G protein-coupled receptor kinase 2
Guanine nucleotide binding protein (G-protein)-coupled receptor kinases (GRKs) specifically phosphorylate the agonist-occupied form of G-protein-coupled receptors such as the beta 2-adrenergic receptor and rhodopsin. The best characterized members of this family include the beta-adrenergic receptor kinase (beta ARK) and rhodopsin kinase. To identify additional members of the GRK family, the polymerase chain reaction was used to amplify human heart cDNA using degenerate oligonucleotide primers from highly conserved regions unique to the GRK family. The isolation has been reported of a cDNA that encodes a 590-amino acid protein kinase, termed GRK5, which has 34.8% and 47.2% amino acid identities with beta ARK and rhodopsin kinase, respectively. Interestingly, GRK5 has an even higher homology with Drosophila GPRK-2 (71.0% identity) and the recently identified human IT11 (69.1% identity). Northern blot analysis of GRK5 with selected human tissues reveals a message of approximately 3 kilobases with highest levels in heart, placenta, lung > skeletal muscle > brain, liver, pancreas > kidney. GRK5, overexpressed in Sf9 insect cells using the baculovirus system, is able to phosphorylate rhodopsin in a light-dependent manner. In addition, GRK5 neither contains a consensus sequence for isoprenylation like rhodopsin kinase nor is activated by G-protein beta gamma subunits like beta ARK1. Thus, GRK5 represents a member of the GRK family that likely has a unique physiological role (Kunapuli, 1993).
G protein-coupled receptor kinases (GRK), such as the beta-adrenergic receptor kinase (beta ARK) and rhodopsin kinase, specifically phosphorylate the activated form of G protein-coupled receptors. To identify additional members of the GRK family, a human heart cDNA library was screened by low stringency hybridization using the catalytic domains of two beta ARK isoforms. A cDNA has been isolated that encodes a 576-amino-acid protein kinase, termed GRK6, that has significant homology with GRK5 (70.1% amino acid identity), IT11 (68.5%), rhodopsin kinase (47.1%), and beta ARK (37.4%). RNA blot analysis of GRK6 with selected human tissues reveals two distinct mRNAs of 3 and 2.4 kilobases with a distribution very similar to that of beta ARK (i.e. brain, skeletal muscle > pancreas > heart, lung, kidney, placenta > liver). GRK6, overexpressed in Sf9 insect cells using the baculovirus system, is able to phosphorylate both the beta 2-adrenergic receptor and rhodopsin in a stimulus-dependent fashion, although it is significantly less active then beta ARK on these substrates. These data extend the family of GRKs and suggest that GRK6 may have a substrate specificity quite distinct from beta ARK and rhodopsin kinase (Benovic, 1993).
A novel member of the family of G protein-coupled receptor kinases (GRKs), named GRK5, has been cloned from bovine taste epithelium. The cDNA sequence predicts a 590-amino acid protein with high overall similarity to rhodopsin kinase. GRK5 mRNA is found most abundantly in lung, heart, retina, and lingual epithelium, but is expressed very little in brain, liver, kidney, or testis. GRK5 expressed in Sf9 cells was purified to apparent homogeneity. GRK5 major autophosphorylation sites were mapped to Ser484 and Thr485. Purified GRK5 phosphorylates rhodopsin in a light-dependent manner and beta 2-adrenergic receptor in an agonist-dependent manner; GRK5 phosphorylates the C-terminal tail regions of both receptor proteins. GRK5 possesses neither a CAAX motif specifying protein prenylation like rhodopsin kinase nor similarity to the G protein beta gamma-subunit binding domain of beta-adrenergic receptor kinases. GRK5 phosphorylation of rhodopsin or beta 2-adrenergic receptor is not stimulated by G protein beta gamma-subunits. The GRK5 protein does not undergo agonist-dependent translocation from cytosol to membranes as do beta-adrenergic receptor kinase and rhodopsin kinase, but rather appears to associate with membranes constitutively. GRK5 thus appears functionally similar to other characterized GRKs, but has distinct regulatory properties which may be important for its cellular function (Premont, 1994).
GRK6 is the most recently identified member of the GRK family and displays higher homology with GRK5 (70.1% amino acid identity) and IT11 (68.5%) compared to beta ARK (37.4%) and rhodopsin kinase (47.1%). To further characterize GRK6, it has been overexpressed in Sf9 cells and purified to homogeneity by sequential chromatography on SP-Sepharose and heparin-Sepharose columns. GRK6 shares a number of in vitro characteristics with GRK5, including potent inhibition by heparin and dextran sulfate, hyperstimulation by polycations, and preference for phosphorylation of non-acidic peptides. Rhodopsin and the beta 2-adrenergic and m2 muscarinic cholinergic receptors serve as stimulus-dependent substrates for GRK6, but with stoichiometries significantly lower than achieved by GRK5 and beta ARK. Additionally, GRK6 does not undergo significant autophosphorylation even though it contains residues identical to those that are autophosphorylated in GRK5 and rhodopsin kinase. These data extend knowledge of a growing family of receptor-specific kinases and suggest that GRK6 has a substrate specificity distinct from beta ARK, rhodopsin kinase, and GRK5 (Loudon, 1994).
Comparison of the deduced amino acid sequence of GRK4 with those of the closely related GRK5 and GRK6 has suggested the apparent loss of 32 codons in the amino-terminal domain and 46 codons in the carboxyl-terminal domain of GRK4. These two regions undergo alternative splicing in the GRK4 mRNA, resulting from the presence or absence of exons filling one or both of these apparent gaps. Each inserted sequence maintains the open reading frame, and the deduced amino acid sequences are similar to corresponding regions of GRK5 and GRK6. Thus, the GRK4 mRNA and the GRK4 protein can exist in any of four distinct variant forms. The human GRK4 gene is composed of 16 exons extending over 75 kilobase pairs of DNA. The two alternatively spliced exons correspond to exons II and XV. The genomic organization of the GRK4 gene is completely distinct from that of the human GRK2 gene, highlighting the evolutionary distance since the divergence of these two genes. Human GRK4 mRNA is expressed highly only in testis, and both alternative exons are abundant in testis mRNA. The four GRK4 proteins have been expressed, and all incorporate [3H]palmitate. GRK4 is capable of augmenting the desensitization of the rat luteinizing hormone/chorionic gonadotropin receptor upon coexpression in HEK293 cells and of phosphorylating the agonist-occupied, purified beta2-adrenergic receptor, indicating that GRK4 is a functional protein kinase (Premont, 1996).
G protein-coupled receptor kinases (GRKs) desensitize G protein-coupled receptors by phosphorylating activated receptors. The six known GRKs have been classified into three subfamilies based on sequence and functional similarities. Examination of the mouse GRK4 subfamily (GRKs 4, 5, and 6) suggests that mouse GRK4 is not alternatively spliced in a manner analogous to human or rat GRK4, whereas GRK6 undergoes extensive alternative splicing to generate three variants with distinct carboxyl termini. Characterization of the mouse GRK 5 and 6 genes reveals that all members of the GRK4 subfamily share an identical gene structure, in which 15 introns interrupt the coding sequence at equivalent positions in all three genes. Surprisingly, none of the three GRK subgroups (GRK1, GRK2/3, and GRK4/5/6) shares even a single intron in common, indicating that these three subfamilies are distinct gene lineages that have been maintained since their divergence over 1 billion years ago. Comparison of the amino acid sequences of GRKs from various mammalian species indicates that GRK2, GRK5, and GRK6 exhibit a remarkably high degree of sequence conservation, whereas GRK1 and particularly GRK4 have accumulated amino acid changes at extremely rapid rates over the past 100 million years. The divergence of individual GRKs at vastly different rates reveals that strikingly different evolutionary pressures apply to the function of the individual GRKs (Premont, 1999).
Tentative identification of the G protein-coupled receptor kinase 2 and 5 (GRK2 and GRK5) sites of phosphorylation of the beta2-adrenergic receptor (betaAR) has been reported based on in vitro phosphorylation of recombinant receptor. Phosphorylated residues identified for GRK2 are threonine 384 and serines 396, 401, and 407. GRK5 phosphorylates these four residues as well as threonine 393 and serine 411. To determine if mutation of these sites alters desensitization, betaARs were constructed in which the threonines and serines of the putative GRK2 and GRK5 sites were substituted with alanines. These constructs were further modified to eliminate the cAMP-dependent protein kinase (PKA) consensus sites. Mutants betaARs were transfected into HEK 293 cells, and standard kinetic parameters were measured following 10 microM epinephrine treatment of cells. The mutant and wild type (WT) receptors are all desensitized 89%-94% after 5 min of epinephrine stimulation and 96%-98% after a 30-min pretreatment. There were no significant changes observed for any of the mutant betaARs relative to the WT in the extent of epinephrine-induced internalization (77%-82% after 30 min). Epinephrine treatment for 1 min induces a rapid increase in the phosphorylation of the GRK5 and PKA- mutant betaARs as well as the WT. It is concluded that sites other than the GRK2 and GRK5 sites identified by in vitro phosphorylation are involved in mediating the major effects of the in vivo GRK-dependent desensitization of the betaAR (Seibold, 1998).
G protein-coupled receptor kinase 5 (GRK5) is a member of a family of enzymes that phosphorylate activated G protein-coupled receptors (GPCR). To address the physiological importance of GRK5-mediated regulation of GPCRs, mice bearing targeted deletion of the GRK5 gene (GRK5-KO) were generated. GRK5-KO mice exhibit mild spontaneous hypothermia as well as pronounced behavioral supersensitivity upon challenge with the nonselective muscarinic agonist oxotremorine. Classical cholinergic responses such as hypothermia, hypoactivity, tremor, and salivation are enhanced in GRK5-KO animals. The antinociceptive effect of oxotremorine is also potentiated and prolonged. Muscarinic receptors in brains from GRK5-KO mice resist oxotremorine-induced desensitization, as assessed by oxotremorine-stimulated [5S]GTPgammaS binding. These data demonstrate that elimination of GRK5 results in cholinergic supersensitivity and impaired muscarinic receptor desensitization and suggest that a deficit of GPCR desensitization may be an underlying cause of behavioral supersensitivity (Gainetdinov, 1999).
Inhibition of G protein-coupled receptor kinases (GRKs) by Ca2+-binding proteins is a general mechanism of GRK regulation. While GRK1 (rhodopsin kinase) is inhibited by the photoreceptor-specific Ca2+-binding protein recoverin, other GRKs can be inhibited by Ca2+-calmodulin. To dissect the mechanism of this inhibition at the molecular level, the GRK domains involved in Ca2+-binding protein interaction have been localized using a series of GST-GRK fusion proteins. GRK1, GRK2, and GRK5, which represent the three known GRK subclasses, were each found to possess two distinct calmodulin-binding sites. These sites have been localized to the N- and C-terminal regulatory regions within domains rich in positively charged and hydrophobic residues. In contrast, the unique N-terminally localized GRK1 site for recoverin has no clearly defined structural characteristics. Interestingly, while the recoverin and calmodulin-binding sites in GRK1 do not overlap, recoverin-GRK1 interaction is inhibited by calmodulin, most likely via an allosteric mechanism. Further analysis of the individual calmodulin sites in GRK5 suggests that the C-terminal site plays the major role in GRK5-calmodulin interaction. While specific mutation within the N-terminal site has no effect on calmodulin-mediated inhibition of GRK5 activity, deletion of the C-terminal site attenuates the effect of calmodulin on GRK5, and the simultaneous mutation of both sites renders the enzyme calmodulin-insensitive. These studies provide new insight into the mechanism of Ca2+-dependent regulation of GRKs (Levay, 1998).
GRK5, a recently identified member of the GRK family, undergoes a rapid phospholipid-stimulated autophosphorylation to a stoichiometry of approximately 2 mol of phosphate/mol of GRK5. The ability of phospholipids to stimulate autophosphorylation is largely blocked by a glutathione S-transferase fusion protein containing the last 102 amino acids of GRK5 (amino acids 489-590), suggesting that this is a primary region involved in GRK5/phospholipid interaction. Phosphoamino acid determination and mutagenesis studies demonstrate that autophosphorylation of GRK5 occurs primarily at residues Ser-484 and Thr-485. Expression and characterization of a mutant GRK5 that does not autophosphorylate (S484A and T485A) reveals that the mutant has a approximately 15-20-fold reduced ability to phosphorylate the beta 2-adrenergic receptor and rhodopsin compared to wild type GRK5. These results suggest that phospholipid-stimulated autophosphorylation may represent a novel mechanism for membrane association and regulation of GRK5 activity (Kunapuli, 1994).
Regulation of GRKs by Ca2+-binding proteins such as calmodulin (CaM) was examined. Gbetagamma-activated GRK2 and GRK3 are inhibited by CaM to similar extents, while a 50-fold more potent inhibitory effect is observed on GRK5. Inhibition by CaM is strictly dependent on Ca2+ and is prevented by the CaM inhibitor CaMBd. Since Gbetagamma, which is a binding target of Ca2+/CaM, is critical for the activation of GRK2 and GRK3, it provides a possible site of interaction between these proteins. However, since GRK5 is Gbetagamma-independent, an alternative mechanism is conceivable. A direct interaction between GRK5 and Ca2+/CaM was revealed using CaM-conjugated Sepharose 4B. This binding does not influence the catalytic activity as demonstrated using the soluble GRK substrate casein. Instead, Ca2+/CaM significantly reduces GRK5 binding to the membrane. The mechanism of GRK5 inhibition appears to be through direct binding to Ca2+/CaM, resulting in inhibition of membrane association and hence receptor phosphorylation. The present study provides the first evidence for a regulatory effect of Ca2+/CaM on some GRK subtypes, thus expanding the range of different mechanisms regulating the functional states of these kinases (Chuang, 1996).
The G protein-coupled receptor kinases (GRKs) phosphorylate agonist occupied G protein-coupled receptors and play an important role in mediating receptor desensitization. The localization of these enzymes to their membrane incorporated substrates is required for their efficient function and appears to be a highly regulated process. Phosphatidylinositol 4, 5-bisphosphate (PIP2) enhances GRK5-mediated beta-adrenergic receptor (betaAR) phosphorylation by directly interacting with this enzyme and facilitating its membrane association. GRK5-mediated phosphorylation of a soluble peptide substrate is unaffected by PIP2, suggesting that the PIP2-enhanced receptor kinase activity arises as a consequence of this membrane localization. The lipid binding site of GRK5 exhibits a high degree of specificity and appears to reside in the amino terminus of this enzyme. Mutation of six basic residues at positions 22, 23, 24, 26, 28, and 29 of GRK5 ablates the ability of this kinase to bind PIP2. This region of the GRK5, which has a similar distribution of basic amino acids to the PIP2 binding site of gelsolin, is highly conserved between members of the GRK4 subfamily (GRK4, GRK5, and GRK6). Indeed, all the members of the GRK4 subfamily exhibit PIP2-dependent receptor kinase activity. The membrane association of betaARK (beta-adrenergic receptor kinase) (GRK2) is mediated, in vitro, by the simultaneous binding of PIP2 and the betagamma subunits of heterotrimeric G proteins to the carboxyl-terminal pleckstrin homology domain of this enzyme. Thus, five members of the GRK family bind PIP2, betaARK (GRK2), betaARK2 (GRK3), GRK4, GRK5, and GRK6. However, the structure, location, and regulation of the PIP2 binding site distinguishes the betaARK (GRK2 and GRK3) and GRK4 (GRK4, GRK5, and GRK6) subfamilies (Pitcher, 1996).
To assess a potential general role for PKC in regulating GRK function, the ability of PKC to phosphorylate GRK5 was characterized. GRK5 can be rapidly and stoichiometrically phosphorylated by PKC in vitro. Intact cell studies reveal that GRK5 is also phosphorylated when transiently expressed in COS-1 cells following treatment with the PKC activator, phorbol 12-myristate 13-acetate. In vitro analysis reveals two major sites of PKC phosphorylation within the C-terminal 26 amino acids of GRK5. GRK5 phosphorylation by PKC dramatically reduces its ability to phosphorylate both receptor (light-activated rhodopsin) and non-receptor (casein and phosvitin) substrates. Kinetic analysis reveals an approximately 5-fold increased Km and approximately 3-fold decreased Vmax for rhodopsin, with no change in the Km for ATP. The reduced affinity of PKC-phosphorylated GRK5 for rhodopsin is also evident in a decreased ability to bind to rhodopsin-containing membranes, while direct binding of GRK5 to phospholipids appears unaltered. These results suggest that PKC might play an important role in modulating the ability of GRK5 to regulate receptor signaling and that GRK phosphorylation by PKC may serve as a disparate mechanism for regulating GRK activity (Pronin, 1997a).
GRK2 and GRK5 can be phosphorylated and either activated or inhibited, respectively, by protein kinase C. Calmodulin, another mediator of calcium signaling, is a potent inhibitor of GRK activity with a selectivity for GRK5. Calmodulin inhibition of GRK5 is mediated via a reduced ability of the kinase to bind to both receptor and phospholipid. Interestingly, calmodulin also activates autophosphorylation of GRK5 at sites distinct from the two major autophosphorylation sites on GRK5. Moreover, calmodulin-stimulated autophosphorylation directly inhibits GRK5 interaction with receptor even in the absence of calmodulin. Using glutathione S-transferase-GRK5 fusion proteins either to inhibit calmodulin-stimulated autophosphorylation or to bind directly to calmodulin, it was determined that an amino-terminal domain of GRK5 (amino acids 20-39) is sufficient for calmodulin binding. This domain is abundant in basic and hydrophobic residues, characteristics typical of calmodulin binding sites, and is highly conserved in GRK4, GRK5, and GRK6. These studies suggest that calmodulin may serve a general role in mediating calcium-dependent regulation of GRK activity (Pronin, 1997b).
GRK2-mediated receptor phosphorylation is preceded by the agonist-dependent membrane association of this enzyme. Previous in vitro studies with purified proteins have suggested that this translocation may be mediated by the recruitment of GRK2 to the plasma membrane by its interaction with the free betagamma subunits of heterotrimeric G proteins (G betagamma). This mechanism operates in intact cells and specificity is imparted by the selective interaction of discrete pools of G betagamma with receptors and GRKs. Treatment of Cos-7 cells transiently overexpressing GRK2 with a beta-receptor agonist promotes a 3-fold increase in plasma membrane-associated GRK2. This translocation of GRK2 is inhibited by the carboxyl terminus of GRK2, a known G betagamma sequestrant. Furthermore, in cells overexpressing both GRK2 and G beta1 gamma2, activation of lysophosphatidic acid receptors leads to the rapid and transient formation of a GRK/G betagamma complex. That G betagamma specificity exists at the level of the GPCR and the GRK is indicated by the observation that a GRK2/G betagamma complex is formed after agonist occupancy of the lysophosphatidic acid and beta-adrenergic but not thrombin receptors. In contrast to GRK2, GRK3 forms a G betagamma complex after stimulation of all three GPCRs. This G betagamma binding specificity of the GRKs is also reflected at the level of the purified proteins. Thus the GRK2 carboxyl terminus binds G beta1 and G beta2 but not G beta3, while the GRK3 fusion protein binds all three G beta isoforms. This study provides a direct demonstration of a role for G betagamma in mediating the agonist-stimulated translocation of GRK2 and GRK3 in an intact cellular system and demonstrates isoform specificity in the interaction of these components (Daaka, 1997).
GRKs are subject to post-translational regulation. For example, GRK5 activity is strongly inhibited by protein kinase C phosphorylation and by Ca2+-calmodulin binding. Ca2+-calmodulin binding also promotes GRK5 autophosphorylation, which further contributes to kinase inhibition. Two important structural domains have been identified in GRK5, a phospholipid binding domain (residues 552-562) and an autoinhibitory domain (residues 563-590), that significantly contribute to GRK5 localization and function. The C-terminal region of GRK5 (residues 563-590) contains residues autophosphorylated in the presence of calmodulin as well as the residues phosphorylated by protein kinase C. Deletion of this domain increases the apparent affinity of GRK5 for receptor substrates 3-4-fold but has no effect on nonreceptor substrates. These findings define residues 563-590 of GRK5 as an autoinhibitory domain with efficacy that is regulated by phosphorylation. Another C-terminal domain in GRK5 that appears to be functionally important is found between residues 552 and 562. Deletion of this region significantly inhibits kinase phosphorylation of membrane-bound receptor substrates but has no effect on soluble substrates. Additional studies reveal that this domain is critical for GRK5 interaction with phospholipids and for the intracellular localization of the kinase. Interestingly, similar regions in GRK4 and GRK6 appear to be palmitoylated (and involved in membrane interaction), suggesting evolutionary conservation of the function of this domain (Pronin, 1998).
G protein-coupled receptor kinases (GRKs) phosphorylate G protein-coupled receptors, thereby terminating receptor signaling. Alpha-actinin potently inhibits all GRK family members. In addition, calcium-bound calmodulin and phosphatidylinositol 4,5-bisphosphate (PIP2), two regulators of GRK activity, coordinate with alpha-actinin to modulate substrate specificity of the GRKs. In the presence of calmodulin and alpha-actinin, GRK5 phosphorylates soluble, but not membrane-incorporated substrates. In contrast, in the presence of PIP2 and alpha-actinin, GRK5 phosphorylates membrane-incorporated, but not soluble substrates. Thus, modulation of alpha-actinin-mediated inhibition of GRKs by PIP2 and calmodulin has profound effects on both GRK activity and substrate specificity (Freeman, 2000).
G-protein-coupled receptor kinase 2 (GRK2) plays a key role in the regulation of G-protein-coupled receptors (GPCRs). GRK2 expression is altered in several pathological conditions, but the molecular mechanisms that modulate GRK2 cellular levels are largely unknown. GRK2 is degraded rapidly by the proteasome pathway. This process is enhanced by GPCR stimulation and is severely impaired in a GRK2 mutant that lacks kinase activity (GRK2-K220R). ß-arrestin function and Src-mediated phosphorylation of GRK2 are critically involved in GRK2 proteolysis. Overexpression of ß-arrestin triggers GRK2-K220R degradation based on its ability to recruit c-Src, since this effect is not observed with ß-arrestin mutants that display an impaired c-Src interaction. The presence of an inactive c-Src mutant or of tyrosine kinase inhibitors strongly inhibits co-transfected or endogenous GRK2 turnover, respectively, and a GRK2 mutant with impaired phosphorylation by c-Src shows a markedly retarded degradation. This pathway for the modulation of GRK2 protein stability puts forward a new feedback mechanism for regulating GRK2 levels and GPCR signaling (Penela, 2001).
These results are consistent with the notion that GRK2-dependent binding of ß-arrestin to GPCRs allows the recruitment of c-Src to the receptor signaling complex at the plasma membrane, leading to phosphorylation of GRK2 on tyrosine residues and its targeting for degradation. This model is in agreement with the rapid ß-arrestin and c-Src recruitment following ß2AR stimulation, and with the agonist-stimulated phosphorylation of GRK2 by c-Src. Under basal conditions, ß-arrestin recruitment to the plasma membrane would be promoted by the activated state of different endogenous GPCRs and/or by the reported basal activity of overexpressed ß2AR. In the presence of GPCR agonists, an acceleration of the GRK2 degradation rate is detected, consistent with a more efficient ß-arrestin and c-Src translocation to the receptor complex. Although detailed knowledge of the sequential assembly of these proteins in a multimolecular complex is lacking, and other molecular interactions may participate in c-Src binding to the receptor complex and GRK2 tyrosine phosphorylation, the proposed model is consistent with the co-immunoprecipitation of ß-arrestin and c-Src, of GRK2 and ß-arrestin and of GRK2 and c-Src. Disruption of the ß-arrestin-c-Src interaction with specific mutants or inhibition of the phosphorylation step by dominant-negative Src or a GRK2 mutant lacking critical phosphorylation sites results, as predicted by this model, in a marked reduction in GRK2 degradation (Penela, 2001).
Persistent stimulation of the beta 1-adrenergic receptor (beta 1AR) engenders, within minutes, diminished responsiveness of the beta 1 AR/adenylyl cyclase signal transduction system. This desensitization remains incompletely defined mechanistically, however. The hypothesis that agonist-induced desensitization of the beta 1AR (like that of the related beta 2AR) involves phosphorylation of the receptor itself, by cAMP-dependent protein kinase (PKA) and the beta-adrenergic receptor kinase (beta ARK1) or other G protein-coupled receptor kinases (GRKs), was tested. Both Chinese hamster fibroblast and 293 cells demonstrate receptor-specific desensitization of the beta 1 AR within 3-5 min. Both cell types also express beta ARK1 and the associated inhibitory proteins beta-arrestin-1 and beta-arrestin-2, as assessed by immunoblotting. Agonist-induced beta 1AR desensitization in 293 cells correlates with a 2 +/- 0.3-fold increase in phosphorylation of the beta 1AR, determined by immunoprecipitation of the beta 1AR from cells metabolically labeled with 32P(i). This agonist-induced beta 1AR phosphorylation derives approximately equally from PKA and GRK activity, as judged by intact cell studies with kinase inhibitors or dominant negative beta ARK1 (K220R) mutant overexpression. Desensitization, likewise, is reduced by only approximately 50% when PKA is inhibited in the intact cells. Overexpression of rhodopsin kinase, beta ARK1, beta ARK2, or GRK5 significantly increases agonist-induced beta 1AR phosphorylation and concomitantly decreases agonist-stimulated cellular cAMP production. Furthermore, purified beta ARK1, beta ARK2, and GRK5 all demonstrate agonist-dependent phosphorylation of the beta 1AR. Consistent with a GRK mechanism, receptor-specific desensitization of the beta 1AR is enhanced by overexpression of beta-arrestin-1 and -2 in transfected 293 cells. It is concluded that rapid agonist-induced desensitization of the beta 1AR involves phosphorylation of the receptor by both PKA and at least beta ARK1 in intact cells. Like the beta 2AR, the beta 1AR appears to bind either beta-arrestin-1 or beta-arrestin-2 and to react with rhodopsin kinase, beta ARK1, beta ARK2, and GRK5 (Freedman, 1995).
Although previous studies have implicated the cytoplasmic tail of the beta2-adrenergic receptor (beta2AR) as the site of GRK-mediated phosphorylation, the identities of the phosphorylated residues were unknown. The sites of GRK2- and GRK5-mediated beta2AR phosphorylation have been identified in this study. The phosphorylation sites of both serine/threonine kinases reside exclusively in a 40-amino acid peptide located at the extreme carboxyl terminus of the beta2AR. Of the seven phosphorylatable residues within this peptide, six are phosphorylated by GRK5 (Thr-384, Thr-393, Ser-396, Ser-401, Ser-407, and Ser-411) and four are phosphorylated by GRK2 (Thr-384, Ser-396, Ser-401, and Ser-407) at equivalent phosphorylation stoichiometries (approximately 1.0 mol Pi/mol receptor). In addition to the GRK5-specific phosphorylation of Thr-393 and Ser-411, differences in the distribution of phosphate between sites are observed for GRK2 and GRK5. Increasing the stoichiometry of GRK2-mediated beta2AR phosphorylation from approximately 1.0 to 5.0 mol Pi/mol receptor increases the stoichiometry of phosphorylation of Thr-384, Ser-396, Ser-401, and Ser-407 rather than increasing the number of phosphoacceptor sites. The location of multiple GRK2 and GRK5 phosphoacceptor sites at the extreme carboxyl terminus of the beta2AR is highly reminiscent of GRK1-mediated phosphorylation of rhodopsin (Fredericks, 1996).
The alpha2-adrenergic receptor (alpha2AR) subtype alpha2C10 undergoes rapid agonist-promoted desensitization which is due to phosphorylation of the receptor. One kinase that has been shown to phosphorylate alpha2C10 in an agonist-dependent manner is the betaAR kinase (betaARK), a member of the family of G protein-coupled receptor kinases (GRKs). In contrast, the alpha2C4 subtype has not been observed to undergo agonist-promoted desensitization or phosphorylation by betaARK. However, the substrate specificities of the GRKs for phosphorylating alpha2AR subtypes are not known. Differential capacities of various GRKs to phosphorylate alpha2C10 and alpha2C4 might be a key factor in dictating in a given cell the presence or extent of agonist-promoted desensitization of these receptors. COS-7 cells were co-transfected with alpha2C10 or alpha2C4 without or with the following GRKs: betaARK, betaARK2, GRK5, or GRK6. Intact cell phosphorylation studies were carried out by labeling cells with 32Pi, exposing some to agonist, and purifying the alpha2AR by immunoprecipitation and SDS-polyacrylamide gel electrophoresis. BetaARK and betaARK2 were both found to phosphorylate alpha2C10 to equal extents. GRK5 and GRK6 did not phosphorylate alpha2C10. In contrast to the findings with alpha2C10, alpha2C4 is not phosphorylated by any of these kinases. Functional studies carried out in transfected HEK293 cells expressing alpha2C10 or alpha2C4 and selected GRKs were consistent with these phosphorylation results. With the marked expression of these receptors, no agonist-promoted desensitization is observed in the absence of GRK co-expression. However, desensitization is imparted to alpha2C10 by co-expression of betaARK but not GRK6, while alpha2C4 failed to desensitize with co-expression of betaARK. These results indicate that short term agonist-promoted desensitization of alpha2ARs by phosphorylation is dependent on both the receptor subtype and the expressed GRK isoform (Jewell-Motz, 1996).
The alpha1B-adrenergic receptor (alpha1BAR), its truncated mutant T368, different G protein-coupled receptor kinases (GRK) and arrestin proteins were transiently expressed in COS-7 or HEK293 cells alone and/or in various combinations. Coexpression of beta-adrenergic receptor kinase (betaARK) 1 (GRK2) or 2 (GRK3) can increase epinephrine-induced phosphorylation of the wild type alpha1BAR above basal as compared to that of the receptor expressed alone. Overexpression of the dominant negative betaARK (K220R) mutant impairs agonist-induced phosphorylation of the receptor. Overexpression of GRK6 can also increase epinephrine-induced phosphorylation of the receptor, whereas GRK5 enhances basal but not agonist-induced phosphorylation of the alpha1BAR. Increasing coexpression of betaARK1 or betaARK2 results in the progressive attenuation of the alpha1BAR-mediated response on polyphosphoinositide (PI) hydrolysis. However, coexpression of betaARK1 or 2 at low levels does not significantly impair the PI response mediated by the truncated alpha1BAR mutant T368, lacking the C terminus, which is involved in agonist-induced desensitization and phosphorylation of the receptor. Similar attenuation of the receptor-mediated PI response is also observed for the wild type alpha1BAR, but not for its truncated mutant, when the receptor is coexpressed with beta-arrestin 1 or beta-arrestin 2. Despite their pronounced effect on phosphorylation of the alpha1BAR, overexpression of GRK5 or GRK6 do not affect the receptor-mediated response. In conclusion, these results provide the first evidence that betaARK1 and 2 as well as arrestin proteins might be involved in agonist-induced regulation of the alpha1BAR. They also identify the alpha1BAR as a potential phosphorylation substrate of GRK5 and GRK6. However, the physiological implications of GRK5- and GRK6-mediated phosphorylation of the alpha1BAR remain to be elucidated (Diviani, 1996).
To explore the potential role played by the GRKs in the regulation of the rat dopamine D1A receptor, whole cell phosphorylation experiments and cAMP assays were performed in 293 cells cotransfected with the receptor alone or with various GRKs (GRK2, GRK3, and GRK5). The agonist-dependent phosphorylation of the rat D1A receptor is substantially increased in cells overexpressing GRK2, GRK3, or GRK5. Moreover, cAMP formation upon receptor activation is differentially regulated in cells overexpressing either GRK2, GRK3, and GRK5 under conditions that elicited similar levels of GRK-mediated receptor phosphorylation. Cells expressing the rat D1A receptor with GRK2 and GRK3 display a rightward shift of the dopamine dose-response curve with little effect on the maximal activation when compared with cells expressing the receptor alone. In contrast, cells expressing GRK5 display a rightward shift in the EC50 value with an additional 40% reduction in the maximal activation when compared with cells expressing the receptor alone. Thus, the dopamine D1A receptor can serve as a substrate for various GRKs and GRK-phosphorylated D1A receptors display a differential reduction of functional coupling to adenylyl cyclase. These results suggest that the cellular complement of G protein-coupled receptor kinases may determine the properties and extent of agonist-mediated responsiveness and desensitization (Tiberi, 1996).
To identify GRK(s) that play a role in homologous desensitization of the thyrotropin (TSH) receptor, thyroid cDNA was amplified by polymerase chain reaction using degenerate oligonucleotide primers from highly conserved regions in GRK family. GRK5 is found in the predominant isoform expressed in the thyroid. Rat GRK5 cDNA was then isolated, which encodes a 590-amino acid protein with 95% homology to human and bovine homologs. Northern blot identified GRK5 mRNA of approximately 3, 8, and 10 kilobases with highest expression levels in lung > heart, kidney, colon > thyroid. In functional studies using a normal rat thyroid FRTL5 cells, overexpression of GRK5 suppresses basal cAMP levels and augments the extent of TSH receptor desensitization, whereas suppression of endogenous GRK5 expression by transfecting the antisense GRK5 construct increases basal cAMP levels and attenuates the extent of receptor desensitization. Although exogenously overexpressed GRK6 also enhances TSH receptor desensitization, it is concluded that GRK5, the predominant GRK isoform in the thyroid, appears to be mainly involved in homologous desensitization of the TSH receptor (Nagayama, 1996).
FSH rapidly desensitizes the FSH-receptor (FSH-R) upon binding. Very little information is available concerning the regulatory proteins involved in this process. The present study investigated whether G protein-coupled receptor kinases (GRKs) and arrestins have a role in FSH-R desensitization, using a mouse Ltk 7/12 cell line stably overexpressing the rat FSH-R as a model. These cells, which express GRK2, GRK3, GRK5, and GRK6 as well as beta-arrestins 1 and 2 are rapidly desensitized in the presence of FSH. Overexpression of GRKs and/or beta-arrestins in Ltk 7/12 cells demonstrate the following: (1) that GRK2, -3, -5, -6a, and -6b inhibit the FSH-R-mediated signaling (from 71% to 96% of maximal inhibition depending on the kinase; (2) that beta-arrestins 1 or 2 also decrease the FSH action when overexpressed (80% of maximal inhibition whereas dominant negative beta-arrestin 2 [319-418] potentiates it 8-fold; (3) that beta-arrestins and GRKs (except GRK6a) exert additive inhibition on FSH-induced response; and (4) that FSH-R desensitization depends upon the endogenous expression of GRKs, since there is potentiation of the FSH response (2- to 3-fold) with antisenses cDNAs for GRK2, -5, and -6, but not GRK3. These results show that the desensitization of the FSH-induced response involves the GRK/arrestin system (Troispoux, 1999).
The Xenopus oocyte expression system to examine the regulation of rat kappa opioid receptor (rKOR) function by G protein receptor kinases (GRKs). Kappa agonists increase 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 is modest. Co-expression of either GRK3 or GRK5 along with beta-arrestin 2 significantly increases the rate of desensitization, whereas addition of either beta-arrestin 2, GRK3, or GRK5 alone has no effect on the KOR desensitization rate. The desensitization is homologous since co-expressed delta opioid receptor-evoked responses are not affected by KOR desensitization. The rate of GRK3/beta-arrestin 2-dependent desensitization is 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)] does not reduce the desensitization rate. Within the C-terminal region, KOR(S369A) substitution significantly attenuates desensitization, whereas the KOR(T363A) and KOR(S356A,T357A) point mutations do not. These results suggest that co-expression of GRK3 or GRK5 and beta-arrestin 2 produce homologous, agonist-induced desensitization of the kappa opioid receptor by a mechanism requiring the phosphorylation of the serine 369 of rKOR (Appleyard, 1999).
Examination of the structure of [Arg(8)]-vasopressin receptors (AVPRs) and oxytocin receptors (OTRs) suggests that G protein-coupled receptor kinases (GRKs) and protein kinase C (PKC) are involved in their signal transduction. To explore the physical association of AVPRs and OTRs with GRKs and PKC, wild types and mutated forms of these receptor subtypes were stably expressed as green fluorescent protein fusion proteins and analyzed by fluorescence, immunoprecipitation, and immunoblotting. Addition of a C-terminal GFP tag does not interfere with ligand binding, internalization, and signal transduction. After agonist stimulation, PKC dissociates from the V(1)R, does not associate with the V(2)R, but associates with the V(3)R and the OTR. After AVP stimulation, only GRK5 briefly associates with AVPRs following a time course that varies with the receptor subtype. No GRK associates with the OTR. Exchanging the V(1)R and V(2)R C termini alters the time course of PKC and GRK5 association. Deletion of the V(1)R C terminus results in no PKC association and a ligand-independent sustained association of GRK5 with the receptor. Deletion of the GRK motif prevents association and reduces receptor phosphorylation. Thus, agonist stimulation of AVP/OT receptors leads to receptor subtype-specific interactions with GRK and PKC through specific motifs present in the C termini of the receptors (Berrada, 2000).
Investigating the parathyroid hormone (PTH) receptor --> inositol phosphate pathway, it has been found that GRKs can inhibit receptor signaling already under nonphosphorylating conditions. GRKs phosphorylated the PTH receptor in membranes and in intact cells; the order of efficacy was GRK2>GRK3>GRK5. Transient transfection of GRKs with the PTH receptor into COS-1 cells inhibits PTH-stimulated inositol phosphate generation. Such an inhibition also is seen with the kinase-negative mutant GRK2-K220R and also for a C-terminal truncation mutant of the PTH receptor that could not be phosphorylated. Several lines of evidence indicate that this phosphorylation-independent inhibition is exerted by an interaction between GRKs and receptors: (1) this inhibition is not mimicked by proteins binding to G proteins, phosducin, and GRK2 C terminus; (2) GRKs cause an agonist-dependent inhibition (= desensitization) of receptor-stimulated G protein GTPase-activity (this effect also is seen with the kinase-inactive GRK2-mutant and the phosphorylation-deficient receptor mutant), and (3) GRKs bind directly to the PTH receptor. These data suggest that signaling by the PTH receptor already is inhibited by the first step of homologous desensitization, the binding of GRKs to the receptors (Dicker, 1999).
Although endothelin-1 can elicit prolonged physiologic responses, accumulating evidence suggests that rapid desensitization affects the primary G protein-coupled receptors mediating these responses, the endothelin A and B receptors (ETA-R and ETB-R). The mechanisms by which this desensitization proceeds remain obscure, however. Because some intracellular domain sequences of the ETA-R and ETB-R differ substantially, the possibility that these receptor subtypes might be differentially regulated by G protein-coupled receptor kinases was examined. Homologous, or receptor-specific, desensitization occurs within 4 min both in the ETA-R-expressing A10 cells and in 293 cells transfected with either the human ETA-R or ETB-R. In 293 cells, this desensitization corresponds temporally with agonist-induced phosphorylation of each receptor. Agonist-induced receptor phosphorylation is not substantially affected by PKC inhibition but is reduced 40% by GRK inhibition, effected by a dominant negative GRK2 mutant. Inhibition of agonist-induced phosphorylation abrogates agonist-induced ETA-R desensitization. Overexpression of GRK2, -5, or -6 in 293 cells augments agonist-induced ET-R phosphorylation approximately 2-fold, but each kinase reduces receptor-promoted phosphoinositide hydrolysis differently. While GRK5 inhibits ET-R signaling by only approximately 25%, GRK2 inhibits ET-R signaling by 80%. Congruent with its superior efficacy in suppressing ET-R signaling, GRK2, but not GRK5, co-immunoprecipitates with the ET-Rs in an agonist-dependent manner. It is concluded that both the ETA-R and ETB-R can be regulated indistinguishably by GRK-initiated desensitization. It is proposed that because of its affinity for ET-Rs, GRK2 is the most likely of the GRKs to initiate ET-R desensitization (Freedman, 1997).
A study was performed of the agonist-dependent sequestration/internalization of dopamine D2 receptor (the long form D2L and short form D2S), which were transiently expressed in COS-7 and HEK 293 cells with or without G-protein-coupled receptor kinases (GRK2 or GRK5). In COS-7 cells expressing D2 receptors alone, virtually no sequestration is observed with or without dopamine (< 4%). When GRK2 is coexpressed, 50% of D2S receptors and 36% of D2L receptors are sequestered by treatment with 10(-4) M dopamine for 2 h, whereas no sequestration is observed in cells expressing the dominant negative form of GRK2 (DN-GRK2). When GRK5 is coexpressed, 36% of D2S receptors are sequestered following the same treatment. The agonist-dependent and GRK2-dependent sequestration of D2S receptors is reduced markedly in the presence of hypertonic medium containing 0.45 M sucrose, suggesting that the sequestration follows the clathrin pathway. Internalization of D2S receptors was also assessed by immunofluorescence confocal microscopy. Translocation of D2 receptors from the cell membrane to intracellular vesicles is observed following the treatment with dopamine from HEK 293 cells only when GRK2 is coexpressed. D2S receptors expressed in HEK 293 cells are phosphorylated by GRK2 in an agonist-dependent manner. These results indicate that the sequestration of D2 receptors occurs only through a GRK-mediated pathway (Ito, 1999).
GRK2 is a microtubule-associated protein and tubulin is identified as a novel GRK2 substrate. GRK2 is associated with microtubules purified from bovine brain, forms a complex with tubulin in cell extracts, and colocalizes with tubulin in living cells. Furthermore, an endogenous tubulin kinase activity that copurifies with microtubules has properties similar to GRK2 and is inhibited by anti-GRK2 monoclonal antibodies. Indeed, GRK2 phosphorylates tubulin in vitro with kinetic parameters very similar to those for phosphorylation of the agonist-occupied beta2-adrenergic receptor, suggesting a functionally relevant role for this phosphorylation event. In a cellular environment, agonist occupancy of GPCRs, which leads to recruitment of GRK2 to the plasma membrane and its subsequent activation, promotes GRK2-tubulin complex formation and tubulin phosphorylation. These findings suggest a novel role for GRK2 as a GPCR signal transducer mediating the effects of GPCR activation on the cytoskeleton (Pitcher, 1998).
G protein-coupled receptor kinases (GRKs) initiate pathways leading to the desensitization of agonist-occupied G-protein-coupled receptors (GPCRs). The cytoskeletal protein actin binds and inhibits GRK5. Actin inhibits the kinase activity directly, reducing GRK5-mediated phosphorylation of both membrane-bound GPCRs and soluble substrates. GRK5 binds actin monomers with a Kd of 0.6 microM and actin filaments with a Kd of 0. 2 microM. Mutation of 6 amino acids near the amino terminus of GRK5 eliminates actin-mediated inhibition of GRK5. Calmodulin binds to the amino terminus of GRK5 and displaces GRK5 from actin. Calmodulin inhibits GRK5-mediated phosphorylation of GPCRs, but not soluble substrates such as casein. Thus in the presence of actin, calmodulin determines the substrate specificity of GRK5 by preferentially allowing phosphorylation of soluble substrates over membrane-bound substrates (Freeman, 1998).
Although the beta-adrenergic receptor kinase (betaARK) mediates agonist-dependent phosphorylation and desensitization of G protein-coupled receptors, recent studies suggest additional cellular functions. During attempts to identify novel betaARK interacting proteins, it was found that the cytoskeletal protein tubulin can specifically bind to a betaARK-coupled affinity column. In vitro analysis demonstrates that betaARK and G protein-coupled receptor kinase-5 (GRK5) are able to stoichiometrically phosphorylate purified tubulin dimers with a preference for beta-tubulin and, under certain conditions, the betaIII-isotype. Examination of the GRK/tubulin binding characteristics revealed that tubulin dimers and assembled microtubules bind GRKs, whereas the catalytic domain of betaARK contains the primary tubulin binding determinants. In vivo interaction of GRK and tubulin is suggested by the following: (1) co-purification of betaARK with tubulin from brain tissue; (2) co-immunoprecipitation of betaARK and tubulin from COS-1 cells; and (3) co-localization of betaARK and GRK5 with microtubule structures in COS-1 cells. In addition, GRK-phosphorylated tubulin is found preferentially associated with the microtubule fraction during in vitro assembly assays suggesting potential functional significance. These results suggest a novel link between the cytoskeleton and GRKs that may be important for regulating GRK and/or tubulin function (Carman, 1998).
G protein-coupled receptor kinases (GRKs) have been principally characterized by their ability to phosphorylate and desensitize G protein-coupled receptors. However, recent studies suggest that GRKs may have more diverse protein/protein interactions in cells. Based on the identification of a consensus caveolin binding motif within the pleckstrin homology domain of GRK2, the direct binding of purified full-length GRK2 to various glutathione S-transferase-caveolin-1 fusion proteins was studied, and a specific interaction of GRK2 with the caveolin scaffolding domain was discovered. Interestingly, analysis of GRK1 and GRK5, which lack a pleckstrin homology domain, revealed in vitro binding properties similar to those of GRK2. Maltose-binding protein caveolin and glutathione S-transferase-GRK fusion proteins were used to map overlapping regions in the N termini of both GRK2 and GRK5 that appear to mediate conserved GRK/caveolin interactions. In vivo association of GRK2 and caveolin was suggested by co-fractionation of GRK2 with caveolin in A431 and NIH-3T3 cells and was further supported by co-immunoprecipitation of GRK2 and caveolin in COS-1 cells. Functional significance for the GRK/caveolin interaction was demonstrated by the potent inhibition of GRK-mediated phosphorylation of both receptor and peptide substrates by caveolin-1 and -3 scaffolding domain peptides. These data reveal a novel mode for the regulation of GRKs that is likely to play an important role in their cellular function (Carman, 1999).
Transgenic mice were generated with cardiac-specific overexpression of the G protein-coupled receptor kinase-5 (GRK5), a serine/threonine kinase most abundantly expressed in the heart compared with other tissues. Animals overexpressing GRK5 show marked beta-adrenergic receptor desensitization in both the anesthetized and conscious state compared with nontransgenic control mice, while the contractile response to angiotensin II receptor stimulation is unchanged. In contrast, the angiotensin II-induced rise in contractility is significantly attenuated in transgenic mice overexpressing the beta-adrenergic receptor kinase-1, another member of the GRK family. These data suggest that myocardial overexpression of GRK5 results in selective uncoupling of G protein-coupled receptors and demonstrate that receptor specificity of the GRKs may be important in determining the physiological phenotype (Rockman, 1996).
Pressure overload cardiac hypertrophy in the mouse is achieved following 7 days of transverse aortic constriction. This is associated with marked beta-adrenergic receptor (beta-AR) desensitization in vivo, as determined by a blunted inotropic response to dobutamine. Extracts from hypertrophied hearts have approximately 3-fold increase in cytosolic and membrane G protein-coupled receptor kinase (GRK) activity. Incubation with specific monoclonal antibodies to inhibit different GRK subtypes showed that the increase in activity can be attributed predominately to the beta-adrenergic receptor kinase (betaARK). Although overexpression of a betaARK inhibitor in hearts of transgenic mice does not alter the development of cardiac hypertrophy, the beta-AR desensitization associated with pressure overload hypertrophy is prevented. To determine whether the induction of betaARK occurs because of a generalized response to cellular hypertrophy, betaARK activity was measured in transgenic mice homozygous for oncogenic ras overexpression in the heart. Despite marked cardiac hypertrophy, no difference in betaARK activity was found in these mice overexpressing oncogenic ras compared with controls. Taken together, these data suggest that betaARK is a central molecule involved in alterations of beta-AR signaling in pressure overload hypertrophy. The mechanism for the increase in betaARK activity appears not to be related to the induction of cellular hypertrophy but to possibly be related to neurohumoral activation (Choi, 1997).
The function of G protein-coupled receptor kinases (GRKs) in the regulation of thrombin-activated signaling in endothelial cells was studied. GRK2, GRK5, and GRK6 isoforms are expressed predominantly in endothelial cells. The function of these isoforms was studied by expressing wild-type and dominant negative (dn) mutants in endothelial cells. The responses to thrombin, which activates intracellular signaling in endothelial cells by cleaving the NH(2) terminus of the G protein-coupled proteinase-activated receptor-1 (PAR-1), was examined. Changes in phosphoinositide hydrolysis and intracellular Ca2+ concentration in response to thrombin were studied as well as the state of endothelial activation. In the latter studies, the transendothelial monolayer electrical resistance, a measure of the loss of endothelial barrier function, was measured in real time. Of the three isoforms, GRK5 overexpression is selective in markedly reducing the thrombin-activated phosphoinositide hydrolysis and increased intracellular Ca2+. GRK5 overexpression also inhibited the thrombin-induced decrease in endothelial monolayer resistance by 75%. These effects of GRK5 overexpression occur in association with the specific increase in the thrombin-induced phosphorylation of PAR-1. In contrast to the effects of GRK5 overexpression, the expression of the dn-GRK5 mutant produces a long-lived increase in intracellular Ca2+ in response to thrombin, whereas dn-GRK2 has no effect. These results indicate the crucial role of the GRK5 isoform in the mechanism of thrombin-induced desensitization of PAR-1 in endothelial cells (Tiruppathi, 2000).
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. The expression of mGluR1a in HEK 293 cells leads to agonist-independent cell death. Since G protein-coupled receptor kinases (GRKs) desensitize a diverse variety of GPCRs, whether GRKs contributes to the regulation of both constitutive and agonist-stimulated mGluR1a activity and thereby prevents mGluR1a-mediated excitotoxicity associated with mGluR1a overactivation was examined. The co-expression of mGluR1a with GRK2 and GRK5, but not GRK4 and GRK6, reduces both constitutive and agonist-stimulated mGluR1a activity. Agonist-stimulated mGluR1a phosphorylation is enhanced by the co-expression of GRK2 and is blocked by two different GRK2 dominant-negative mutants. Furthermore, GRK2-dependent mGluR1a desensitization protects against mGluR1a-mediated cell death, at least in part by blocking mGluR1a-stimulated apoptosis. These 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 (Dale, 2000).
The glucose-dependent insulinotropic polypeptide receptor (GIPR) is a member of class II G protein-coupled receptors. Recent studies have suggested that desensitization of the GIPR might contribute to impaired insulin secretion in type II diabetic patients, but the molecular mechanisms of GIPR signal termination are unknown. Using HEK L293 cells stably transfected with GIPR complementary DNA (L293-GIPR), the mechanisms of GIPR desensitization were investigated. GIP dose dependently increased intracellular cAMP levels in L293-GIPR cells, but this response is abolished (65%) by cotransfection with G protein-coupled receptor kinase 2 (GRK2), but not with GRK5 or GRK6. Beta-arrestin-1 transfection also induces a significantly decrease in GIP-stimulated cAMP production, and this effect is greater with cotransfection of both GRK2 and beta-arrestin-1 than with either alone. In betaTC3 cells, expression of GRK2 or beta-arrestin-1 attenuates GIP-induced insulin release and cAMP production, whereas glucose-stimulated insulin secretion is not affected. GRK2 and beta-arrestin-1 messenger RNAs are expressed endogenously in betaTC3 and L293 cells. Overexpression of GRK2 enhances agonist-induced GIPR phosphorylation, but receptor endocytosis is not affected by cotransfection with GRKs or beta-arrestin-1. These results suggest a potential role for GRK2/beta-arrestin-1 system in modulating GIP-mediated insulin secretion in pancreatic islet cells. Furthermore, GRK-mediated receptor phosphorylation is not required for endocytosis of the GIPR (Tseng, 2000).
The myometrial beta-adrenergic receptor (beta-AR)-adenylyl cyclase pathway is markedly desensitized at the end of pregnancy in the rat. Whether changes in the amount and/or the activity of G protein-coupled receptor kinase (GRK) occurs at the same period of pregnancy was tested. Using Northern and Western blotting, GRK2, GRK5, GRK6, and a small amount of GRK3 were identified in late pregnant rat myometrium. GRK activity, as measured by in vitro phosphorylation of rhodopsin, is detected in both cytosolic and plasma membrane fractions. Interestingly, in the 6-10 h preceding parturition, there is a substantial increase (+190%) of myometrial membrane-associated GRK activity. This is associated with an increase in membrane GRK2 immunoreactivity. Such alterations occur concomitantly with uncoupling of beta-AR, as assessed by quantification of high-affinity binding receptors. These data suggest that GRK activity increase may be one of the mechanisms underlying alterations in the coupling between beta-AR and adenylyl cyclase and may thus contribute to the initiation of myometrial contractions at term (Simon, 2001).
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