Tyr1009 and Tyr1021 in the platelet-derived growth factor beta-receptor mediate agonist triggered calcium signalling.
Calcium signalling was studied in porcine aortic endothelial cells stably transfected with wild type or mutants of the human platelet-derived growth factor (PDGF) beta-receptor and fibroblast growth factor (FGF) receptor-1 (FGFR1). Phospholipase C-gamma (PLC-gamma) has a consensus binding site at phosphorylated Tyr1021 in the PDGF beta-receptor. The phosphorylated tyrosine at 1009 is a binding site for Syp/PTP1D, an adaptor molecule mediating Grb2/RAS signalling. Also, Tyr1009 has been shown to be a minor binding site for PLC-gamma; however previous data have indicated that it does not have any functional significance in PLC-gamma signalling. The concentration of cytoplasmic calcium ([Ca2+]i) was measured by microfluorometry and digital imaging. About 72% of the cells transfected with wild type PDGF beta-receptor responded to a challenge with PDGF-BB. Mutants in which both Tyr1009 and Tyr1021 in the PDGF beta-receptor were exchanged for phenylalanine totally lacked [Ca2+]i responses. However, in those with a single mutation at Tyr1009 or Tyr1021, 36% and 12% of the cells responded, respectively. In cells transfected with FGFR1 or FGFchim, with the kinase insert of FGFR1 replaced by the insert of the PDGF beta-receptor, a [Ca2+]i increases was observed in similar proportions of cells. The amplitudes of the growth factor-induced [Ca2+]i responses was comparable in the different transfectants. Thrombin, activating a G-protein coupled receptor, triggered [Ca2+]i peaks more rapidly, and in a higher proportion of cells compared to the growth factors. The present data indicate that both Tyr1009 and Tyr1021 alone and in cooperation mediate PDGF-BB triggered calcium signalling.
Ridefelt
P
P
Department of Clinical Laboratory Sciences, Uppsala University, Sweden. peter.ridefelt@klinkem.uu.se
Siegbahn
A
A
D000595:Amino Acid Sequence; D000818:Animals; D000077214:Becaplermin; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D016384:Consensus Sequence; D004730:Endothelium, Vascular / Q000187:drug effects / Q000502:physiology*; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D051966:Phospholipase C gamma; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology*; D017354:Point Mutation; D020574:Proto-Oncogene Proteins c-sis; D020797:Receptor, Platelet-Derived Growth Factor beta; D017468:Receptors, Fibroblast Growth Factor / Q000502:physiology; D017479:Receptors, Platelet-Derived Growth Factor / Q000819:agonists / Q000737:chemistry / Q000502:physiology*; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D013552:Swine; D013917:Thrombin / Q000494:pharmacology; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism; D014443:Tyrosine
D007527:Isoenzymes; D010982:Platelet-Derived Growth Factor; D020574:Proto-Oncogene Proteins c-sis; D017468:Receptors, Fibroblast Growth Factor; D011994:Recombinant Proteins; D016222:Fibroblast Growth Factor 2; D000077214:Becaplermin; D014443:Tyrosine; D020797:Receptor, Platelet-Derived Growth Factor beta; D017479:Receptors, Platelet-Derived Growth Factor; D010738:Type C Phospholipases; D051966:Phospholipase C gamma; D013917:Thrombin; D002118:Calcium