Accumulating evidence suggests that receptor protein-tyrosine kinases, like the platelet-derived growth factor receptor-␤ (PDGFR␤) 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 agonistdependent phosphorylation and desensitization of multiple heptahelical receptors. In testing whether GRK2 could phosphorylate and desensitize the PDGFR␤, we first found by phosphoamino acid analysis that cells expressing GRK2 could serine-phosphorylate the PDGFR␤ in an agonist-dependent manner. Augmentation or inhibition of GRK2 activity in cells, respectively, reduced or enhanced tyrosine phosphorylation of the PDGFR␤ but not the EGFR. Either overexpressed in cells or as a purified protein, GRK2 demonstrated agonist-promoted serine phosphorylation of the PDGFR␤ and, unexpectedly, the EGFR as well. Because GRK2 did not phosphorylate a kinase-dead (K634R) PDGFR␤ mutant, GRK2-mediated PDGFR␤ phosphorylation required receptor tyrosine kinase activity, as does PDGFR␤ ubiquitination. Agonist-induced ubiquitination of the PDGFR␤, but not the EGFR, was enhanced in cells overexpressing GRK2. Nevertheless, GRK2 overexpression did not augment PDGFR␤ down-regulation. Like the vast majority of GRK2 substrates, the PDGFR␤, 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 PDGFR␤, 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.

Toll-like receptor 4 (TLR4) promotes vascular inflammatory disorders such as neointimal hyperplasia and atherosclerosis. TLR4 triggers NFB signaling through the ubiquitin ligase TRAF6 (tumor necrosis factor receptor-associated factor 6). TRAF6 activity can be impeded by deubiquitinating enzymes like ubiquitin-specific protease 20 (USP20), which can reverse TRAF6 autoubiquitination, and by association with the multifunctional adaptor protein ␤-arrestin2. Although ␤-arrestin2 effects on TRAF6 suggest an anti-inflammatory role, physiologic ␤-arrestin2 promotes inflammation in atherosclerosis and neointimal hyperplasia. We hypothesized that anti-and proinflammatory dimensions of ␤-arrestin2 activity could be dictated by ␤-arrestin2's ubiquitination status, which has been linked with its ability to scaffold and localize activated ERK1/2 to signalosomes. With purified proteins and in intact cells, our protein interaction studies showed that TRAF6/USP20 association and subsequent USP20-mediated TRAF6 deubiquitination were ␤-arrestin2-dependent. Generation of transgenic mice with smooth muscle cell-specific expression of either USP20 or its catalytically inactive mutant revealed anti-inflammatory effects of USP20 in vivo and in vitro. Carotid endothelial denudation showed that antagonizing smooth muscle cell USP20 activity increased NFB activation and neointimal hyperplasia. We found that ␤-arrestin2 ubiquitination was promoted by TLR4 and reversed by USP20. The association of USP20 with ␤-arrestin2 was augmented when ␤-arrestin2 ubiquitination was prevented and reduced when ␤-arrestin2 ubiquitination was rendered constitutive. Constitutive ␤-arrestin2 ubiquitination also augmented NFB activation. We infer that pro-and anti-inflammatory activities of ␤-arrestin2 are determined by ␤-arrestin2 ubiquitination and that changes in USP20 expression and/or activity can therefore regulate inflammatory responses, at least in part, by defining the ubiquitination status of ␤-arrestin2.␤-Arrestin2 (␤arr2) is an ϳ46-kDa multifunctional scaffolding protein that was discovered originally for its ability to desensitize G protein-mediated signaling evoked by seventransmembrane receptors (7TMRs) 4 (1, 2). However, ␤arr2 modulates the signaling and/or endocytosis of not only most 7TMRs but also of several receptor protein tyrosine kinases, cytokine receptors, ion channel receptors, and the LDL receptor (3, 4). Both the endocytic and signaling functions of ␤arr2 are intertwined with its ubiquitination, which in turn is stimulus-driven and regulated by specific E3 ubiquitin ligases or deubiquitinases (DUBs) (4). ␤arr2 not only undergoes dynamic ubiquitination/deubiquitination but also recruits E3 ubiquitin ligases to other substrates. Indeed, ␤arr2 is integral to the ubiquitination of cell surface receptors, channels, and non-receptor proteins (4, 5). However, thus far there is no clear demonstration that ␤arr2 can scaffold a DUB to specific substrates and affect signal transduction by mediating deubiquitination.A role in deubiquitination c...

Objective G protein-coupled receptor kinase-5 (GRK5) is a widely expressed Ser/Thr kinase that regulates several atherogenic receptors, and may activate or inhibit NFκB. This study sought to determine whether and by what mechanisms GRK5 affects atherosclerosis. Methods and Results Grk5−/−/Apoe−/− mice developed 50% greater aortic atherosclerosis than Apoe−/− mice, and demonstrated greater proliferation of macrophages and smooth muscle cells (SMCs) in atherosclerotic lesions. In Apoe−/− mice, carotid interposition grafts from Grk5−/− mice demonstrated greater up-regulation of cell adhesion molecules than grafts from WT mice, and subsequently more atherosclerosis. By comparing Grk5−/− with WT cells, we found that GRK5 desensitized two key atherogenic receptor tyrosine kinases: the PDGF receptor-β (PDGFRβ) in SMCs, by augmenting ubiquitination/degradation; the colony stimulating factor-1 receptor (CSF-1R) in macrophages, by reducing CSF-1-induced tyrosyl phosphorylation. GRK5 activity in monocytes also reduced migration promoted by the 7-transmembrane receptor for MCP-1 (CCR2). Whereas GRK5 diminished NFκB-dependent gene expression in SMCs and endothelial cells, it had no effect on NFκB activity in macrophages. Conclusions GRK5 attenuates atherosclerosis through multiple cell type-specific mechanisms, including reduction of SMC and endothelial cell NFκB activity, and desensitization of receptor-specific signaling through the monocyte CCR2, macrophage CSF-1R, and the SMC PDGFRβ.

G protein-coupled receptor kinase-2 (GRK2) can phosphorylate and desensitize the platelet-derived growth factor receptor-␤ (PDGFR␤) in heterologous cellular systems. To determine whether GRK2 regulates the PDGFR␤ in physiologic systems, we examined PDGFR␤ signaling in mouse embryonic fibroblasts from GRK2-null and cognate wild type mice. To discern a mechanism by which GRK2-mediated phosphorylation can desensitize the PDGFR␤, but not the epidermal growth factor receptor (EGFR), we investigated effects of GRK2-mediated phosphorylation on the association of the PDGFR␤ with the Na ؉ /H ؉ exchanger regulatory factor (NHERF), a protein shown to potentiate dimerization of the PDGFR␤, but not the EGFR. Physiologic expression of GRK2 diminished (a) phosphoinositide hydrolysis elicited through the PDGFR␤ but not heterotrimeric G proteins; (b) Akt activation evoked by the PDGFR␤ but not the EGFR; and (c) PDGF-induced tyrosyl phosphorylation of the PDGFR␤ itself. PDGFR␤ desensitization by physiologically expressed GRK2 correlated with a 2.5-fold increase in PDGF-promoted PDGFR␤ seryl phosphorylation. In 293 cells, GRK2 overexpression reduced PDGFR␤/ NHERF association by 60%. This effect was reproduced by S1104D mutation of the PDGFR␤, which also diminished PDGFR␤ activation and signaling (like the S1104A mutation) to an extent equivalent to that achieved by GRK2-mediated PDGFR␤ phosphorylation. GRK2 overexpression desensitized only the wild type but not the S1104A PDGFR␤. We conclude that GRK2-mediated PDGFR␤ seryl phosphorylation plays an important role in desensitizing the PDGFR␤ in physiologic systems. Furthermore, this desensitization appears to involve GRK2-mediated phosphorylation of PDGFR␤ Ser 1104

Lysosomal degradation of ubiquitinated β2-adrenergic receptors (β2ARs) serves as a major mechanism of long-term desensitization in response to prolonged agonist stimulation. Surprisingly, the βAR antagonist carvedilol also induced ubiquitination and lysosomal trafficking of both endogenously expressed β2ARs in vascular smooth muscle cells (VSMCs) and overexpressed Flag-β2ARs in HEK-293 cells. Carvedilol prevented β2AR recycling, blocked recruitment of Nedd4 E3 ligase, and promoted the dissociation of the deubiquitinases USP20 and USP33. Using proteomics approaches (liquid chromatography–tandem mass spectrometry), we identified that the E3 ligase MARCH2 interacted with carvedilol-bound β2AR. The association of MARCH2 with internalized β2ARs was stabilized by carvedilol and did not involve β-arrestin. Small interfering RNA–mediated down-regulation of MARCH2 ablated carvedilol-induced ubiquitination, endocytosis, and degradation of endogenous β2ARs in VSMCs. These findings strongly suggest that specific ligands recruit distinct E3 ligase machineries to activated cell surface receptors and direct their intracellular itinerary. In response to β blocker therapy with carvedilol, MARCH2 E3 ligase activity regulates cell surface β2AR expression and, consequently, its signaling.

Smooth muscle cell (SMC) proliferation and migration are substantially controlled by the platelet-derived growth factor receptor-␤ (PDGFR␤), which can be regulated by the Ser/Thr kinase G protein-coupled receptor kinase-2 (GRK2). In mouse aortic SMCs, however, we found that prolonged PDGFR␤ activation engendered down-regulation of GRK5, but not GRK2; moreover, GRK5 and PDGFR␤ were coordinately up-regulated in SMCs from atherosclerotic arteries. With SMCs from GRK5 knock-out and cognate wild type mice (five of each), we found that physiologic expression of GRK5 increased PDGF-promoted PDGFR␤ seryl phosphorylation by 3-fold and reduced PDGFR␤-promoted phosphoinositide hydrolysis, thymidine incorporation, and overall PDGFR␤ tyrosyl phosphorylation by ϳ35%. Physiologic SMC GRK5 activity also increased PDGFR␤ association with the phosphatase Shp2 (8-fold), enhanced phosphorylation of PDGFR␤ Tyr 1009 (the docking site for Shp2), and reduced phosphorylation of PDGFR␤ Tyr 1021. Consistent with having increased PDGFR␤-associated Shp2 activity, GRK5-expressing SMCs demonstrated greater PDGF-induced Src activation than GRK5-null cells. GRK5-mediated desensitization of PDGFR␤ inositol phosphate signaling was diminished by Shp2 knock-down or impairment of PDGFR␤/Shp2 association. In contrast to GRK5, physiologic GRK2 activity did not alter PDGFR␤/Shp2 association. Finally, purified GRK5 effected agonist-dependent seryl phosphorylation of partially purified PDGFR␤s. We conclude that GRK5 mediates the preponderance of PDGF-promoted seryl phosphorylation of the PDGFR␤ in SMCs, and, through mechanisms involving Shp2, desensitizes PDGFR␤ inositol phosphate signaling and enhances PDGFR␤-triggered Src activation.

Objective Kalirin is a multifunctional protein that contains two guanine nucleotide exchange factor (GEF) domains for the GTPases Rac1 and RhoA. Variants of KALRN have been associated with atherosclerosis in humans, but Kalirin’s activity has been characterized almost exclusively in the CNS. We therefore tested the hypothesis that Kalirin functions as a RhoGEF in arterial smooth muscle cells (SMCs). Methods and Results Kalirin-9 protein is expressed abundantly in aorta and bone marrow, as well as in cultured SMCs, endothelial cells, and macrophages. Moreover, arterial Kalirin was up-regulated during early atherogenesis in apolipoprotein E-deficient mice. In cultured SMCs, signaling was affected similarly in three models of Kalirin loss-of-function: heterozygous Kalrn deletion, Kalirin RNAi, and treatment with the Kalirin RhoGEF-1 inhibitor 1-(3-nitrophenyl)-1H-pyrrole-2,5-dione. With reduced Kalirin function, SMCs showed normal RhoA activation but diminished Rac1 activation, assessed as reduced Rac-GTP levels, p21-activated kinase autophosphorylation, and SMC migration. Kalrn−/+ SMCs proliferated 30% less rapidly than WT SMCs. Neointimal hyperplasia engendered by carotid endothelial denudation was ~60% less in Kalrn−/+ and SMC-specific Kalrn−/+ mice than in control mice. Conclusions Kalirin functions as a GEF for Rac1 in SMCs, and promotes SMC migration and proliferation both in vitro and in vivo.

G protein-coupled receptor kinase-2 (GRK2) serinephosphorylates the platelet-derived growth factor receptor-␤ (PDGFR␤), and thereby diminishes signaling by the receptor. Because activation of GRK2 may involve phosphorylation of its N-terminal tyrosines by cSrc, we tested whether the PDGFR␤ itself could tyrosine-phosphorylate and activate GRK2. To do so, we used wild type (WT) and Y857F mutant PDGFR␤s in HEK cells, which lack endogenous PDGFRs. The Y857F PDGFR␤ autophosphorylates normally but does not phosphorylate exogenous substrates. Although PDGFstimulated Y857F and WT PDGFR␤s activated c-Src equivalently, the WT PDGFR␤ tyrosine-phosphorylated GKR2 60-fold more than the Y857F PDGFR␤ in intact cells. With purified GRK2 and either WT or Y857F PDGFR␤s immunoprecipitated from HEK cells, GRK2 tyrosyl phosphorylation was PDGF-dependent and required the WT PDGFR␤, even though the WT and Y857F PDGFR␤s autophosphorylated equivalently. This PDGFR␤-mediated GRK2 tyrosyl phosphorylation enhanced GRK2 activity: GRK2-mediated seryl phosphorylation of the PDGFR␤ was 9-fold greater for the WT than for the Y857F in response to PDGF, but equivalent when GRK2 was activated by sequential stimulation of ␤ 2 -adrenergic and PDGF-␤ receptors. Furthermore, both PDGFR␤-mediated GRK2 tyrosyl phosphorylation and GRK2-mediated PDGFR␤ seryl phosphorylation were reduced ϳ50% in intact cells by mutation to phenylalanine of three tyrosines in the N-terminal domain of GRK2. We conclude that the activated PDGFR␤ itself phosphorylates GRK2 tyrosyl residues and thereby activates GRK2, which then serine-phosphorylates and desensitizes the PDGFR␤.As a receptor protein-tyrosine kinase, the platelet-derived growth factor receptor-␤ (PDGFR␤) 1 triggers cellular proliferation, migration, and survival (1) but also contributes to atherosclerosis (2-4) and malignant neoplasia (5, 6). Agonist-induced dimerization of the PDGFR␤ enables receptor activation consequent to autophosphorylation (7), followed by recruitment to the PDGFR␤ of various signaling proteins, and tyrosyl phosphorylation of PDGFR␤ substrates (1). In order for the PDGFR␤ to phosphorylate these "exogenous" substrates, however, the PDGFR␤ must be autophosphorylated on Tyr 857 , located in the PDGFR␤ kinase activation loop (8).Regulatory constraints on PDGFR␤ signaling include tyrosyl dephosphorylation (9, 10), degradation and down-regulation of cellular PDGFR␤s (11, 12), and agonist-induced phosphorylation of the PDGFR␤ on serine residues (13-15). In fibroblasts, the preponderance of this PDGFR␤ seryl phosphorylation appears to be mediated by GRK2 (13), a ubiquitous allosteric kinase that also phosphorylates activated G protein-coupled (heptahelical) receptors and thereby initiates their desensitization (16). We have demonstrated GRK2-mediated PDGFR␤ seryl phosphorylation with purified kinase preparations (14), as well as by comparing PDGFR␤ seryl phosphorylation in GRK2-null, cognate WT, and GRK2 "add-back" fibroblasts (13). GRK2-mediated PDGFR␤ phosphorylation diminishes PDGFR␤ tyrosyl ph...