regulates GRK2-mediated VPAC(2) receptor desensitization in gastric smooth muscle cells. Vasoactive intestinal peptide induced VPAC(2) receptor phosphorylation, internalization, and desensitization in both freshly dispersed and cultured smooth muscle cells. Costimulation with ACh in the presence of M(2) receptor antagonist (i.e., activation of M(3) receptors) inhibited VPAC(2) receptor phosphorylation, internalization, and desensitization. Inhibition was blocked by the selective protein kinase C (PKC) inhibitor bisindolylmaleimide, suggesting that the inhibition was mediated by PKC, derived from M(3) receptor activation. Similar results were obtained by direct activation of PKC with phorbol myristate acetate. In the presence of the M(2) receptor antagonist, ACh induced phosphorylation of Raf kinase inhibitory protein (RKIP), increased RKIP-GRK2 association, decreased RKIP-Raf-1 association, and stimulated ERK1/2 activity, suggesting that, upon phosphorylation by PKC, RKIP dissociates from its known target Raf to associate with, and block the activity of, GRK2. In muscle cells expressing RKIP(S153A), which lacks the PKC phosphorylation site, RKIP phosphorylation was blocked and the inhibitory effect of ACh on VPAC(2) receptor phosphorylation, internalization, and desensitization and the stimulatory effect on ERK1/2 activation were abolished. This study identified a novel mechanism of cross-regulation of G(s)-coupled receptor phosphorylation and internalization by G(q)-coupled receptors. The mechanism involved phosphorylation of RKIP by PKC, switching RKIP from association with Raf-1 to association with, and inhibition of, GRK2.     

Cross-regulation of VPAC2 receptor internalization by m2 receptors via c-Src-mediated phosphorylation of GRK2

The aim of the study was to examine the mechanisms by which ACh, acting via m2 receptors, regulates GRK2-mediated VPAC(2) receptor desensitization in gastric smooth muscle cells. VIP induced VPAC(2) receptor phosphorylation and internalization in freshly dispersed smooth muscle cells. Co-stimulation with acetylcholine (ACh), in the presence of m3 receptor antagonist, 4-DAMP, augmented VPAC(2) receptor phosphorylation and internalization. The m2 receptor antagonist methoctramine or the c-Src inhibitor PP2 blocked the effect of ACh, suggesting that the augmentation was mediated by c-Src, derived from m2 receptor activation. ACh induced activation of c-Src and phosphorylation of GRK2 and the effects of ACh were blocked by methoctramine, PP2, or by uncoupling of m2 receptors from G(i3) with pertussis toxin. In conclusion, we identified a novel mechanism of cross-regulation of GRK2-mediated phosphorylation and internalization of G(s)-coupled VPAC(2) receptors by G(i)-coupled m2 receptors via tyrosine phosphorylation of GRK2 and stimulation of GRK2 activity.     

Regulation of membrane targeting of the G protein-coupled receptor kinase 2 by protein kinase A and its anchoring protein AKAP79

The beta2 adrenergic receptor (beta2AR) undergoes desensitization by a process involving its phosphorylation by both protein kinase A (PKA) and G protein-coupled receptor kinases (GRKs). The protein kinase A-anchoring protein AKAP79 influences beta2AR phosphorylation by complexing PKA with the receptor at the membrane. Here we show that AKAP79 also regulates the ability of GRK2 to phosphorylate agonist-occupied receptors. In human embryonic kidney 293 cells, overexpression of AKAP79 enhances agonist-induced phosphorylation of both the beta2AR and a mutant of the receptor that cannot be phosphorylated by PKA (beta2AR/PKA-). Mutants of AKAP79 that do not bind PKA or target to the beta2AR markedly inhibit phosphorylation of beta2AR/PKA-. We show that PKA directly phosphorylates GRK2 on serine 685. This modification increases Gbetagamma subunit binding to GRK2 and thus enhances the ability of the kinase to translocate to the membrane and phosphorylate the receptor. Abrogation of the phosphorylation of serine 685 on GRK2 by mutagenesis (S685A) or by expression of a dominant negative AKAP79 mutant reduces GRK2-mediated translocation to beta2AR and phosphorylation of agonist-occupied beta2AR, thus reducing subsequent receptor internalization. Agonist-stimulated PKA-mediated phosphorylation of GRK2 may represent a mechanism for enhancing receptor phosphorylation and desensitization.     

GRK3 mediates desensitization of CRF1 receptors: a potential mechanism regulating stress adaptation

Potential G protein-coupled receptor kinase (GRK) and protein kinase A (PKA) mediation of homologous desensitization of corticotropin-releasing factor type 1 (CRF1) receptors was investigated in human retinoblastoma Y-79 cells. Inhibition of PKA activity by PKI(5-22) or H-89 failed to attenuate homologous desensitization of CRF1 receptors, and direct activation of PKA by forskolin or dibutyryl cAMP failed to desensitize CRF-induced cAMP accumulation. However, treatment of permeabilized Y-79 cells with heparin, a nonselective GRK inhibitor, reduced homologous desensitization of CRF1 receptors by approximately 35%. Furthermore, Y-79 cell uptake of a GRK3 antisense oligonucleotide (ODN), but not of a random or mismatched ODN, reduced GRK3 mRNA expression by approximately 50% without altering GRK2 mRNA expression and inhibited homologous desensitization of CRF1 receptors by approximately 55%. Finally, Y-79 cells transfected with a GRK3 antisense cDNA construct exhibited an approximately 50% reduction in GRK3 protein expression and an ~65% reduction in homologous desensitization of CRF1 receptors. We conclude that GRK3 contributes importantly to the homologous desensitization of CRF1 receptors in Y-79 cells, a brain-derived cell line.     

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.     

Transient hypoxia differentially decreases GRK2 protein levels in CHO cells stably expressing the m1 mAChR

G protein-coupled kinase 2 (GRK2) has a key role in regulating signaling activities of a variety of G protein-coupled receptors (GPCRs). Several recent studies have directly implicated GRK2 phosphorylation in desensitization of GPCRs. In addition, binding by G(betagamma) or phosphorylation by PKC or c-Src [corrected] has been shown to activate or enhance GRK2 activity, respectively. Conversely, the calcium binding protein calmodulin or the serine/threonine kinase ERK has been implicated in inhibiting GRK2 activity. However, with the exception of a recent report indicating that activation of beta2-adrenergic receptor results in the ubiquitination and rapid degradation of GRK2, very little is known about cellular mechanisms that alter the protein levels of GRK2 [corrected]. Here, we report a novel serendipitous observation regarding alteration of GRK2 [corrected] protein levels. Exposure of CHO cells stably expressing the m1 muscarinic acetylcholine receptor (mAChR) to transient hypoxia caused near ablation of the GRK2 protein. In contrast, GRK2 protein levels remained unchanged in the parental CHO cells or in CHO cells stably expressing the m2 mAChR when exposed to transient hypoxia. The present study reports a novel observation that is unveiled by transient hypoxia in which GRK2 protein levels are altered by cellular mechanisms involving the m1 mAChR.     

Retrovirally mediated transfer of a G protein-coupled receptor kinase (GRK) dominant-negative mutant enhances endogenous calcitonin receptor signaling in Chinese hamster ovary cells. GRK inhibition enhances expression of receptors and receptor mRNA

G protein-coupled receptor kinases (GRKs) initiate pathways leading to agonist-dependent phosphorylation and desensitization of G protein-coupled receptors. However, the role of GRKs in modulation of signaling properties of native receptors has not been clearly defined. Here we addressed this question by generating Chinese hamster ovary (CHO) cells stably expressing a dominant-negative mutant of GRK2 (DN-GRK2), K220R, using retrovirally mediated gene transfer, and we assessed function of the endogenously expressed calcitonin (CT) receptors. We found that CT-mediated responses were prominently enhanced in CHO cells expressing DN-GRK2 compared with mock-infected control CHO cells with approximately 3-fold increases in CT-promoted cAMP production in whole cells and adenylyl cyclase activity in membrane fractions. CT-promoted phosphoinositide hydrolysis was also enhanced in DN-GRK2 cells. The number of CT receptors was increased approximately 3-fold in DN-GRK2 cells, as assessed by (125)I-salmon CT-specific binding, and this was associated with increased CT receptor mRNA levels. These results indicate that DN-GRK2 has multiple consequences for CT receptor signaling, but a primary effect is an increase in CT receptor mRNA and receptor number and, in turn, enhanced CT receptor signaling. As such, our findings provide a mechanistic basis for previous observations regarding agonist-promoted down-regulation of CT receptors and for resistance and escape from response to CT in vitro and in vivo. Moreover, the data suggest that blunting of receptor desensitization by DN-GRK2 blocks a GRK-mediated tonic inhibition of CT receptor expression and response. We speculate that GRKs play a similar role for other G protein-coupled receptors as well.     

EGF transregulates opioid receptors through EGFR-mediated GRK2 phosphorylation and activation

G protein-coupled receptor (GPCR) kinases (GRKs) are key regulators of GPCR function. Here we demonstrate that activation of epidermal growth factor receptor (EGFR), a member of receptor tyrosine kinase family, stimulates GRK2 activity and transregulates the function of G protein-coupled opioid receptors. Our data showed that EGF treatment promoted DOR internalization induced by DOR agonist and this required the intactness of GRK2-phosphorylation sites in DOR. EGF stimulation induced the association of GRK2 with the activated EGFR and the translocation of GRK2 to the plasma membrane. After EGF treatment, GRK2 was phosphorylated at tyrosyl residues. Mutational analysis indicated that EGFR-mediated phosphorylation occurred at GRK2 N-terminal tyrosyl residues previously shown as c-Src phosphorylation sites. However, c-Src activity was not required for EGFR-mediated phosphorylation of GRK2. In vitro assays indicated that GRK2 was a direct interactor and a substrate of EGFR. EGF treatment remarkably elevated DOR phosphorylation in cells expressing the wild-type GRK2 in an EGFR tyrosine kinase activity-dependent manner, whereas EGF-stimulated DOR phosphorylation was greatly decreased in cells expressing mutant GRK2 lacking EGFR tyrosine kinase sites. We further showed that EGF also stimulated internalization of mu-opioid receptor, and this effect was inhibited by GRK2 siRNA. These data indicate that EGF transregulates opioid receptors through EGFR-mediated tyrosyl phosphorylation and activation of GRK2 and propose GRK2 as a mediator of cross-talk from RTK to GPCR signaling pathway.     

Overexpression of wild-type and catalytically inactive forms of GRK2 and GRK6 fails to alter the agonist-induced phosphorylation of the C5a receptor (CD88): evidence that GRK6 is autophosphorylated in COS-7 cells.

The G protein-coupled receptor kinase family comprises six members (GRK1 to GRK6) that phosphorylate and desensitize a number of agonist-occupied G protein-coupled receptors. Overexpression of the dominant negative mutant GRK2-K220R is often accompanied by an inhibition of the agonist-mediated phosphorylation of G protein-coupled receptors. In the case of the C5a receptor (C5aR), the overexpression of wild-type GRK2 or GRK6 as well as of catalytically inactive forms of these kinases (GRK2-K220R and GRK6-K215R) failed to increase or to inhibit the agonist-mediated phosphorylation of C5aR, respectively. Replacement of Lys215 by an arginine residue in GRK6 yielded a protein with a relative molecular mass of 63 kDa, whereas wild-type GRK6 had a relative molecular mass of 66 kDa on polyacrylamide gel. The mutations S484D and T485D in the catalytically inactive mutant GRK6-K215R resulted in a protein (GRK6-RDD) with the same electrophoretic mobility as wild-type GRK6. Furthermore, in the absence of phosphatase inhibitors, GRK6 was rapidly converted into the 63 kDa species, whereas GRK6-RDD was not. Overepression of GRK6-RDD failed to alter the agonist-mediated phosphorylation of C5aR. Taken together, the results suggest that C5aR is not a substrate for either GRK2 or GRK6 and that GRK6 is very likely autophosphorylated on Ser484 and Thr485 in vivo.     

Inhibition of Galphaq-dependent PLC-beta1 activity by PKG and PKA is mediated by phosphorylation of RGS4 and GRK2

In smooth muscle of the gut, G_q-coupled receptor agonists activate preferentially PLC-1 to stimulate phosphoinositide (PI) hydrolysis and inositol 1,4,5-trisphosphate (IP₃) generation and induce IP₃-dependent Ca²⁺ release. Inhibition of Ca²⁺ mobilization by cAMP- (PKA) and cGMP-dependent (PKG) protein kinases reflects inhibition of PI hydrolysis by both kinases and PKG-specific inhibitory phosphorylation of IP₃ receptor type I. The mechanism of inhibition of PLC-1-dependent PI hydrolysis has not been established. Neither G_q nor PLC-1 was directly phosphorylated by PKA or PKG in gastric smooth muscle cells. However, both kinases 1) phosphorylated regulator of G protein signaling 4 (RGS4) and induced its translocation from cytosol to plasma membrane, 2) enhanced ACh-stimulated association of RGS4 and G_q·GTP and intrinsic G_q·GTPase activity, and 3) inhibited ACh-stimulated PI hydrolysis. RGS4 phosphorylation and inhibition of PI hydrolysis were blocked by selective PKA and PKG inhibitors. Expression of RGS4(S52A), which lacks a PKA/PKG phosphorylation site, blocked the increase in GTPase activity and the decrease in PI hydrolysis induced by PKA and PKG. Blockade of PKA-dependent effects was only partial. Selective phosphorylation of G protein-coupled receptor kinase 2 (GRK2), which contains a RGS domain, by PKA augmented ACh-stimulated GRK2:G_q·GTP association; both effects were blocked in cells expressing GRK2(S685A), which lacks a PKA phosphorylation site. Inhibition of PI hydrolysis induced by PKA was partly blocked in cells expressing GRK2(S685A) and completely blocked in cells coexpressing GRK2(S685A) and RGS4(S52A) or G_q(G188S), a G_q mutant that binds GRK2 but not RGS4. The results demonstrate that inhibition of PLC-1-dependent PI hydrolysis by PKA is mediated via stimulatory phosphorylation of RGS4 and GRK2, leading to rapid inactivation of G_q·GTP. PKG acts only via phosphorylation of RGS4. regulators of G protein signaling; G protein-coupled receptor kinase 2; phospholipase C; cAMP-dependent protein kinase; cGMP-dependent protein kinase     

Agonist-induced formation of opioid receptor-G protein-coupled receptor kinase (GRK)-G beta gamma complex on membrane is required for GRK2 function in vivo

G protein-coupled receptor kinases (GRKs) catalyze agonist-induced receptor phosphorylation on the membrane and initiate receptor desensitization. Previous in vitro studies have shown that the binding of GRK to membrane-associated G beta gamma subunits plays an important role in translocation of GRK2 from the cytoplasm to the plasma membrane. The current study investigated the role of the interaction of GRK2 with the activated delta-opioid receptor (DOR) and G beta gamma subunits in the membrane translocation and function of GRK2 using intact human embryonic kidney 293 cells. Our results showed that agonist treatment induced GRK2 binding to DOR, GRK2 translocation to the plasma membrane, and DOR phosphorylation in cells expressing the wild-type DOR but not the mutant DOR lacking the carboxyl terminus, which contains all three GRK2 phosphorylation sites. DORs with the GRK2 phosphorylation sites modified (M3) or with the acidic residues flanking phosphorylation sites mutated (E355Q/D364N) failed to be phosphorylated in response to agonist stimulation. Agonist-induced GRK2 membrane translocation and GRK-receptor association were observed in cells expressing M3 but not E355Q/D364N. Moreover, over-expression of G beta gamma subunits promoted GRK2 binding to DOR, whereas over-expression of transducin alpha or the carboxyl terminus of GRK2 blocked binding. Further study demonstrated that agonist stimulation induced the formation of a complex containing DOR, GRK2, and G beta gamma subunits in the cell and that agonist-stimulated formation of this complex is essential for the stable localization of GRK2 on the membrane and for its catalytic activity in vivo.     

Roles of phosphorylation-dependent and -independent mechanisms in the regulation of histamine H2 receptor by G protein-coupled receptor kinase 2

It is widely assumed that G protein-coupled receptor kinase 2 (GRK2)-mediated specific inhibition of G protein-coupled receptors (GPCRs) response involves GRK-mediated receptor phosphorylation followed by β-arrestin binding and subsequent uncoupling from the heterotrimeric G protein. It has recently become evident that GRK2-mediated GPCRs regulation also involves phosphorylation-independent mechanisms. In the present study we investigated whether the histamine H2 receptor (H2R), a Gα(s)-coupled GPCR known to be desensitized by GRK2, needs to be phosphorylated for its desensitization and/or internalization and resensitization. For this purpose we evaluated the effect of the phosphorylating-deficient GRK2K220R mutant on H2R signaling in U937, COS7, and HEK293T cells. We found that although this mutant functioned as dominant negative concerning receptor internalization and resensitization, it desensitized H2R signaling in the same degree as the GRK2 wild type. To identify the domains responsible for the kinase-independent receptor desensitization, we co-transfected the receptor with constructions encoding the GRK2 RGS-homology domain (RH) and the RH or the kinase domain fused to the pleckstrin-homology domain. Results demonstrated that the RH domain of GRK2 was sufficient to desensitize the H2R. Moreover, disruption of RGS functions by the use of GRK2D110A/K220R double mutant, although coimmunoprecipitating with the H2R, reversed GRK2K220R-mediated H2R desensitization. Overall, these results indicate that GRK2 induces desensitization of H2R through a phosphorylation-independent and RGS-dependent mechanism and extends the GRK2 RH domain-mediated regulation of GPCRs beyond Gα(q)-coupled receptors. On the other hand, GRK2 kinase activity proved to be necessary for receptor internalization and the resulting resensitization.     

Selective Reduction in A2 Adenosine Receptor Desensitization Following Antisense-Induced Suppression of G Protein-Coupled Receptor Kinase 2 Expression

Phosphorylation of G protein-coupled receptors (GPCRs) by GPCR kinases (GRKs) is considered to play a critical role in the desensitization of responses mediated by these receptors. To explore the role of GRK2 in A2 adenosine receptor desensitization, we attempted to reduce specifically GRK2 expression in NG108-15 cells by stable transfection with an antisense rat GRK2 cDNA sequence. This yielded up to a 69% loss of GRK2 when compared with plasmid-transfected control cells, which correlated with a reduction in kinase activity when measured by the ability of cell lysates to promote light-dependent phosphorylation of rhodopsin. Levels of GRK3 were the same in antisense and plasmid-transfected controls. On addition of the A2 adenosine receptor agonist 5'-(N-ethylcarboxamido)adenosine, cyclic AMP accumulation was greater in GRK2 antisense cells as compared with plasmid control cells. In contrast, cyclic AMP accumulation via agonist stimulation of either IP-prostanoid or secretin receptors or by addition of forskolin was not significantly different among all clones examined. The increase in A2 adenosine receptor response could not be explained by changes in A2A adenosine receptor expression, as assessed by ligand binding experiments with the radioligand 2-3H-labelled 4-[2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-++ +ylamino]ethyl]phenol ([3H]ZM241385). These data show for the first time a direct correlation between expression of GRK2 and desensitization of natively expressed A2 adenosine receptors in intact cells, suggesting that GRK2 plays a major role in the regulation of these receptors. Key Words: G protein-coupled receptor kinase-G protein-coupled receptor-Antisense-NG108-15 cells-A2 adenosine receptors-Desensitization.     

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.     

Characterization of the carboxyl terminal-truncated endothelin B receptor coexpressed with G protein-coupled receptor kinase 2.

The role of phosphorylation of the C-terminal tail of endothelin B receptor (ETBR) in agonist-induced desensitization was investigated, using a mutant lacking C-terminal 40 amino acids (delta 40 ETBR). In cells expressing the wild type or delta 40 ETBR, ET-1 caused rapid desensitization of calcium responses. The wild type ETBR was phosphorylated by biotinylated ET-1, and the phosphorylation was markedly enhanced by coexpression with G protein-coupled receptor kinase 2 (GRK2). However, delta 40 ETBR was not phosphorylated regardless of coexpression with GRK2. On the other hand, ET-1-induced IP3 formation in these cells was decreased by coexpression with GRK2 or catalytically inactive Lys220Arg GRK2 to the similar extent. The present study demonstrates the presence of phosphorylation-independent desensitization mechanism in delta 40 ETBR and suggests that GRK2 might play a role other than that as a kinase.     

Desensitization of the Y1 cell adrenocorticotropin receptor: evidence for a restricted heterologous mechanism implying a role for receptor-effector complexes

Receptor desensitization provides a potential mechanism for the regulation of adrenocortical adrenocorticotropin (ACTH) responsiveness. Using the mouse adrenocortical Y1 cell line we demonstrate that ACTH effectively desensitizes the cAMP response of its own receptor, the melanocortin 2 receptor (MC2R), in these cells with a maximal effect between 30 and 60 min. Neither forskolin nor isoproterenol (in Y1 cells stably transfected with the beta(2)-adrenergic receptor) desensitize this ACTH response. ACTH desensitizes its receptor at concentrations at which only a fraction of receptors are occupied, implying that this mechanism acts on agonist-unoccupied receptors. Y1 cells express G protein-coupled receptor kinase (GRK) 2 and 5, but stable expression of a dominant negative GRK2 (K220W) only marginally reduces the desensitization by ACTH. The protein kinase A (PKA) inhibitor, H89, extinguishes almost the entire desensitization response over the initial 30-min period at all concentrations of ACTH. A mutant MC2R in which the single consensus PKA phosphorylation site has been mutated (S208A) when expressed in MC2R-negative Y6 cells is also unable to desensitize. These data imply a heterologous, PKA-dependent, mode of desensitization, which is restricted to agonist-occupied and -unoccupied MC2R, possibly as a consequence of receptor/effector complexes that functionally compartmentalize this receptor.     

Phosphorylation-independent desensitization of metabotropic glutamate receptor 5 by G protein-coupled receptor kinase 2 in HEK 293 cells

The metabotropic glutamate receptor 5 (mGluR5) is one of the important excitatory neurotransmitter receptors in the central nervous system, and its desensitization by G protein-coupled receptor kinases (GRKs) plays an important role in neuron protection against receptor overstimulation. It is reported that GRK2 could down-regulate the mGluR5 signaling in both HEK 293 cells and neurons. However, whether GRK2-mediated mGluR5 desensitization is phosphorylation dependent remains controversial. Here, we demonstrated that the signal intensity and kinetics of mGluR5 desensitization was inhibited or changed by GRK2 in HEK 293 cells. By using the catalytically inactive GRK2 mutant K220R, and the receptor mutants that lack potential phosphorylation sites in the C-terminal tail, we demonstrated that the GRK2-mediated mGluR5 desensitization was phosphorylation-independent. Furthermore, overexpression of an N-terminal regulator of G protein signaling (RGS) homology (RH) domain of GRK2 was sufficient to attenuate the mGluR5 signaling, whereas the expression of GRK2 D110A mutant devoid in Gαq binding was unable to inhibit mGluR5 signaling. In summary, this study provides evidence that GRK2 mediates phosphorylationindependent mGluR5 desensitization via the interaction between the RGS domain and Gαq in HEK 293 cells.     

Differential regulation of β2-adrenoceptor and adenosine A2B receptor signalling by GRK and arrestin proteins in arterial smooth muscle

Generation of cAMP through G_s-coupled G protein-coupled receptor (GPCR) [e.g. β₂-adrenoceptor (β₂AR), adenosine A_2B receptor (A_2BR)] activation, induces arterial smooth muscle relaxation, counteracting the actions of vasoconstrictors. G_s-coupled GPCR signalling is regulated by G protein-coupled receptor kinases (GRK) and arrestin proteins, and dysregulation of Gs/GPCR signalling is thought play a role in the development of hypertension, which may be a consequence of enhanced GRK2 and/or arrestin expression. However, despite numerous studies indicating that β₂AR and A_2BR can be substrates for GRK/arrestin proteins, currently little is known regarding GRK/arrestin regulation of these endogenous receptors in arterial smooth muscle. Here, endogenous GRK isoenzymes and arrestin proteins were selectively depleted using RNA-interference in rat arterial smooth muscle cells (RASM) and the consequences of this for β₂AR- and A_2BR-mediated adenylyl cyclase (AC) signalling were determined by assessing cAMP accumulation. GRK2 or GRK5 depletion enhanced and prolonged β₂AR/AC signalling, while combined deletion of GRK2/5 has an additive effect. Conversely, activation of AC by A_2BR was regulated by GRK5, but not GRK2. β₂AR desensitization was attenuated following combined GRK2/GRK5 knockdown, but not by depletion of individual GRKs, arrestins, or by inhibiting PKA. Arrestin3 (but not arrestin2) depletion enhanced A_2BR-AC signalling and attenuated A_2BR desensitization, while β₂AR-AC signalling was regulated by both arrestin isoforms. This study provides a first demonstration of how different complements of GRK and arrestin proteins contribute to the regulation of signalling and desensitization of these important receptors mediating vasodilator responses in arterial smooth muscle.     

Phosphorylation-independent regulation of metabotropic glutamate receptor signaling by G protein-coupled receptor kinase 2

The accepted paradigm for G protein-coupled receptor kinase (GRK)-mediated desensitization of G protein-coupled receptors involves GRK-mediated receptor phosphorylation followed by the binding of arrestin proteins. Although GRKs contribute to metabotropic glutamate receptor 1 (mGluR1) inactivation, beta-arrestins do not appear to be required for mGluR1 G protein uncoupling. Therefore, we investigated whether the phosphorylation of serine and threonine residues localized within the C terminus of mGluR1a is sufficient to allow GRK2-mediated attenuation of mGluR1a signaling. We find that the truncation of the mGluR1a C-terminal tail prevents mGluR1a phosphorylation and that GRK2 does not contribute to the phosphorylation of an mGluR1 splice variant (mGluR1b). However, mGluR1a-866Delta- and mGluR1b-stimulated inositol phosphate formation is attenuated following GRK2 expression. The expression of the GRK2 C-terminal domain to block membrane translocation of endogenous GRK2 increases mGluR1a-866Delta- and mGluR1b-stimulated inositol phosphate formation, presumably by blocking membrane translocation of GRK2. In contrast, expression of the kinase-deficient GRK2-K220R mutant inhibits inositol phosphate formation by these unphosphorylated receptors. Expression of the GRK2 N-terminal domain (residues 45-185) also attenuates both constitutive and agonist-stimulated mGluR1a, mGluR1a-866Delta, and mGluR1b signaling, and the GRK2 N terminus co-precipitates with mGluR1a. Taken together, our observations indicate that attenuation of mGluR1 signaling by GRK2 is phosphorylation-independent and that the interaction of the N-terminal domain of GRK2 with mGluR1 contributes to the regulation of mGluR1 G protein coupling.     

Different kinases desensitize the human delta-opioid receptor (hDOP-R) in the neuroblastoma cell line SK-N-BE upon peptidic and alkaloid agonists

In a previous work, we described a differential desensitization of the human δ-opioid receptor (hDOP-R) by etorphine (a non-selective and alkaloid agonist) and δ-selective and peptidic agonists (DPDPE ([d-Pen^2,5]enkephalin) and deltorphin I (Tyr-d-Ala-Phe-Asp-Val-Val-Gly-NH₂)) in the neuroblastoma cell line SK-N-BE (Allouche et al., Eur. J. Pharmacol., 371, 235, 1999). In the present study, we explored the putative role of different kinases in this differential regulation.

First, selective chemical inhibitors of PKA, PKC and tyrosine kinases were used and we showed a significant reduction of etorphine-induced opioid receptor desensitization by the bisindolylmaleimide I (PKC inhibitor) while genistein (tyrosine kinase inhibitor) was potent to impair desensitization induced by the different agonists. When the PKA was inhibited by H89 pretreatment, no modification of opioid receptor desensitization was observed whatever the agonist used.

Second, we further studied the role of G protein-coupled receptor kinases (GRKs) and by using western-blot experiments we observed that only the GRK2 isoform was expressed in the SK-N-BE cells. Next, the neuroblastoma cells were transfected with the wild type GRK2 or its dominant negative mutant GRK2-K220R and the inhibition on cAMP level was determined in naïve and agonist-pretreated cells. We showed that over-expression of GRK2-K220R totally abolished etorphine-induced receptor desensitization while no effect was observed with peptidic agonists and over-expression of GRK2 selectively impaired cAMP inhibition promoted by etorphine suggesting that this kinase was involved in the regulation of hDOP-R activated only by etorphine.

Third, correlation between functional experiments and phosphorylation of the hDOP-R after agonist activation was assessed by western-blot using the specific anti-phospho-DOP-R Ser³⁶³ antibody. While all agonists were potent to increase phosphorylation of opioid receptor, we showed no impairment of receptor phosphorylation level after PKC inhibitor pretreatment. Upon agonist activation, no enhancement of receptor phosphorylation was observed when the GRK2 was over-expressed while the GRK2-K220R partially reduced the hDOP-R Ser³⁶³ phosphorylation only after peptidic agonists pretreatment.

In conclusion, hDOP-R desensitization upon etorphine exposure relies on the GRK2, PKC and tyrosine kinases while DPDPE and deltorphin I mediate desensitization at least via tyrosine kinases. Although the Ser³⁶³ was described as the primary phosphorylation site of the mouse DOP-R, we observed no correlation between desensitization and phosphorylation of this amino acid.