Jiao-Hui Wu

Phosphorylation of the Platelet-derived Growth Factor Receptor-β 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-β (PDGFRβ) 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 PDGFRβ, we first found by phosphoamino acid analysis that cells expressing GRK2 could serine-phosphorylate the PDGFRβ in an agonist-dependent manner. Augmentation or inhibition of GRK2 activity in cells, respectively, reduced or enhanced tyrosine phosphorylation of the PDGFRβ but not the EGFR. Either overexpressed in cells or as a purified protein, GRK2 demonstrated agonist-promoted serine phosphorylation of the PDGFRβ and, unexpectedly, the EGFR as well. Because GRK2 did not phosphorylate a kinase-dead (K634R) PDGFRβ mutant, GRK2-mediated PDGFRβ phosphorylation required receptor tyrosine kinase activity, as does PDGFRβ ubiquitination. Agonist-induced ubiquitination of the PDGFRβ, but not the EGFR, was enhanced in cells overexpressing GRK2. Nevertheless, GRK2 overexpression did not augment PDGFRβ down-regulation. Like the vast majority of GRK2 substrates, the PDGFRβ, 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 PDGFRβ, 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.

The Adaptor Protein β-Arrestin2 Enhances Endocytosis of the Low Density Lipoprotein Receptor

Endocytosis of the low density lipoprotein (LDL) receptor (LDLR) in coated pits employs the clathrin adaptor protein ARH. Similarly, agonist-dependent endocytosis of heptahelical receptors in coated pits employs the clathrin adaptor β-arrestin proteins. In mice fed a high fat diet, we found that homozygous deficiency of β-arrestin2 increased total and LDL plus intermediate-density lipoprotein cholesterol levels by 23 and 53%, respectively (p < 0.05), but had no effect on high density lipoprotein cholesterol levels. We therefore tested whether β-arrestins could affect the constitutive endocytosis of the LDLR. When overexpressed in cells, β-arrestin1 and β-arrestin2 each associated with the LDLR, as judged by co-immunoprecipitation, and augmented LDLR endocytosis by ∼70%, as judged by uptake of fluorescent LDL. However, physiologic expression levels of only β-arrestin2, and not β-arrestin1, enhanced endogenous LDLR endocytosis (by 65%) in stably transfected β-arrestin1/β-arrestin2 double-knockout mouse embryonic fibroblasts (MEFs). Concordantly, when RNA interference was used to suppress expression of β-arrestin2, but not β-arrestin1, LDLR endocytosis was reduced. Moreover, β-arrestin2–/– MEFs demonstrated LDLR endocytosis that was 50% less than cognate wild type MEFs. In fusion protein pull-down assays, β-arrestin2 bound to the LDLR cytoplasmic tail stoichiometrically, and binding was abolished by mutation of LDLR Tyr807 to Ala. Mutation of LDLR cytoplasmic tail Ser833 to Asp enhanced both the affinity of LDLR fusion protein binding to β-arrestin2, and the efficiency of LDLR endocytosis in cells expressing β-arrestin2 physiologically. We conclude that β-arrestin2 can bind to and enhance endocytosis of the LDLR, both in vitro and in vivo, and may thereby influence lipoprotein metabolism.

MARCH2 promotes endocytosis and lysosomal sorting of carvedilol-bound β2-adrenergic receptors

The β2-adrenergic receptor antagonist carvedilol recruits MARCH2, a unique E3 ubiquitin ligase, to promote receptor endocytosis and lysosomal trafficking.

Regulation of the Platelet-derived Growth Factor Receptor-β by G Protein-coupled Receptor Kinase-5 in Vascular Smooth Muscle Cells Involves the Phosphatase Shp2

Smooth muscle cell (SMC) proliferation and migration are substantially controlled by the platelet-derived growth factor receptor-β (PDGFRβ), 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 PDGFRβ activation engendered down-regulation of GRK5, but not GRK2; moreover, GRK5 and PDGFRβ 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 PDGFRβ seryl phosphorylation by 3-fold and reduced PDGFRβ-promoted phosphoinositide hydrolysis, thymidine incorporation, and overall PDGFRβ tyrosyl phosphorylation by ∼35%. Physiologic SMC GRK5 activity also increased PDGFRβ association with the phosphatase Shp2 (8-fold), enhanced phosphorylation of PDGFRβ Tyr1009 (the docking site for Shp2), and reduced phosphorylation of PDGFRβ Tyr1021. Consistent with having increased PDGFRβ-associated Shp2 activity, GRK5-expressing SMCs demonstrated greater PDGF-induced Src activation than GRK5-null cells. GRK5-mediated desensitization of PDGFRβ inositol phosphate signaling was diminished by Shp2 knock-down or impairment of PDGFRβ/Shp2 association. In contrast to GRK5, physiologic GRK2 activity did not alter PDGFRβ/Shp2 association. Finally, purified GRK5 effected agonist-dependent seryl phosphorylation of partially purified PDGFRβs. We conclude that GRK5 mediates the preponderance of PDGF-promoted seryl phosphorylation of the PDGFRβ in SMCs, and, through mechanisms involving Shp2, desensitizes PDGFRβ inositol phosphate signaling and enhances PDGFRβ-triggered Src activation.

Human Umbilical Cord Blood–Derived Endothelial Cells Reendothelialize Vein Grafts and Prevent Thrombosis

Objective—To accelerate vein graft reendothelialization and reduce vein graft thrombosis by infusing human umbilical cord blood-derived endothelial cells (hCB-ECs) because loss of endothelium contributes to vein graft thrombosis and neointimal hyperplasia. Methods and Results—Under steady flow conditions in vitro, hCB-ECs adhered to smooth muscle cells 2.5 to 13 times more than ECs derived from peripheral blood or human aorta (P<0.05). Compared with peripheral blood and human aorta ECs, hCB-ECs had 1.4-fold more cell surface &agr;5&bgr;1 integrin heterodimers per cell (P<0.05) and proliferated on fibronectin 4- to 10-fold more rapidly (P<0.05). Therefore, we used hCB-ECs to enhance reendothelialization of carotid interposition vein grafts implanted in NOD.CB17-Prkdcscid/J mice. Two weeks postoperatively, vein grafts from hCB-EC-treated mice demonstrated approximately 55% reendothelialization and no luminal thrombosis. In contrast, vein grafts from sham-treated mice demonstrated luminal thrombosis in 75% of specimens (P<0.05) and only approximately 14% reendothelialization. In vein grafts from hCB-EC-treated mice, 33±10% of the endothelium was of human origin, as judged by human major histocompatibility class I expression. Conclusion—The hCB-ECs adhere to smooth muscle cells under flow conditions in vitro, accelerate vein graft reendothelialization in vivo, and prevent vein graft thrombosis. Thus, hCB-ECs offer novel therapeutic possibilities for vein graft disease.

Tumor Necrosis Factor Receptor-2 Signaling Attenuates Vein Graft Neointima Formation by Promoting Endothelial Recovery

Objective—Inflammation appears intricately linked to vein graft arterialization. We have previously shown that tumor necrosis factor (TNF) receptor-1 (TNFR1, p55) signaling augments vein graft neointimal hyperplasia (NH) and remodeling through its effects on vascular smooth muscle cells (SMCs). In this study we examined the role of TNFR2 (p75) signaling in vein graft arterialization. Methods and Results—Inferior vena cava-to-carotid artery interposition grafting was performed between p75−/− and congenic (C57B1/6J) wild-type (WT) mice. Six weeks postoperatively, neointimal and medial dimensions were greater in p75−/− grafts placed into p75−/− recipients (by 42% or 60%, respectively; P<0.05), when compared with WT veins grafted into WT recipients. Relative to WT vein grafts, p75 deficiency augmented early (2-week-old) graft vascular cell adhesion molecule (VCAM)-1 expression (by 2.4-fold, P<0.05), increased endothelial cell apoptosis (2-fold), and delayed graft re-endothelialization. Both cellular proliferation in early, and collagen I content of mature (6-week-old) vein grafts were increased (by 70% and 50%, respectively) in p75−/− grafts. P75 deficiency augmented TNF-induced apoptosis of cultured endothelial cells, but did not affect TNF-stimulated SMC proliferation or migration induced by co-cultured macrophages. Conclusions—TNF signaling via p75 reduces vein graft neointimal hyperplasia through mechanisms involving reduction of adhesion molecule expression and endothelial cell apoptosis.

G Protein–Coupled Receptor Kinase-5 Attenuates Atherosclerosis by Regulating Receptor Tyrosine Kinases and 7-Transmembrane Receptors

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 nuclear factor-&kgr;B (NF-&kgr;B). 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 upregulation of cell adhesion molecules than grafts from wild-type mice and, subsequently, more atherosclerosis. By comparing Grk5−/− with wild-type cells, we found that GRK5 desensitized 2 key atherogenic receptor tyrosine kinases: the platelet-derived growth factor receptor-&bgr; in SMCs, by augmenting ubiquitination/degradation; and 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 monocyte chemoattractant protein-1 CC chemokine receptor-2. Whereas GRK5 diminished NF-&kgr;B-dependent gene expression in SMCs and endothelial cells, it had no effect on NF-&kgr;B activity in macrophages. Conclusion—GRK5 attenuates atherosclerosis through multiple cell type-specific mechanisms, including reduction of SMC and endothelial cell NF-&kgr;B activity and desensitization of receptor-specific signaling through the monocyte CC chemokine receptor-2, macrophage CSF-1R, and the SMC platelet-derived growth factor receptor-&bgr;.

Kalirin Promotes Neointimal Hyperplasia by Activating Rac in Smooth Muscle Cells

Objective—Kalirin is a multifunctional protein that contains 2 guanine nucleotide exchange factor 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 central nervous system. We therefore tested the hypothesis that Kalirin functions as a Rho-guanine nucleotide exchange factor in arterial smooth muscle cells (SMCs). Approach 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 upregulated during early atherogenesis in apolipoprotein E-deficient mice. In cultured SMCs, signaling was affected similarly in 3 models of Kalirin loss-of-function: heterozygous Kalrn deletion, Kalirin RNAi, and treatment with the Kalirin Rho-guanine nucleotide exchange factor -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 wild-type SMCs. Neointimal hyperplasia engendered by carotid endothelial denudation was ≈60% less in Kalrn–/+ and SMC-specific Kalrn–/+ mice than in control mice. Conclusion—Kalirin functions as a guanine nucleotide exchange factor for Rac1 in SMCs, and promotes SMC migration and proliferation both in vitro and in vivo.

Reciprocal Regulation of the Platelet-Derived Growth Factor Receptor-β and G Protein-Coupled Receptor Kinase 5 by Cross-Phosphorylation: Effects on Catalysis

Signaling by the platelet-derived growth factor receptor-β (PDGFRβ) is diminished when the PDGFRβ is phosphorylated on seryl residues by G protein-coupled receptor kinase-5 (GRK5), but mechanisms for GRK5 activation by the PDGFRβ remain obscure. We therefore tested whether the PDGFRβ is able to tyrosine-phosphorylate and thereby activate GRK5. Purified GRK5 was tyrosine-phosphorylated by the wild-type PDGFRβ to a stoichiometry of 0.8 mol phosphate/mol GRK5, an extent ∼5 times greater than observed with a Y857F PDGFRβ mutant that fails to phosphorylate exogenous substrates but autophosphorylates and activates Src normally. The degree of PDGFRβ-mediated phosphorylation of GRK5 correlated with GRK5 activity, as assessed by seryl phosphorylation of the PDGFRβ in purified protein preparations, in intact cells expressing a tyrosine-to-phenylalanine GRK5 mutant, and in GRK5 peptide phosphorylation assays. However, tyrosyl phosphorylation of GRK5 was not necessary for GRK5-mediated phosphorylation of the β2-adrenergic receptor, even though β2-adrenergic receptor activation promoted tyrosyl phosphorylation of GRK5 in smooth muscle cells. Phosphorylation of the PDGFRβ by GRK5 in smooth muscle cells or in purified protein preparations reduced PDGFRβ-mediated peptide phosphorylation. In contrast, phosphorylation of GRK5 by the PDGFRβ enhanced the Vmax of GRK5-mediated peptide phosphorylation, by 3.4-fold, without altering the GRK5 KM for peptide. We conclude that GRK5 tyrosyl phosphorylation is required for the activation of GRK5 by the PDGFRβ, but not by the β2-adrenergic receptor, and that by activating GRK5, the PDGFRβ triggers its own desensitization.

C-X-C Motif Chemokine Receptor 3 Splice Variants Differentially Activate Beta-Arrestins to Regulate Downstream Signaling Pathways

Biased agonism, the ability of different ligands for the same receptor to selectively activate some signaling pathways while blocking others, is now an established paradigm for G protein-coupled receptor signaling. One group of receptors in which endogenous bias is critical is the chemokine system, consisting of over 50 ligands and 20 receptors that bind one another with significant promiscuity. We have previously demonstrated that ligands for the same receptor can cause biased signaling responses. The goal of this study was to identify mechanisms that could underlie biased signaling between different receptor splice variants. The C-X-C motif chemokine receptor 3 (CXCR3) has two splice variants, CXCR3A and CXCR3B, which differ by 51 amino acids at its N-terminus. Consistent with an earlier study, we found that C-X-C motif chemokine ligands 4, 9, 10, and 11 all activated Gαi at CXCR3A, while at CXCR3B these ligands demonstrated no measurable Gαi or Gαs activity. β-arrestin (βarr) was recruited at a reduced level to CXCR3B relative to CXCR3A, which was also associated with differences in βarr2 conformation. βarr2 recruitment to CXCR3A was attenuated by both G protein receptor kinase (GRK) 2/3 and GRK5/6 knockdown, while only GRK2/3 knockdown blunted recruitment to CXCR3B. Extracellular regulated kinase 1/2 phosphorylation downstream from CXCR3A and CXCR3B was increased and decreased, respectively, by βarr1/2 knockout. The splice variants also differentially activated transcriptional reporters. These findings demonstrate that differential splicing of CXCR3 results in biased responses associated with distinct patterns of βarr conformation and recruitment. Differential splicing may serve as a common mechanism for generating biased signaling and provides insights into how chemokine receptor signaling can be modulated post-transcriptionally.