Coupling of endothelin receptors to the ERK/MAP kinase pathway

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Abstract
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 Gα 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 Gαq, but not of Gαi or Gαs, 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‐β 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 Gαq/phospholipase C‐β/Ca2+/Src signaling cascade is necessary for efficient coupling of ET receptors to the ERK/MAPK pathway.