The platelet-derived growth factor receptor-beta phosphorylates and activates G protein-coupled receptor kinase-2. A mechanism for feedback inhibition.
G protein-coupled receptor kinase-2 (GRK2) serine-phosphorylates the platelet-derived growth factor receptor-beta (PDGFRbeta), and thereby diminishes signaling by the receptor. Because activation of GRK2 may involve phosphorylation of its N-terminal tyrosines by c-Src, we tested whether the PDGFRbeta itself could tyrosine-phosphorylate and activate GRK2. To do so, we used wild type (WT) and Y857F mutant PDGFRbetas in HEK cells, which lack endogenous PDGFRs. The Y857F PDGFRbeta autophosphorylates normally but does not phosphorylate exogenous substrates. Although PDGF-stimulated Y857F and WT PDGFRbetas activated c-Src equivalently, the WT PDGFRbeta tyrosine-phosphorylated GKR2 60-fold more than the Y857F PDGFRbeta in intact cells. With purified GRK2 and either WT or Y857F PDGFRbetas immunoprecipitated from HEK cells, GRK2 tyrosyl phosphorylation was PDGF-dependent and required the WT PDGFRbeta, even though the WT and Y857F PDGFRbetas autophosphorylated equivalently. This PDGFRbeta-mediated GRK2 tyrosyl phosphorylation enhanced GRK2 activity: GRK2-mediated seryl phosphorylation of the PDGFRbeta 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 beta2-adrenergic and PDGF-beta receptors. Furthermore, both PDGFRbeta-mediated GRK2 tyrosyl phosphorylation and GRK2-mediated PDGFRbeta seryl phosphorylation were reduced approximately 50% in intact cells by mutation to phenylalanine of three tyrosines in the N-terminal domain of GRK2. We conclude that the activated PDGFRbeta itself phosphorylates GRK2 tyrosyl residues and thereby activates GRK2, which then serine-phosphorylates and desensitizes the PDGFRbeta.
Department of Medicine (Cardiology), Duke University Medical Center, Durham, North Carolina 27710, USA.
Jiao-Hui
JH
Wu
Robi
R
Goswami
Luke K
LK
Kim
William E
WE
Miller
Karsten
K
Peppel
Neil J
NJ
Freedman
D000818:Animals; D002417:Cattle; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D025461:Feedback, Physiological / Q000502:physiology*; D054774:G-Protein-Coupled Receptor Kinase 5; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008954:Models, Biological; D009154:Mutation; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D020797:Receptor, Platelet-Derived Growth Factor beta / Q000235:genetics / Q000378:metabolism*; D018343:Receptors, Adrenergic, beta-2 / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism; D051552:beta-Adrenergic Receptor Kinases; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
D018343:Receptors, Adrenergic, beta-2; D014443:Tyrosine; D012694:Serine; D020797:Receptor, Platelet-Derived Growth Factor beta; D019061:src-Family Kinases; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D051552:beta-Adrenergic Receptor Kinases; D054774:G-Protein-Coupled Receptor Kinase 5; C517898:GRK5 protein, human; D019204:GTP-Binding Proteins