G protein-coupled receptor kinase 5 contains a DNA-binding nuclear localization sequence

G protein-coupled receptor kinases (GRKs) mediate desensitization of agonist-occupied G protein-coupled receptors (GPCRs). Here we report that GRK5 contains a DNA-binding nuclear localization sequence (NLS) and that its nuclear localization is regulated by GPCR activation, results that suggest potential nuclear functions for GRK5. As assessed by fluorescence confocal microscopy, transfected and endogenous GRK5 is present in the nuclei of HEp2 cells. Mutation of basic residues in the catalytic domain of GRK5 (between amino acids 388 and 395) results in the nuclear exclusion of the mutant enzyme (GRK5(Delta)(NLS)), demonstrating that GRK5 contains a functional NLS. The nuclear localization of GRK5 is subject to dynamic regulation. Calcium ionophore treatment or activation of Gq-coupled muscarinic-M3 receptors promotes the nuclear export of the kinase in a Ca(2+)/calmodulin (Ca(2+)/CaM)-dependent fashion. Ca(2+)/CaM binding to the N-terminal CaM binding site of GRK5 mediates this effect. Furthermore, GRK5, but not GRK5(Delta)(NLS) or GRK2, binds specifically and directly to DNA in vitro. Consistent with their presence in the nuclei of transfected cells, all the GRK4, but not GRK2, subfamily members contain putative NLSs. These results suggest that the GRK4 subfamily of GRKs may play a signaling role in the nucleus and that GRK4 and GRK2 subfamily members perform divergent cellular functions.     

Identification of a neuronal calcium sensor (NCS-1) possibly involved in the regulation of receptor phosphorylation

Abstract

Persistent stimulation of G protein-coupled receptors by agonists leads rapidly to reduced responses, a phenomenon described as desensitization. It involves primarily the phosphorylation of receptor sites by specific kinases of the G protein-coupled receptor kinase (GRK) family. The β-adrenergic receptor kinase 1 (GRK2) desensitizes agonist-activated β₂-adrenergic receptors, whereas rhodopsin kinase (GRK1) phosphorylates and inactivates photon-activated rhodopsin. Little is known about the role of calcium in desensitization. Here we report the characterization of a novel neuronal calcium sensor (NCS) named NCS-1 possibly involved in the regulation of receptor phosphorylation. NCS-1 is a new member of the EF-hand superfamily, which includes calmodulin, troponin C, parvalbumin, and recoverins. By Northern analysis and in situ hybridization, we discovered that NCS-1 is specifically expressed in the central and peripheral nervous systems. Chick NCS-1 has 72% of amino acid identity with Drosophila frequenin, a protein found in the nervous system and at the motor nerve terminals of neuromuscular junctions. By analogy with the reported function for two other members of the NCS family, we discuss whether G protein-coupled receptors or GRKs are the targets of neuronal calcium sensors.     

Regulation of the methylation status of G protein-coupled receptor kinase 1 (rhodopsin kinase)

Rhodopsin kinase (GRK1) is a member of G protein-coupled receptor kinase family and a key enzyme in the quenching of photolysed rhodopsin activity and desensitisation of the rod photoreceptor neurons. Like some other rod proteins involved in phototransduction, GRK1 is posttranslationally modified at the C terminus by isoprenylation (farnesylation), endoproteolysis and α-carboxymethylation. In this study, we examined the potential mechanisms of regulation of GRK1 methylation status, which have remained unexplored so far. We found that considerable fraction of GRK1 is endogenously methylated. In isolated rod outer segments, its methylation is inhibited and demethylation stimulated by low-affinity nucleotide binding. This effect is not specific for ATP and was observed in the presence of a non-hydrolysable ATP analogue AMP-PNP, GTP and other nucleotides, and thus may involve a site distinct from the active site of the kinase. GRK1 demethylation is inhibited in the presence of Ca(2+) by recoverin. This inhibition requires recoverin myristoylation and the presence of the membranes, and may be due to changes in GRK1 availability for processing enzymes upon its redistribution to the membranes induced by recoverin/Ca(2+). We hypothesise that increased GRK1 methylation in dark-adapted rods due to elevated cytoplasmic Ca(2+) levels would further increase its association with the membranes and recoverin, providing a positive feedback to efficiently suppress spurious phosphorylation of non-activated rhodopsin molecules and thus maximise senstivity of the photoreceptor. This study provides the first evidence for dynamic regulation of GRK1 α-carboxymethylation, which might play a role in the regulation of light sensitivity and adaptation in the rod photoreceptors.     

Recoverin as a redox-sensitive protein

Recoverin is a member of the neuronal calcium sensor (NCS) family of EF-hand calcium binding proteins. In a visual cycle of photoreceptor cells, recoverin regulates activity of rhodopsin kinase in a Ca2+-dependent manner. Like all members of the NSC family, recoverin contains a conserved cysteine (Cys38) in nonfunctional EF-hand 1. This residue was shown to be critical for activation of target proteins in some members of the NCS family but not for interaction of recoverin with rhodopsin kinase. Spectrophotometric titration of Ca2+-loaded recoverin gave 7.6 for the pKa value of Cys38 thiol, suggesting partial deprotonation of the thiol in vivo conditions. An ability of recoverin to form a disulfide dimer and thiol-oxidized monomer under mild oxidizing conditions was found using SDS-PAGE in reducing and nonreducing conditions and Ellman's test. Both processes are reversible and modulated by Ca2+. Although formation of the disulfide dimer takes place only for Ca2+-loaded recoverin, accumulation of the oxidized monomer proceeds more effectively for apo-recoverin. The Ca2+ modulated susceptibility of the recoverin thiol to reversible oxidation may be of potential importance for functioning of recoverin in photoreceptor cells.     

Atomic Structure of GRK5 Reveals Distinct Structural Features Novel for G Protein-coupled Receptor Kinases

G protein-coupled receptor kinases (GRKs) are members of the protein kinase A, G, and C families (AGC) and play a central role in mediating G protein-coupled receptor phosphorylation and desensitization. One member of the family, GRK5, has been implicated in several human pathologies, including heart failure, hypertension, cancer, diabetes, and Alzheimer disease. To gain mechanistic insight into GRK5 function, we determined a crystal structure of full-length human GRK5 at 1.8 Å resolution. GRK5 in complex with the ATP analog 5′-adenylyl β,γ-imidodiphosphate or the nucleoside sangivamycin crystallized as a monomer. The C-terminal tail (C-tail) of AGC kinase domains is a highly conserved feature that is divided into three segments as follows: the C-lobe tether, the active-site tether (AST), and the N-lobe tether (NLT). This domain is fully resolved in GRK5 and reveals novel interactions with the nucleotide and N-lobe. Similar to other AGC kinases, the GRK5 AST is an integral part of the nucleotide-binding pocket, a feature not observed in other GRKs. The AST also mediates contact between the kinase N- and C-lobes facilitating closure of the kinase domain. The GRK5 NLT is largely displaced from its previously observed position in other GRKs. Moreover, although the autophosphorylation sites in the NLT are >20 Å away from the catalytic cleft, they are capable of rapid cis-autophosphorylation suggesting high mobility of this region. In summary, we provide a snapshot of GRK5 in a partially closed state, where structural elements of the kinase domain C-tail are aligned to form novel interactions to the nucleotide and N-lobe not previously observed in other GRKs.     

Fission yeast homolog of neuronal calcium sensor-1 (Ncs1p) regulates sporulation and confers calcium tolerance

The neuronal calcium sensor (NCS) proteins (e.g. recoverin, neurocalcins, and frequenin) are expressed at highest levels in excitable cells, and some of them regulate desensitization of G protein-coupled receptors. Here we present NMR analysis and genetic functional studies of an NCS homolog in fission yeast (Ncs1p). Ncs1p binds three Ca2+ ions at saturation with an apparent affinity of 2 microm and Hill coefficient of 1.9. Analysis of NMR and fluorescence spectra of Ncs1p revealed significant Ca2+-induced protein conformational changes indicative of a Ca2+-myristoyl switch. The amino-terminal myristoyl group is sequestered inside a hydrophobic cavity of the Ca2+-free protein and becomes solvent-exposed in the Ca2+-bound protein. Subcellular fractionation experiments showed that myristoylation and Ca2+ binding by Ncs1p are essential for its translocation from cytoplasm to membranes. The ncs1 deletion mutant (ncs1Delta) showed two distinct phenotypes: nutrition-insensitive sexual development and a growth defect at high levels of extracellular Ca2+ (0.1 m CaCl(2)). Analysis of Ncs1p mutants lacking myristoylation (Ncs1p(G2A)) or deficient in Ca2+ binding (Ncs1p(E84Q/E120Q/E168Q)) revealed that Ca2+ binding was essential for both phenotypes, while myristoylation was less critical. Exogenous cAMP, a key regulator for sexual development, suppressed conjugation and sporulation of ncs1Delta, suggesting involvement of Ncs1p in the adenylate cyclase pathway turned on by the glucose-sensing G protein-coupled receptor Git3p. Starvation-independent sexual development of ncs1Delta was also complemented by retinal recoverin, which controls Ca2+-regulated desensitization of rhodopsin. In contrast, the Ca2+-intolerance of ncs1Delta was not affected by cAMP or recoverin, suggesting that the two ncs1Delta phenotypes are mechanistically independent. We propose that Schizosaccharomyces pombe Ncs1p negatively regulates sporulation perhaps by controlling Ca2+-dependent desensitization of Git3p.     

Involvement of the recoverin C-terminal segment in recognition of the target enzyme rhodopsin kinase

NCS (neuronal Ca2+ sensor) proteins belong to a family of calmodulin-related EF-hand Ca2+-binding proteins which, in spite of a high degree of structural similarity, are able to selectively recognize and regulate individual effector enzymes in a Ca2+-dependent manner. NCS proteins vary at their C-termini, which could therefore serve as structural control elements providing specific functions such as target recognition or Ca2+ sensitivity. Recoverin, an NCS protein operating in vision, regulates the activity of rhodopsin kinase, GRK1, in a Ca2+-dependent manner. In the present study, we investigated a series of recoverin forms that were mutated at the C-terminus. Using pull-down assays, surface plasmon resonance spectroscopy and rhodopsin phosphorylation assays, we demonstrated that truncation of recoverin at the C-terminus significantly reduced the affinity of recoverin for rhodopsin kinase. Site-directed mutagenesis of single amino acids in combination with structural analysis and computational modelling of the recoverin-kinase complex provided insight into the protein-protein interface between the kinase and the C-terminus of recoverin. Based on these results we suggest that Phe3 from the N-terminal helix of rhodopsin kinase and Lys192 from the C-terminal segment of recoverin form a cation-π interaction pair which is essential for target recognition by recoverin. Taken together, the results of the present study reveal a novel rhodopsin-kinase-binding site within the C-terminal region of recoverin, and highlights its significance for target recognition and regulation.     

G protein-coupled receptor kinase 2 (GRK2) is a Rho-activated scaffold protein for the ERK MAP kinase cascade

The G protein-coupled receptor kinases (GRKs) are best known for their role in phosphorylating and desensitising G protein-coupled receptors (GPCRs). The GRKs also regulate signalling downstream of other families of receptors and have a number of non-receptor substrates and binding partners. Here we identify RhoA_GTP and Raf1 as novel binding partners of GRK2 and report a previously unsuspected function for this kinase. GRK2 is a RhoA effector that serves as a RhoA-activated scaffold protein for the ERK MAP kinase cascade. The ability of GRK2 to bind to Raf1, MEK1 and ERK2 is dependent on RhoA_GTP binding to the catalytic domain of the kinase. Exogenous GRK2 has previously been shown to increase ERK activation downstream of the epidermal growth factor receptor (EGFR). Here we find that GRK2-mediated ERK activation downstream of the EGFR is Rho-dependent and that treatment with EGF promotes RhoA_GTP binding and ERK scaffolding by GRK2. Depletion of GRK2 expression by RNAi reveals that GRK2 is required for EGF-induced, Rho- and ERK-dependent thymidine incorporation in vascular smooth muscle cells (VSMCs). We therefore hypothesise that Rho-dependent ERK MAPK scaffolding by GRK2 downstream of the EGFR may have an important role in the vasculature, where increased levels of both GRK2 and RhoA have been associated with hypertension.     

G protein-coupled receptor kinase 5 phosphorylates nucleophosmin and regulates cell sensitivity to polo-like kinase 1 inhibition

G protein-coupled receptor kinases (GRKs) phosphorylate activated G protein-coupled receptors, leading to their desensitization and endocytosis. GRKs have also been implicated in phosphorylating other classes of proteins and can localize in a variety of cellular compartments, including the nucleus. Here, we attempted to identify potential nuclear substrates for GRK5. Our studies reveal that GRK5 is able to interact with and phosphorylate nucleophosmin (NPM1) both in vitro and in intact cells. NPM1 is a nuclear protein that regulates a variety of cell functions including centrosomal duplication, cell cycle control, and apoptosis. GRK5 interaction with NPM1 is mediated by the N-terminal domain of each protein, and GRK5 primarily phosphorylates NPM1 at Ser-4, a site shared with polo-like kinase 1 (PLK1). NPM1 phosphorylation by GRK5 and PLK1 correlates with the sensitivity of cells to undergo apoptosis with cells having higher GRK5 levels being less sensitive and cells with lower GRK5 being more sensitive to PLK1 inhibitor-induced apoptosis. Taken together, our results demonstrate that GRK5 phosphorylates Ser-4 in nucleophosmin and regulates the sensitivity of cells to PLK1 inhibition.     

Photoreceptor calcium sensor proteins in detergent-resistant membrane rafts are regulated via binding to caveolin-1

Rod cell membranes contain cholesterol-rich detergent-resistant membrane (DRM) rafts, which accumulate visual cascade proteins as well as proteins involved in regulation of phototransduction such as rhodopsin kinase and guanylate cyclases. Caveolin-1 is the major integral component of DRMs, possessing scaffolding and regulatory activities towards various signaling proteins. In this study, photoreceptor Ca²⁺-binding proteins recoverin, NCS1, GCAP1, and GCAP2, belonging to neuronal calcium sensor (NCS) family, were recognized as novel caveolin-1 interacting partners. All four NCS proteins co-fractionate with caveolin-1 in DRMs, isolated from illuminated bovine rod outer segments. According to pull-down assay, surface plasmon resonance spectroscopy and isothermal titration calorimetry data, they are capable of high-affinity binding to either N-terminal fragment of caveolin-1 (1–101), or its short scaffolding domain (81–101) via a novel structural site. In recoverin this site is localized in C-terminal domain in proximity to the third EF-hand motif and composed of aromatic amino acids conserved among NCS proteins. Remarkably, the binding of NCS proteins to caveolin-1 occurs only in the absence of calcium, which is in agreement with higher accessibility of the caveolin-1 binding site in their Ca²⁺-free forms. Consistently, the presence of caveolin-1 produces no effect on regulatory activity of Ca²⁺-saturated recoverin or NCS1 towards rhodopsin kinase, but upregulates GCAP2, which potentiates guanylate cyclase activity being in Ca²⁺-free conformation. In addition, the interaction with caveolin-1 decreases cooperativity and augments affinity of Ca2 + binding to recoverin apparently by facilitating exposure of its myristoyl group. We suggest that at low calcium NCS proteins are compartmentalized in photoreceptor rafts via binding to caveolin-1, which may enhance their activity or ensure their faster responses on Ca²⁺-signals thereby maintaining efficient phototransduction recovery and light adaptation.     

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.     

Chemical genetic engineering of G protein-coupled receptor kinase 2

G protein-coupled receptor kinases (GRKs) play a pivotal role in receptor regulation. Efforts to study the acute effects of GRKs in intact cells have been limited by a lack of specific inhibitors. In the present study we have developed an engineered version of GRK2 that is specifically and reversibly inhibited by the substituted nucleotide analog 1-naphthyl-PP1 (1Na-PP1), and we explored GRK2 function in regulated internalization of the mu-opioid receptor (muOR). A previously described method that conferred analog sensitivity on various kinases, by introducing a space-creating mutation in the conserved active site, failed when applied to GRK2 because the corresponding mutation (L271G) rendered the mutant kinase (GRK2-as1) catalytically inactive. A sequence homology-based approach was used to design second-site suppressor mutations. A C221V second-site mutation produced a mutant kinase (GRK2-as5) with full functional activity and analog sensitivity as compared with wild-type GRK2 in vitro and in intact cells. The role of GRK2-as5 activity in the membrane trafficking of the muOR was also characterized. Morphine-induced internalization was completely blocked when GRK2-as5 activity was inhibited before morphine application. However, inhibition of GRK2-as5 during recycling and reinternalization of the muOR did not attenuate these processes. These results suggest there is a difference in the GRK requirement for initial ligand-induced internalization of a G protein-coupled receptor compared with subsequent rounds of reinternalization.     

Constitutively active rhodopsin mutants causing night blindness are effectively phosphorylated by GRKs but differ in arrestin-1 binding

The effects of activating mutations associated with night blindness on the stoichiometry of rhodopsin interactions with G protein-coupled receptor kinase 1 (GRK1) and arrestin-1 have not been reported. Here we show that the monomeric form of WT rhodopsin and its constitutively active mutants M257Y, G90D, and T94I, reconstituted into HDL particles are effectively phosphorylated by GRK1, as well as two more ubiquitously expressed subtypes, GRK2 and GRK5. All versions of arrestin-1 tested (WT, pre-activated, and constitutively monomeric mutants) bind to monomeric rhodopsin and show the same selectivity for different functional forms of rhodopsin as in native disc membranes. Rhodopsin phosphorylation by GRK1 and GRK2 promotes arrestin-1 binding to a comparable extent, whereas similar phosphorylation by GRK5 is less effective, suggesting that not all phosphorylation sites on rhodopsin are equivalent in promoting arrestin-1 binding. The binding of WT arrestin-1 to phospho-opsin is comparable to the binding to its preferred target, P-Rh*, suggesting that in photoreceptors arrestin-1 only dissociates after opsin regeneration with 11-cis-retinal, which converts phospho-opsin into inactive phospho-rhodopsin that has lower affinity for arrestin-1. Reduced binding of arrestin-1 to the phospho-opsin form of G90D mutant likely contributes to night blindness caused by this mutation in humans.     

Beta 2-adrenergic receptor stimulated,G protein-coupled receptor kinase 2 mediated,phosphorylation of ribosomal protein P2

G protein-coupled receptor kinases are well characterized for their ability to phosphorylate and desensitize G protein-coupled receptors (GPCRs). In addition to phosphorylating the beta2-adrenergic receptor (beta2AR) and other receptors, G protein-coupled receptor kinase 2 (GRK2) can also phosphorylate tubulin, a nonreceptor substrate. To identify novel nonreceptor substrates of GRK2, we used two-dimensional gel electrophoresis to find cellular proteins that were phosphorylated upon agonist-stimulation of the beta2AR in a GRK2-dependent manner. The ribosomal protein P2 was identified as an endogenous HEK-293 cell protein whose phosphorylation was increased following agonist stimulation of the beta2AR under conditions where tyrosine kinases, PKC and PKA, were inhibited. P2 along with its other family members, P0 and P1, constitutes a part of the elongation factor-binding site connected to the GTPase center in the 60S ribosomal subunit. Phosphorylation of P2 is known to regulate protein synthesis in vitro. Further, P2 and P1 are shown to be good in vitro substrates for GRK2 with K(M) values approximating 1 microM. The phosphorylation sites in GRK2-phosphorylated P2 are identified (S102 and S105) and are identical to the sites known to regulate P2 activity. When the 60S subunit deprived of endogenous P1 and P2 is reconstituted with GRK2-phosphorylated P2 and unphosphorylated P1, translational activity is greatly enhanced. These findings suggest a previously unrecognized relationship between GPCR activation and the translational control of gene expression mediated by GRK2 activation and P2 phosphorylation and represent a potential novel signaling pathway responsible for P2 phosphorylation in mammals.     

Development of a yeast bioassay to characterize G protein-coupled receptor kinases. Identification of an NH2-terminal region essential for receptor phosphorylation

G protein-coupled receptor kinases (GRKs) specifically bind and phosphorylate the agonist-occupied form of G protein-coupled receptors. To further characterize the mechanism of GRK/receptor interaction, we developed a yeast-based bioassay using strains engineered to functionally express the somatostatin receptor subtype 2 and exhibit agonist-dependent growth. Here, we demonstrate that agonist-promoted growth was effectively inhibited by co-expression with either wild type GRK2 or GRK5, whereas catalytically inactive forms of these kinases were without effect. In an effort to identify residues involved in receptor interaction, we generated a pool of GRK5 mutants and then utilized the bioassay to identify mutants selectively deficient in inhibiting agonist-promoted growth. This resulted in the identification of a large number of mutants, several of which were expressed, purified, and characterized in more detail. Two of the mutants, GRK5-L3Q/K113R and GRK5-T10P, were defective in receptor phosphorylation and also exhibited a partial defect in phospholipid binding and phospholipid-stimulated autophosphorylation of the kinase. In contrast, these mutants had wild type activity in phosphorylating the non-receptor substrate tubulin. To further characterize the function of the NH2-terminal region of GRK5, we generated a deletion mutant lacking residues 2-14 and found that this mutant was also severely impaired in receptor phosphorylation and phospholipid-promoted autophosphorylation. In addition, an NH2-terminal 14-amino acid peptide from GRK5 selectively inhibited receptor phosphorylation by GRK5 but had minimal effect on GRK2 activity. Based on these findings, we propose a model whereby the extreme NH2 terminus of GRK5 mediates phospholipid binding and is required for optimal receptor phosphorylation.     

Phosphorylation-independent regulation of metabotropic glutamate receptor 1 signaling requires g protein-coupled receptor kinase 2 binding to the second intracellular loop

Metabotropic glutamate receptors (mGluRs) are members of a unique class of G protein-coupled receptors (class III) that include the calcium-sensing and gamma-aminobutyric acid type B receptors. The activity of mGluRs is regulated by second messenger-dependent protein kinases and G protein-coupled receptor kinases (GRKs). The attenuation of both mGluR1a and mGluR1b signaling by GRK2 is phosphorylation- and beta-arrestin-independent and requires the concomitant association of GRK2 with both the receptor and Galpha(q/11). G protein interactions are mediated, in part, by the mGluR1 intracellular second loop, but the domains required for GRK2 binding are unknown. In the present study, we showed that GRK2 binds to the second intracellular loop of mGluR1a and mGluR1b and also to the mGluR1a carboxyl-terminal tail. Alanine scanning mutagenesis revealed a discrete domain within loop 2 that contributes to GRK2 binding, and the mutation of either lysine 691 or 692 to an alanine within this domain resulted in a loss of GRK2 binding to both mGluR1a and mGluR1b. Mutation of either Lys(691) or Lys(692) prevented GRK2-mediated attenuation of mGluR1b signaling, whereas the mutation of only Lys(692) prevented GRK2-mediated inhibition of mGluR1a signaling. Thus, the mGluR1a carboxyl-terminal tail may also be involved in regulating the signaling of the mGluR1a splice variant. Taken together, our findings indicated that kinase binding to an mGluR1 domain involved in G protein-coupling is essential for the phosphorylation-independent attenuation of signaling by GRK2.     

PolyADP-ribosylation is required for long-term memory formation in mammals

Recoverin is suggested to inhibit rhodopsin kinase (GRK1) at high [Ca²⁺] in the dark state of the photoreceptor cell. Decreasing [Ca²⁺] terminates inhibition and facilitates phosphorylation of illuminated rhodopsin (Rh*). When recoverin formed a complex with GRK1, it did not interfere with the phosphorylation of a C-terminal peptide of rhodopsin (S338-A348) by GRK1. Furthermore, while GRK1 competed with transducin on interaction with rhodopsin and thereby suppressed GTPase activity of transducin, recoverin in the complex with GRK1 did not influence this competition. Constructs of GRK1 that encompass its N-terminal, catalytic or C-terminal domains were used in pull-down assays and surface plasmon resonance analysis to monitor interaction. Ca²⁺-recoverin bound to the N-terminus of GRK1, but did not bind to the other constructs. GRK1 interacted with rhodopsin also by its N-terminus in a light-dependent manner. No interaction was observed with the C-terminus. We conclude that inhibition of GRK1 by recoverin is not the result of their direct competition for the same docking site on Rh*, although the interaction sites of GRK1/Rh* and GRK1/recoverin partially overlap. The N-terminus of GRK1 is recognized by Rh* leading to a conformational change which moves the C-terminus of Rh* into the catalytic kinase groove. Ca²⁺-recoverin interacting with the N-terminus of GRK1 prevents this conformational change and thus blocks Rh* phosphorylation by GRK1.     

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

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

Role of G protein-coupled receptor kinases in the homologous desensitization of the human and mouse melanocortin 1 receptors

The melanocortin 1 receptor, a G protein-coupled receptor positively coupled to adenylyl cyclase, is a key regulator of epidermal melanocyte proliferation and differentiation and a determinant of human skin phototype and skin cancer risk. Despite its potential importance for regulation of pigmentation, no information is available on homologous desensitization of this receptor. We found that the human melanocortin 1 receptor (MC1R) and its mouse ortholog (Mc1r) undergo homologous desensitization in melanoma cells. Desensitization is not dependent on protein kinase A, protein kinase C, calcium mobilization, or MAPKs, but is agonist dose-dependent. Both melanoma cells and normal melanocytes express two members of the G protein-coupled receptor kinase (GRK) family, GRK2 and GRK6. Cotransfection of the receptor and GRK2 or GRK6 genes in heterologous cells demonstrated that GRK2 and GRK6 impair agonist-dependent signaling by MC1R or Mc1r. However, GRK6, but not GRK2, was able to inhibit MC1R agonist-independent constitutive signaling. Expression of a dominant negative GRK2 mutant in melanoma cells increased their cAMP response to agonists. Agonist-stimulated cAMP production decreased in melanoma cells enriched with GRK6 after stable transfection. Therefore, GRK2 and GRK6 seem to be key regulators of melanocortin 1 receptor signaling and may be important determinants of skin pigmentation.