Overexpression of G Protein–Coupled Receptor Kinase-2 in Smooth Muscle Cells Attenuates Mitogenic Signaling via G Protein–Coupled and Platelet-Derived Growth Factor Receptors

Peppel, Karsten; Jacobson, Anne; Huang, Xuewei; Murray, John P.; Oppermann, Martin; Freedman, Neil J.

doi:10.1161/01.cir.102.7.793

Cited by 53 publications

(88 citation statements)

References 24 publications

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“…Recombinant Adenoviruses and Plasmids-The cDNA encoding bovine GRK5 was inserted into the plasmid pSKAC, and adenoviruses were produced in 293 cells, purified on CsCl gradients, and titered as we described previously (9). Plasmids encoding the N-terminal FLAG TM -tagged human PDGFR␤ (FPDGFR␤) (7), bovine GRK2 (21), and bovine GRK5 (21) have been described previously.…”

Section: Methodsmentioning

confidence: 99%

“…Cell Culture and Adenovirus Infections-New Zealand White rabbits, grk5 Ϫ/Ϫ and littermate WT mice (14) were sacrificed to make thoracic aortic SMCs, as previously described (9). HEK 293 cells and embryo fibroblasts (MEFs) from grk2 Ϫ/Ϫ and littermate WT mice were grown and transfected as described (8).…”

Section: Methodsmentioning

confidence: 99%

“…SMCs were used only through passage 7. Infections of SMCs with recombinant adenoviruses were performed at equivalent multiplicities of infection (from 50 to 100), and cells were assayed 48 -72 h after infection, as we described previously (9). In GRK5 "add-back" experiments with grk5 Ϫ/Ϫ SMCs, assays were performed 6 h after adenovirus infection (when GRK5 expression levels were equivalent to those obtaining in WT SMCs).…”

Section: Methodsmentioning

confidence: 99%

“…We have recently demonstrated that the PDGFR␤ undergoes agonistdependent seryl phosphorylation by G protein-coupled receptor kinase-2 (GRK2), a ubiquitously expressed Ser/Thr kinase (7,8). GRK2-mediated phosphorylation of the PDGFR␤ results in diminished PDGFR␤ signaling, or desensitization, through mechanisms related to reduced PDGFR␤/Na ϩ /H ϩ exchanger regulatory factor association (8), reduced PDGFR␤ tyrosyl phosphorylation (7)(8)(9), and PDGFR␤ hyperubiquitination (7). This PDGFR␤ desensitization manifests as diminished second messenger signaling (9) and phosphoinositide 3-kinase activation (10) in the short term, and manifests as diminished SMC migration (10), thymidine incorporation (9,10), and proliferation (9) in the medium to long term.…”

mentioning

confidence: 99%

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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

Goswami²,

Cai

et al. 2006

Journal of Biological Chemistry

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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.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

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

Goswami²,

Cai

et al. 2006

Journal of Biological Chemistry

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“…GRK2 inhibits TGFmediated cell growth arrest and apoptosis in human hepatocarcinoma cells (4). On the other hand, GRK2 attenuates thyroid stimulating hormone-and PDGF-dependent proliferation of thyroid cancer cell lines (5) and smooth muscle cells (6), respectively, whereas it increases mitogenic signaling pathways in response to EGF in osteoblasts (7) or upon activation of the Smoothened receptor in fibroblasts (8). Increased GRK2 levels also potentiate migration of epithelial cells toward fibronectin and sphingosine-1-phosphate (9).…”

mentioning

confidence: 99%

G protein–coupled receptor kinase 2 (GRK2) modulation and cell cycle progression

Penela

Rivas

Salcedo

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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Cell cycle progression requires changes in the activity or levels of a variety of key signaling proteins. G protein-coupled receptor kinase 2 (GRK2) plays a central role in G protein-coupled receptor regulation. Recent research is uncovering its involvement in additional cellular functions, but the potential role of GRK2 in the cell cycle has not been addressed. We report that GRK2 protein levels are transiently down-regulated during the G2/M transition by a mechanism involving CDK2-mediated phosphorylation of GRK2 at Serine670, which triggers binding to the prolyl-isomerase Pin1 and subsequent degradation. Prevention of GRK2 phosphorylation at S670 impedes normal GRK2 down-regulation and markedly delays cell cycle progression. Interestingly, we find that endogenous GRK2 down-regulation is prevented on activation of the G2/M checkpoint by doxorubicin and that stabilized GRK2 levels in such conditions inversely correlate with the p53 response and the induction of apoptosis, suggesting that GRK2 participates in the regulatory network controlling cell cycle arrest and survival in such conditions. cyclin-dependent kinase 2 | G2/M checkpoint | Pin1 | degradation | p53

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Growth inhibition of human hepatocellular carcinoma cells by overexpression of G‐protein‐coupled receptor kinase 2

Wei

Hurtt

Ciccarelli

et al. 2012

Journal Cellular Physiology

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Hepatocellular carcinoma (HCC) is one of the deadliest forms of human liver cancer and does not respond well to conventional therapies. Novel effective treatments are urgently in need. G-protein-coupled kinase 2 (GRK2) is unique serine/threonine kinase that involves in many signaling pathways and regulates various essential cellular processes. Altered levels of GRK2 have been linked with several human diseases including cancer. In this study, we investigated a novel approach for HCC treatment by inducing overexpression of GRK2 in human HCC cells. We found that overexpression of GRK2 through recombinant adenovirus transduction inhibits the growth of human HCC cells. BrdU incorporation assay showed that the growth inhibition caused by elevated GRK2 level was due to reduced cell proliferation but not apoptosis. To examine the anti-proliferative function of increased GRK2 level, we performed cell cycle analysis using propidium iodide staining. We found that the proliferation suppression was associated with G2/M phase cell cycle arrest by the wild-type GRK2 but not its kinase-dead K220R mutant. Furthermore, increased levels of wild-type GRK2 induced upregulation of phosphor-Ser15 p53 and cyclin B1 in a dose-dependent manner. Our data indicate that the anti-proliferative function of elevated GRK2 is associated with delayed cell cycle progression and is GRK2 kinase activity-dependent. Enforced expression of GRK2 in human HCC by molecular delivery may offer a potential therapeutic approach for the treatment of human liver cancer.

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Overexpression of G Protein–Coupled Receptor Kinase-2 in Smooth Muscle Cells Attenuates Mitogenic Signaling via G Protein–Coupled and Platelet-Derived Growth Factor Receptors

Abstract: GRK2 overexpression diminishes SMC mitogenic signaling and proliferation stimulated by PDGF or agonists for several GPCRs. Gene transfer of GRK2 may therefore be therapeutically useful for neointimal hyperplasia.

Cited by 53 publications

References 24 publications

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

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

G protein–coupled receptor kinase 2 (GRK2) modulation and cell cycle progression

Growth inhibition of human hepatocellular carcinoma cells by overexpression of G‐protein‐coupled receptor kinase 2

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