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

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

Agonist-dependent phosphorylation of the G protein-coupled receptor kinase 2 (GRK2) by Src tyrosine kinase

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 co-expressed 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 beta(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 approximately 5-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.     

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

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

MAPK-dependent degradation of G protein-coupled receptor kinase 2

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 beta-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 beta-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.     

Molecular mechanisms involved in epidermal growth factor receptor-mediated inhibition of dopamine D3 receptor signaling

The phenomenon wherein the signaling by a given receptor is regulated by a different class of receptors is termed transactivation or crosstalk. Crosstalk between receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs) is highly diverse and has unique functional implications because of the distinct structural features of the receptors and the signaling pathways involved. The present study used the epidermal growth factor receptor (EGFR) and dopamine D₃ receptor (D₃R), which are both associated with schizophrenia, as the model system to study crosstalk between RTKs and GPCRs. Loss-of-function approaches were used to identify the cellular components involved in the tyrosine phosphorylation of G protein-coupled receptor kinase 2 (GRK2), which is responsible for EGFR-induced regulation of the functions of D₃R. SRC proto-oncogene (Src, non-receptor tyrosine kinase), heterotrimeric G protein Gβγ subunit, and endocytosis of EGFR were involved in the tyrosine phosphorylation of GRK2. In response to EGF treatment, Src interacted with EGFR in a Gβγ-dependent manner, resulting in the endocytosis of EGFR. Internalized EGFR in the cytosol mediated Src/Gβγ-dependent tyrosine phosphorylation of GRK2. The binding of tyrosine-phosphorylated GRK2 to the T142 residue of D₃R resulted in uncoupling from G proteins, endocytosis, and lysosomal downregulation. This study identified the molecular mechanisms involved in the EGFR-mediated regulation of the functions of D₃R, which can be extended to the crosstalk between other RTKs and GPCRs.     

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

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

Tyrosine phosphorylation of G-protein-coupled-receptor kinase 2 (GRK2) by c-Src modulates its interaction with Galphaq

G-protein-coupled-receptor kinase 2 (GRK2) plays a key role in the modulation of G-protein-coupled-receptor (GPCR) signaling by both phosphorylating agonist-occupied GPCRs and by directly binding to activated Galphaq subunits, inhibiting downstream effectors activation. The GRK2/Galphaq interaction involves the N-terminal region of the kinase that displays homology to regulators of G-protein signaling (RGS) proteins. We have previously reported that upon GPCR stimulation, GRK2 can be phosphorylated by c-Src on tyrosine residues that are present in the RGS-homology (RH) region of this kinase. Here, we demonstrate that c-Src kinase activity increases the interaction between GRK2 and Galphaq. Tyrosine phosphorylation of GRK2 appears to be critically involved in the modulation of this interaction since the stimulatory effect of c-Src is not observed with a GRK2 mutant with impaired tyrosine phosphorylation (GRK2 Y13,86,92F), whereas a mutant that mimics GRK2 tyrosine phosphorylation in these residues displays an increased interaction with Galphaq. As evidence for a physiological role of this modulatory mechanism, activation of the muscarinic receptor M1, a Galphaq-coupled receptor, promotes an increase in GRK2/Galphaq co-immunoprecipitation that parallels the enhanced GRK2 phosphorylation on tyrosine residues. Moreover, c-Src activation enhances inhibition of the Galphaq/phospholipase Cbeta signaling pathway in intact cells, in a GRK2-tyrosine-phosphorylation-dependent manner. Our results suggest a feedback mechanism by which phosphorylation of GRK2 by c-Src increases both GRK2 kinase activity towards GPCRs and its specific interaction with Galphaq subunits, leading to a more rapid switch off of Galphaq-mediated signaling.     

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

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

Activation of tyrosine kinase of EGFR induces Gbetagamma-dependent GRK-EGFR complex formation

This study demonstrated that activation of tyrosine kinase of epidermal growth factor receptor (EGFR) induces its association with G protein-coupled receptor kinase 2 (GRK2). Immunoprecipitation experiments showed that EGF stimulation increased GRK2 binding to EGFR complex in HEK293 cells coexpressing EGFR and GRK2. The EGF-induced GRK2-EGFR complex formation was greatly reduced by perturbation of EGFR and Src tyrosine kinase activity. Furthermore, studies with GRK2 mutants showed that neither catalytic activity nor the N-terminal domain of GRK2 was required for EGF-induced GRK2-EGFR complex formation. However, overexpression of Gbetagamma scavengers blocked EGF-induced formation of GRK2-EGFR complex.     

c-Src trafficking and co-localization with the EGF receptor promotes EGF ligand-independent EGF receptor activation and signaling

c-Src is a non-receptor tyrosine kinase that associates with both the plasma membrane and endosomal compartments. In many human cancers, especially breast cancer, c-Src and the EGF receptor (EGFR) are overexpressed. Dual overexpression of c-Src and EGFR correlates with a Src-dependent increase in activation of EGFR, and synergism between these two tyrosine kinases increases the mitogenic activity of EGFR. Despite extensive studies of the functional interaction between c-Src and EGFR, little is known about the interactions in the trafficking pathways for the two proteins and how that influences signaling. Given the synergism between c-Src and EGFR, and the finding that EGFR is internalized and can signal from endosomes, we hypothesized that c-Src and EGFR traffic together through the endocytic pathway. Here we use a regulatable c-SrcGFP fusion protein that is a bona fide marker for c-Src to show that c-Src undergoes constitutive macropinocytosis from the plasma membrane into endocytic compartments. The movement of c-Src was dependent on its tyrosine kinase activity. Stimulation of cells with EGF revealed that c-Src traffics into the cell with activated EGFR and that c-Src expression and kinase activity prolongs EGFR activation. Surprisingly, even in the absence of EGF addition, c-Src expression induced activation of EGFR and of EGFR-mediated downstream signaling targets ERK and Shc. These data suggest that the synergy between c-Src and EGFR also occurs as these two kinases traffic together, and that their co-localization promotes EGFR-mediated signaling.     

Regulation of epidermal growth factor receptor internalization by G protein-coupled receptors

The epidermal growth factor (EGF) receptor (EGFR) plays a central role in regulating cell proliferation, differentiation, and migration. Cellular responses to EGF are dependent upon the amount of EGFR present on the cell surface. Stimulation with EGF induces sequestration of the receptor from the plasma membrane and its subsequent downregulation. Recently, internalization of the EGFR was also shown to be required for mitogenic signaling via the activation of MAP kinases. Therefore, mechanisms regulating internalization of the EGFR represent an important facet for the control of cellular response. Here, we demonstrate that EGFR is removed from the cell surface not only following stimulation with EGF, but also in response to stimulation of G protein-coupled lysophosphatidic acid (LPA) and beta2 adrenergic (beta2AR) receptors. Using a FLAG epitope-tagged EGFR to quantitate receptor internalization, we show that incubation with EGF, LPA, or isoproterenol (ISO) causes the time-dependent loss of cell surface EGFR. Internalization of EGFR by these ligands involves the tyrosine kinase activity of the receptor itself and c-Src, as well as the GTPase activity of dynamin. Unexpectedly, we find that internalization of the EGFR by EGF is dependent upon Gbetagamma and beta-arrestin proteins; expression of minigenes encoding the carboxyl terminii of the G protein-coupled receptor kinase 2, or beta-arrestin1, attenuates LPA-, ISO-, and EGF-mediated internalization of EGFR. Thus, G protein-coupled receptors can control the function of the EGFR by regulating its endocytosis.     

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

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

Phosphorylation of the platelet-derived growth factor receptor-beta and epidermal growth factor receptor by G protein-coupled receptor kinase-2. Mechanisms for selectivity of desensitization

Accumulating evidence suggests that receptor protein-tyrosine kinases, like the platelet-derived growth factor receptor-beta (PDGFRbeta) and epidermal growth factor receptor (EGFR), may be desensitized by serine/threonine kinases. One such kinase, G protein-coupled receptor kinase-2 (GRK2), is known to mediate agonist-dependent phosphorylation and desensitization of multiple heptahelical receptors. In testing whether GRK2 could phosphorylate and desensitize the PDGFRbeta, we first found by phosphoamino acid analysis that cells expressing GRK2 could serine-phosphorylate the PDGFRbeta in an agonist-dependent manner. Augmentation or inhibition of GRK2 activity in cells, respectively, reduced or enhanced tyrosine phosphorylation of the PDGFRbeta but not the EGFR. Either overexpressed in cells or as a purified protein, GRK2 demonstrated agonist-promoted serine phosphorylation of the PDGFRbeta and, unexpectedly, the EGFR as well. Because GRK2 did not phosphorylate a kinase-dead (K634R) PDGFRbeta mutant, GRK2-mediated PDGFRbeta phosphorylation required receptor tyrosine kinase activity, as does PDGFRbeta ubiquitination. Agonist-induced ubiquitination of the PDGFRbeta, but not the EGFR, was enhanced in cells overexpressing GRK2. Nevertheless, GRK2 overexpression did not augment PDGFRbeta down-regulation. Like the vast majority of GRK2 substrates, the PDGFRbeta, but not the EGFR, activated heterotrimeric G proteins allosterically in membranes from cells expressing physiologic protein levels. We conclude that GRK2 can phosphorylate and desensitize the PDGFRbeta, perhaps through mechanisms related to receptor ubiquitination. Specificity of GRK2 for receptor protein-tyrosine kinases, expressed at physiologic levels, may be determined by the ability of these receptors to activate heterotrimeric G proteins, among other factors.     

Beta-arrestin- and c-Src-dependent degradation of G-protein-coupled receptor kinase 2

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 find that beta-arrestin function and Src-mediated phosphorylation of GRK2 are critically involved in GRK2 proteolysis. Overexpression of beta-arrestin triggers GRK2-K220R degradation based on its ability to recruit c-Src, since this effect is not observed with beta-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.     

Dissociation of focal adhesion kinase and paxillin tyrosine phosphorylation induced by bombesin and lysophosphatidic acid from epidermal growth factor receptor transactivation in Swiss 3T3 cells

Tyrosine phosphorylation of the nonreceptor tyrosine kinase p125 focal adhesion kinase (FAK) and the adapter protein paxillin is rapidly increased by multiple agonists, including bombesin (BOM) and lysophosphatidic acid (LPA), through heptahelical G protein-coupled receptors (GPCRs). The pathways involved remain incompletely understood. The experiments presented here were designed to test the role of epidermal growth factor receptor (EGFR) transactivation in the rapid increase of tyrosine phosphorylation of FAK and paxillin induced by GPCR agonists. Our results show that treatment with the selective EGFR tyrosine kinase inhibitor AG 1478, at concentrations that completely blocked the increase in tyrosine phosphorylation of these proteins induced by EGF, did not affect the stimulation of tyrosine phosphorylation of either FAK or paxillin induced by multiple GPCR agonists including LPA, BOM, vasopressin, bradykinin, and endothelin. Similar results were obtained when Swiss 3T3 cells were treated with another highly specific inhibitor of the EGF receptor kinase activity, PD-158780. Collectively, our results clearly dissociate EGFR transactivation from the tyrosine phosphorylation of FAK and paxillin induced by multiple GPCR agonists.     

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

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

Multiple scaffolding functions of {beta}-arrestins in the degradation of G protein-coupled receptor kinase 2

G protein-coupled receptor kinase 2 (GRK2) plays a fundamental role in the regulation of G protein-coupled receptors (GPCRs), and changes in GRK2 expression levels can have an important impact on cell functions. GRK2 is known to be degraded by the proteasome pathway. We have shown previously that β-arrestins participate in enhanced kinase turnover upon GPCR stimulation by facilitating GRK2 phosphorylation by c-Src or by MAPK or by recruiting the Mdm2 E3 ubiquitin ligase to the receptor complex. In this report, we have investigated how such diverse β-arrestin scaffold functions are integrated to modulate GRK2 degradation. Interestingly, we found that in the absence of GPCR activation, β-arrestins do not perform an adaptor role for GRK2/Mdm2 association, but rather compete with GRK2 for direct Mdm2 binding to regulate basal kinase turnover. Upon agonist stimulation, β-arrestins-mediated phosphorylation of GRK2 at serine 670 by MAPK facilitates Mdm2-mediated GRK2 degradation, whereas c-Src-dependent phosphorylation would support the action of an undetermined β-arrestin-recruited ligase in the absence of GPCR activation. The ability of β-arrestins to play different scaffold functions would allow coordination of both Mdm2-dependent and -independent processes aimed at the specific modulation of GRK2 turnover in different signaling contexts.     

Signal characteristics of G protein-transactivated EGF receptor.

The epidermal growth factor receptor (EGFR) tyrosine kinase recently was identified as providing a link to mitogen-activated protein kinase (MAPK) in response to G protein-coupled receptor (GPCR) agonists in Rat-1 fibroblasts. This cross-talk pathway is also established in other cell types such as HaCaT keratinocytes, primary mouse astrocytes and COS-7 cells. Transient expression of either Gq- or Gi-coupled receptors in COS-7 cells allowed GPCR agonist-induced EGFR transactivation, and lysophosphatidic acid (LPA)-generated signals involved the docking protein Gab1. The increase in SHC tyrosine phosphorylation and MAPK stimulation through both Gq- and Gi-coupled receptors was reduced strongly upon selective inhibition of EGFR function. Inhibition of phosphoinositide 3-kinase did not affect GPCR-induced stimulation of EGFR tyrosine phosphorylation, but inhibited MAPK stimulation, upon treatment with both GPCR agonists and low doses of EGF. Furthermore, the Src tyrosine kinase inhibitor PP1 strongly interfered with LPA- and EGF-induced tyrosine phosphorylation and MAPK activation downstream of EGFR. Our results demonstrate an essential role for EGFR function in signaling through both Gq- and Gi-coupled receptors and provide novel insights into signal transmission downstream of EGFR for efficient activation of the Ras/MAPK pathway.     

Reactive oxygen species mediate Met receptor transactivation by G protein-coupled receptors and the epidermal growth factor receptor in human carcinoma cells

Cross-communication between the Met receptor tyrosine kinase and the epidermal growth factor receptor (EGFR) has been proposed to involve direct association of both receptors and EGFR kinase-dependent phosphorylation. Here, we demonstrate that in human hepatocellular and pancreatic carcinoma cells the Met receptor becomes tyrosine phosphorylated not only upon EGF stimulation but also in response to G protein-coupled receptor (GPCR) agonists. Whereas specific inhibition of the EGFR kinase activity blocked EGF- but not GPCR agonist-induced Met receptor transactivation, it was abrogated in the presence of a reducing agent or treatment of cells with a NADPH oxidase inhibitor. Both GPCR ligands and EGF are further shown to increase the level of reactive oxygen species within the cell. Interestingly, stimulation of the Met receptor by either GPCR agonists, EGF or its cognate ligand HGF, resulted in release of Met-associated beta-catenin and in its Met-dependent translocation into the nucleus, as analyzed by small interfering RNA-mediated knockdown of the Met receptor. Our results provide a new molecular explanation for cell surface receptor cross-talk involving the Met receptor and thereby link the wide diversity of GPCRs and the EGFR to the oncogenic potential of Met signaling in human carcinoma cells.     

G protein-coupled receptor kinase 2-mediated phosphorylation of ezrin is required for G protein-coupled receptor-dependent reorganization of the actin cytoskeleton

G protein-coupled receptor kinase 2 (GRK2) phosphorylates and desensitizes activated G protein-coupled receptors (GPCRs). Here, we identify ezrin as a novel non-GPCR substrate of GRK2. GRK2 phosphorylates glutathione S-transferase (GST)-ezrin, but not an ezrin fusion protein lacking threonine 567 (T567), in vitro. These results suggest that T567, the regulatory phosphorylation site responsible for maintaining ezrin in its active conformation, represents the principle site of GRK2-mediated phosphorylation. Two lines of evidence indicate that GRK2-mediated ezrin-radixinmoesin (ERM) phosphorylation serves to link GPCR activation to cytoskeletal reorganization. First, in Hep2 cells muscarinic M1 receptor (M1MR) activation causes membrane ruffling. This ruffling response is ERM dependent and is accompanied by ERM phosphorylation. Inhibition of GRK2, but not rho kinase or protein kinase C, prevents ERM phosphorylation and membrane ruffling. Second, agonist-induced internalization of the beta2-adrenergic receptor (beta2AR) and M1MR is accompanied by ERM phosphorylation and localization of phosphorylated ERM to receptor-containing endocytic vesicles. The colocalization of internalized beta2AR and phosphorylated ERM is not dependent on Na+/H+ exchanger regulatory factor binding to the beta2AR. Inhibition of ezrin function impedes beta2AR internalization, further linking GPCR activation, GRK activity, and ezrin function. Overall, our results suggest that GRK2 serves not only to attenuate but also to transduce GPCR-mediated signals.