[D-Arg(1),D-Trp(5,7,9),Leu(11)]Substance P inhibits bombesin-induced mitogenic signal transduction mediated by both G(q) and G(12) in Swiss 3T3cells.

Substance P (SP) analogues including [d-Arg(1),d-Trp(5,7,9), Leu(11)]SP are broad spectrum neuropeptide antagonists and potential anticancer agents, but their mechanism of action is not fully understood. Here, we examined the mechanism of action of [d-Arg(1), d-Trp(5,7,9),Leu(11)]SP as an inhibitor of G protein-coupled receptor (GPCR)-mediated signal transduction and cellular DNA synthesis in Swiss 3T3 cells. Addition of [d-Arg(1),d-Trp(5,7,9), Leu(11)]SP, at 10 micrometer, caused a striking rightward shift in the dose-response curves of DNA synthesis induced by bombesin, bradykinin, or vasopressin and markedly inhibited the activation of p42(mapk) (ERK-2) and p44(mapk) (ERK-1) induced by these GPCR agonists. In addition, this SP analogue also prevented the protein kinase C-dependent activation of protein kinase D induced by these agonists. [d-Arg(1),d-Trp(5,7,9),Leu(11)]SP, at a concentration (10 micrometer) that inhibited these G(q)-mediated events, also prevented GPCR agonist-induced responses mediated through the G proteins of the G(12) subfamily. These include bombesin-induced assembly of focal adhesions, formation of parallel arrays of actin stress fibers, increase in the tyrosine phosphorylation of focal adhesion kinase (FAK), p130(Cas), and paxillin, and formation of a complex between FAK and Src. We conclude that [d-Arg(1),d-Trp(5,7,9),Leu(11)]SP acts as a mitogenic antagonist of neuropeptide GPCRs blocking signal transduction via both G(q) and G(12).