Coupling of endothelin receptors to the ERK/MAP kinase pathway -: Roles of palmitoylation and Gαq

Endothelins are potent mitogens that stimulate extracellular signal-regulated kinases (ERK/MAP kinases) through their cognate G-protein-coupled receptors, ETA and ETB. To address the role of post-translational ET receptor modifications such as acylation on ERK activation and to identify relevant downstream effectors coupling the ET receptor to the ERK signaling cascades we have constructed a panel of palmitoylation-deficient ET receptor mutants with differential Got protein binding capacity. Endothelin-1 stimulation of wild-type ETA or ETB induced a fivefold to sixfold increase in ERK in COS-7 and CHO cells whereas full-length nonpalmitoylated ETA and ETB mutants failed to stimulate ERK. A truncated ETB lacking the C-terminal tail domain including putative phosphorylation and arrestin binding site(s) but retaining the critical palmitoylation site(s) was still able to fully stimulate ERK activation. Using mutated ET receptors with selective G-protein-coupling we found that endothelin-induced stimulation of G alpha (q), but not of G alpha (i) or G alpha (s), is essential for endothelin-mediated ERK activation. Inhibition of protein kinases A and C or epidermal growth factor receptor kinase failed to prevent ETA- and ETB-mediated ERK activation whereas blockage of phospholipase C-P completely abrogated endothelin-promoted ERK activation through ETA and ETB in recombinant COS-7 and native C6 cells. Complex formation of Ca2+ or inhibition of Src family tyrosine kinases prevented ET-1-induced ERK-2 activation in C6-cells. Our results indicate that endothelin-promoted ERK/MAPK activation criticially depends on palmitoylation but not on phosphorylation of ET receptors, and that the G alpha (q)/phospholipase C-beta /Ca2+/Src signaling cascade is necessary for efficient coupling of ET receptors to the ERK/MAPK pathway.