Ca2+ channel activation by platelet-derived growth factor-induced tyrosine phosphorylation and Ras guanine triphosphate-binding proteins in rat glomerular mesangial cells.
We investigated the signaling pathways mediating 1-pS Ca2+ channel activation by PDGF in cultured rat mesangial cells. In cell-attached patches, intrapipette PDGF-BB (PDGF B chain homodimer isoform) (50 ng/ml) dramatically stimulates channel activity (P < 0.003, n = 6). Tyrosine kinase inhibition (100 microM genistein or 10 microM tryphostin 9) abolished PDGF-induced channel activation (P < 0.02, n = 6). In excised patches, the effect of tyrosine kinase inhibition could be reversed by 200 microM GTPgammaS (P < 0.02, n = 4). In contrast, 200 microM GDPbetaS inhibited PDGF-induced channel activity (P < 0.04, n = 6). Pertussis toxin (250 ng/ml) had no effect on PDGF-induced channel activity (P = 0.45, n = 6). When excised patches were exposed to anti-Ras antibody (5 microg/ml), PDGF-induced channel activity was abolished (P < 0.002, n = 11). Western immunoblots revealed that PDGF-BB binding stimulates the formation of a membrane-bound complex consisting of growth factor receptor-binding protein 2, son of sevenless, and the PDGF-beta receptor. Complex formation was abolished by genistein. In mesangial cells, the intrinsic tyrosine kinase activity of the PDGF-beta receptor stimulates the formation of a membrane-bound growth factor receptor-binding protein 2/son of sevenless/PDGF-beta receptor complex and activation of the pertussis toxin-insensitive GTP-binding protein, p21-Ras, which leads to the opening of 1-pS Ca2+ channels.
Ma
H
H
Department of Medicine, Renal Division, Emory University School of Medicine, Altanta, Georgia 30322, USA.
Matsunaga
H
H
Li
B
B
Schieffer
B
B
Marrero
M B
MB
Ling
B N
BN
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D015220:Calcium Channels / Q000187:drug effects*; D051380:GRB2 Adaptor Protein; D019204:GTP-Binding Proteins / Q000502:physiology; D005920:Glomerular Mesangium / Q000378:metabolism*; D008297:Male; D008565:Membrane Proteins / Q000502:physiology; D010766:Phosphorylation; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology*; D011505:Protein-Tyrosine Kinases / Q000502:physiology; D011506:Proteins / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D020664:Son of Sevenless Proteins; D014443:Tyrosine / Q000378:metabolism*; D018631:ras Proteins / Q000502:physiology
D048868:Adaptor Proteins, Signal Transducing; D015220:Calcium Channels; D051380:GRB2 Adaptor Protein; C495950:Grb2 protein, rat; D008565:Membrane Proteins; D010982:Platelet-Derived Growth Factor; D011506:Proteins; D020664:Son of Sevenless Proteins; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D019204:GTP-Binding Proteins; D018631:ras Proteins