Selective amplification of endothelin-stimulated inositol 1,4,5-trisphosphate and calcium signaling by v-src transformation of rat-1 fibroblasts.
The effects of the expression of the protein tyrosine kinase pp60v-src on endothelin- and thrombin-stimulated inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) production and calcium responses were investigated in Rat-1 fibroblasts. The ability of endothelin-1 to induce the accumulation of these second messengers was dramatically amplified by v-src transformation, with 6- and 3-fold enhancements of the peak Ins(1,4,5)P3 and peak calcium responses, respectively. In contrast, thrombin-dependent responses were slightly reduced following v-src transformation, demonstrating that the augmentation of endothelin-stimulated signal transduction is a selective effect. The magnitude of the stimulated accumulation of Ins(1,4,5)P3 presumably depends upon both the functional activation of phospholipase C to produce Ins(1,4,5)P3, and the activity of the enzymes that metabolize Ins(1,4,5)P3. Although the metabolism of Ins(1,4,5)P3 was strikingly altered by expression of pp60v-src, with a bias towards the production of higher inositol polyphosphates that is consistent with an activated Ins(1,4,5)P3 3-kinase, this change could not account for the marked increase in endothelin-stimulated signaling induced by v-src transformation. This suggests that an effect of pp60v-src is expressed at the level of the plasma membrane, through an interaction with one or more components in the receptor/guanine nucleotide binding protein (G protein)/phospholipase C system that transduces the endothelin signal into Ins(1,4,5)P3 production. Preparation of membranes from normal and v-src-transformed cells showed that, while there was no change in the number of high-affinity endothelin binding sites, the release of Ins(1,4,5)P3 in response to guanine nucleotides and endothelin-1 was significantly increased following v-src transformation. In contrast, the Ins(1,4,5)P3 responses to thrombin and high Ca2+ concentrations were unaffected by transformation. Thus the selective interactions within the G protein system that couples the endothelin receptor to phospholipase C are potential sites at which the v-src transformation process may act to amplify endothelin-dependent Ins(1,4,5)P3 production.
Mattingly
R R
RR
Department of Pharmacology, University of Virginia School of Medicine, Charlottesville 22908.
Wasilenko
W J
WJ
Woodring
P J
PJ
Garrison
J C
JC
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002413:Cations, Divalent; D002471:Cell Transformation, Neoplastic; D016232:Endothelins / Q000494:pharmacology*; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D016391:Genes, src; D015544:Inositol 1,4,5-Trisphosphate / Q000096:biosynthesis / Q000378:metabolism*; D007457:Iodine Radioisotopes; D007700:Kinetics; D051381:Rats; D015290:Second Messenger Systems; D015398:Signal Transduction; D013379:Substrate Specificity; D013917:Thrombin / Q000494:pharmacology
D002413:Cations, Divalent; D016232:Endothelins; D007457:Iodine Radioisotopes; D015544:Inositol 1,4,5-Trisphosphate; D013917:Thrombin; D002118:Calcium