P.Penela and A.Elorza contributed equally to this work 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. We recently have described that 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). In this report, we ®nd that b-arrestin function and Src-mediated phosphorylation of GRK2 are critically involved in GRK2 proteolysis. Overexpression of b-arrestin triggers GRK2-K220R degradation based on its ability to recruit c-Src, since this effect is not observed with b-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.

GRK2 is a member of the G protein-coupled receptor kinase (GRK) family, which phosphorylates the activated form of a variety of G protein-coupled receptors (GPCR) and plays an important role in GPCR modulation. It has been recently reported that stimulation of the mitogen-activated protein kinase cascade by GPCRs involves tyrosine phosphorylation of docking proteins mediated by members of the Src tyrosine kinase family. In this report, we have investigated the possible role of c-Src in modulating GRK2 function. We demonstrate that c-Src can directly phosphorylate GRK2 on tyrosine residues, as shown by in vitro experiments with purified proteins. The phosphorylation reaction exhibits an apparent K m for GRK2 of 12 nM, thus suggesting a physiological relevance in living cells. Consistently, overexpression of the constitutively active c-Src Y527F mutant in COS-7 cells leads to tyrosine phosphorylation of coexpressed GRK2. In addition, GRK2 can be detected in phosphotyrosine immunoprecipitates from HEK-293 cells transiently transfected with this Src mutant. Interestingly, phosphotyrosine immunoblots reveal a rapid and transient increase in GRK2 phosphorylation upon agonist stimulation of ␤ 2 -adrenergic receptors co-transfected with GRK2 and wild type c-Src in COS-7 cells. This tyrosine phosphorylation is maximal within 5 min of isoproterenol stimulation and reaches values of ϳ5-fold over basal conditions. Furthermore, GRK2 phosphorylation on tyrosine residues promotes an increased kinase activity toward its substrates. Our results suggest that GRK2 phosphorylation by c-Src is inherent to GPCR activation and put forward a new mechanism for the regulation of GPCR signaling. Activation of G protein-coupled receptors (GPCRs)1 triggers a process termed desensitization, initiated by agonist-dependent phosphorylation of the receptor by specific G protein-coupled receptor kinases (GRKs) (1). This phosphorylation event leads to the recruitment of cytosolic proteins known as ␤-arrestins to the receptor-signaling complex, with the subsequent uncoupling from heterotrimeric G proteins and loss of receptor responsiveness (1-3). GRK2 is a ubiquitous member of the GRK family that has been shown to phosphorylate different GPCRs (4, 5). Recent evidence indicates that GRK2 and ␤-arrestin not only promote receptor uncoupling but also directly participate in GPCR sequestration, thus triggering receptor dephosphorylation and recycling to the plasma membrane (reviewed in Refs. 6 -8). On the other hand, the recently unveiled role of GRK2 in the phosphorylation of other nonreceptor substrates such as tubulin (9, 10) further indicates the relevance of this kinase in GPCR-mediated signaling.Consistent with an important physiological role, GRK2 activity and subcellular localization appear to be subject to complex regulatory processes that involve the interaction of diverse domains of the kinase with G protein ␤␥ subunits, several lipids, anchoring proteins, and the agonist-bound form of receptors. The interaction of GRK2 with ␤␥ subunits thro...

G protein-coupled receptor kinase 2 (GRK2) is a key modulator of G protein-coupled receptors (GPCR). Altered expression of GRK2 has been described to occur during pathological conditions characterized by impaired GPCR signaling. We have reported recently that GRK2 is rapidly degraded by the proteasome pathway and that ␤-arrestin function and Src-mediated phosphorylation are involved in targeting GRK2 for proteolysis. In this report, we show that phosphorylation of GRK2 by MAPK also triggers GRK2 turnover by the proteasome pathway. Modulation of MAPK activation alters the degradation of transfected or endogenous GRK2, and a GRK2 mutant that mimics phosphorylation by MAPK shows an enhanced degradation rate, thus indicating a direct effect of MAPK on GRK2 turnover. Interestingly, MAPK-mediated modulation of wild-type GRK2 stability requires ␤-arrestin function and is facilitated by previous phosphorylation of GRK2 on tyrosine residues by c-Src. Consistent with an important physiological role, interfering with this GRK2 degradation process results in altered GPCR responsiveness. Our data suggest that both c-Src and MAPK-mediated phosphorylation would contribute to modulate GRK2 degradation, and put forward the existence of new feedback mechanisms connecting MAPK cascades and GPCR signaling.G protein-coupled receptors (GPCR) 1 detect a broad spectrum of extracellular signals at the plasma membrane, thereby modulating key cellular functions as diverse as growth, differentiation, inflammation, or neurotransmission (1, 2). Agonistoccupied receptors promote the activation and dissociation of heterotrimeric G proteins into ␣ and ␤␥ subunits, both of which regulate a wide variety of effector systems. In addition, agonist stimulation also leads to the deactivation of GPCR signaling (desensitization) by triggering receptor phosphorylation by specific G protein-coupled receptor kinases (GRKs) and binding of the cytosolic proteins ␤-arrestins to the phosphorylated receptor (3, 4). GRK2 is a ubiquitous member of the GRK family, which has been shown to modulate a variety of GPCRs (5, 6).The same regulatory molecules that contribute to receptor uncoupling from G proteins also regulate GPCR endocytosis, intracellular trafficking, and resensitization and participate in the modulation of mitogen-activated protein kinase (MAPK) cascades by GPCR (7). Thus, ␤-arrestins mediate the recruitment of clathrin and ␤ 2 -adaptin to allow for receptor internalization and also act as scaffold molecules by bridging receptors with signaling proteins such as c-Src, thus facilitating the activation of the ERK/MAPK cascade by GPCR (8, 9). In addition, the isoforms of ␤-arrestins can directly interact with components of two different MAPK cascades bringing these molecules into close proximity with the receptor complex (10, 11). On the other hand, GRK2 has been shown to interact with the ARF modulator GIT, with phosphatidylinositol 3Ј-OH kinase ␥, and the G protein ␣ q subunit (12) and to phosphorylate nonreceptor substrates such as tubulin, synucleins...

A variety of G protein-coupled receptors (GPCRs) are phosphorylated by G protein-coupled receptor kinase 2 (GRK2). This event promotes the binding of regulatory proteins termed beta-arrestins to GPCRs, leading to uncoupling from G proteins and receptor internalization. Recent data indicate that GRK2 and beta-arrestins also play an important role in the stimulation of the extracellular signal-regulated kinases (ERK)/mitogen-activated protein kinase (MAPK) cascade by GPCRs. In this report, we have investigated the existence of functional interactions between GRK2 and MAPK. We show that activation of beta(2)-adrenergic receptors (beta(2)-AR) promotes the rapid association of GRK2 and MAPK in living cells, as assessed by coimmunoprecipitation experiments in COS-7 cells transfected with beta(2)-AR, GRK2, and an epitope-tagged MAPK. Coimmunoprecipitation of MAPK and GRK2 is blocked by inhibition of the MAPK cascade and is not observed upon activation of MAPK in the absence of beta(2)-AR stimulation, thus indicating that both an active MAPK and agonist occupancy of GPCR are required for the association to occur. Interestingly, we have found that purified ERK1/MAPK can directly phosphorylate the C-terminal domain of GRK2, and that the phosphorylation process is favored by the presence of Gbetagamma-subunits or an activated receptor. Furthermore, GRK2 phosphorylation by MAPK leads to a decreased activity of GRK2 toward GPCR. Taken together, our results suggest that stimulation of GPCRs promotes the rapid association of GRK2 and MAPK leading to modulation of GRK2 functionality, thus putting forward a new feedback mechanism for the regulation of GPCR signaling.

G protein-coupled receptor kinase 2 (GRK2) plays a key role in determining the rate and extent of G protein-coupled receptor (GPCR) desensitization and resensitization. Recent data indicate that GRK2 activity, subcellular distribution and expression are tightly regulated. The important physiological function of GRK2 as a modulator of the efficacy of GPCR signal transduction systems is exemplified by its relevance in cardiovascular physiopathology as well as by its emerging role in the regulation of chemokine receptors.z 1998 Federation of European Biochemical Societies.Key words: G protein-coupled receptor kinase; Desensitization; Cardiovascular; Chemokine receptorThe cellular responses to agonists acting through G proteincoupled receptors (GPCRs) are usually rapidly attenuated, a process that is critical to prevent the uncontrolled stimulation of cells. Mechanisms of attenuation include removal of agonist from the extracellular £uid and receptor desensitization, endocytosis and down-regulation. Receptor phosphorylation by speci¢c G protein-coupled receptor kinases (GRKs) plays a key role in triggering rapid desensitization [1^5]. The GRKmediated phosphorylation of the agonist-occupied receptor promotes the binding of a member of the family of uncoupling proteins termed L-arrestins, resulting in the uncoupling of the receptor from G proteins.There are six known members of the GRK family, GRK1 to GRK6, which share a number of structural and functional similarities [2,4,6,7] and di¡er in both an N-terminal domain of largely unknown function, and a C-terminal domain of variable length, that contains speci¢c determinants for membrane attachment. GRK2 is a ubiquitous member of the GRK family which has been shown to phosphorylate a variety of GPCRs (see [3] for a recent review). Recent evidence indicates that, besides promoting receptor uncoupling, GRK2 would also directly participate in GPCR sequestration, thus triggering receptor dephosphorylation and recycling [5,8^10]. Moreover, GRK2 and L-arrestin have been suggested to participate in the regulation of the mitogen-activated protein kinase cascade by GPCR [11]. This fact, together with the recently reported phosphorylation of tubulin by GRK2 [12], further stresses the relevance of this kinase in GPCR signaling. This review focuses on the mechanisms of regulation of GRK2 activity and subcellular distribution, as well as on some physiological processes in which an important participation of this kinase has recently been unveiled. Modulation of GRK2 subcellular distribution and activityGRK2 activity and subcellular localization appear to be subject to complex regulatory processes. Although GRK2 was initially described as a soluble, cytosolic enzyme that transiently translocates to the plasma membrane upon receptor activation, recent data indicate that several GRK2 pools exist inside the cells: cytosolic, plasma membrane-bound and microsomal membrane-bound [11,13^15]. Interestingly, GRK2 activity is regulated upon interaction with several proteins and lipids ...

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The G protein-coupled receptor (GPCR) kinase GRK2 phosphorylates G protein-coupled receptors in an agonist-dependent manner. GRK2 activity is modulated through interactions of diverse domains of the kinase with G protein ␤␥ subunits, several lipids, anchoring proteins, and activated receptors. We report that kinase activity toward either GPCR (rhodopsin) or a synthetic peptide substrate is enhanced in the presence of GST-GRK2 fusion proteins or peptides corresponding to either N-or C-terminal sequences of GRK2. This direct stimulatory action of intrinsic domains on GRK2 activity does not add to the effect of other regulators, such as G␤␥ subunits, and strongly suggests the existence of some mode of autoregulation. The existence of regulatory intramolecular interactions in GRK2 is supported by the facts that a C-terminal peptide protects the N-terminal region from proteolytic cleavage and that two domains of GRK2 independently coexpressed in cells associate as assessed by immunoprecipitation. Molecular modeling suggests that intramolecular interactions among the N-terminal, C-terminal and kinase domains would keep GRK2 in a constrained conformation characteristic of an inactive, basal state. Our model proposes that disruption of such intramolecular contacts by intermolecular interactions with regulatory proteins (mimicked by exogenously added kinase fragments in vitro) would promote the conformational changes required to bring about GRK2 translocation and activation.