Post-translational modifications such as phosphorylation and palmitoylation play important roles for the function and regulation of receptors coupled to heterotrimeric guanyl nucleotide binding proteins. Here we demonstrate that the human endothelin receptor A (ET(A)) incorporates [H-3]palmitate, Mutation of a cluster of five cysteine residues present in the cytoplasmic tail of ET(A) into serine or alanine residues completely prevented palmitoylation of the receptor. The ligand binding affinity of the non-palmitoylated ET(A) mutants was essentially unchanged as compared to the palmitoylated wild type ET(A) suggesting that the replacement of the cysteine residues did not alter the overall structure of the receptor, Furthermore, the ligand-induced stimulation of adenylyl cyclase by the mutant ET(A) was unaffected by the mutation, In contrast, the mutated non-palmitoylated receptors but not the wild type receptor failed to stimulate phosphatidylinositol hydrolysis by phospholipase C activation upon challenge by endothelin-1. Furthermore, the mutant receptors failed to stimulate the ligand-induced transient increase in the cytoplasmic calcium seen with the wild type ET(A), Endothelin-1 induced mitogenic stimuli via the wild type receptors but not through the mutated receptors suggesting an important role for phospholipase C in this signal transduction pathway, The differential regulation of distinct signal transduction pathways by post-translational modification suggests that palmitoylation of the ET(A) provides a novel mechanism of modulating ET(A) receptor activity.
