Phosphorylation of the Platelet-derived Growth Factor Receptor-β by G Protein-coupled Receptor Kinase-2 Reduces Receptor Signaling and Interaction with the Na+/H+ Exchanger Regulatory Factor

Abstract

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

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

“…HEK 293 cells and embryo fibroblasts (MEFs) from grk2 Ϫ/Ϫ and littermate WT mice were grown and transfected as described (8). The grk5 Ϫ/Ϫ mice used in this study were hybrids of the 129/SvJ and C57Bl/6J lines and were generated by mating grk5 Ϫ/ϩ mice.…”

“…Immunoblotting and Immunoprecipitations-These assays used antibodies and procedures described previously (7)(8)(9). Immunoprecipitation (IP) of endogenous PDGFR␤s used either rabbit or goat anti-PDGFR␤ IgG (Santa Cruz Biotechnology), whereas IP of transfected (N-terminal epitope-tagged) PDGFR␤ constructs (WT and Y1009F) used anti-FLAG M2-agarose (Sigma).…”

“…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).…”

“…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.…”

Peptide substrates for G protein‐coupled receptor kinase 2

“…The RESA peptide was designed based on the observation that most peptide substrates of GRK2 possess acidic residues on the N-terminal side of the Ser/Thr phosphate acceptor residue. Reported GRK2 phosphorylation targets include b-tubulin [11,15,16], synucleins (a and b) [17], Nedd4 [18], Nedd4-2 [18], b2-adrenergic receptor [19], epithelial Na + channels [20], phosducin [21], rhodopsin [22], ribosomal protein P2 [23], downstream regulatory element antagonist modulator (DREAM) [24] (MAPK) [25], phosphodiesterase c (PDEc) [26], Smad2 [27], ezrin/ radixin [28,29], and platelet-derived growth factor receptor-b (PDGFR-b) [30]. Phosphorylation of substrates by GRK2 can be enhanced in the presence of G protein bc subunits and negatively charged phospholipids (e.g., phosphatidylserine) [13,16,31].…”