Protein kinase C‐dependent coupling of rat endothelin receptor type A and Gs proteins

Among GPCRs, the endothelin‐A (ET‐A) receptor differs because it couples with several heterotrimeric G proteins: G q/11 , G s , Gi/o, G z , G 12/13 . Multiple coupling of this receptor has been observed in several cell types, including anterior pituitary cells. The mechanism by which this receptor couples to several G proteins is still unknown. Here we examined the potential role of protein kinase C (PKC) in G s activation by the receptors. The coupling of the rat ET‐A receptor to cAMP accumulation pathway was dramatically abolished in cells pretreated with Gö6983 and Ro31‐8425, a PKC specific inhibitor, suggesting a role of this signaling pathway in receptor‐mediated G s activation. In a search for a residue responsible for PKC effects, we focused on four putative intracellular PKC phosphorylation sites at S289, S373, T416 and S420, and the latter three sites are conserved in all mammalian ETA receptors. Those residues were substituted with alanine and mutants were analyzed. Those mutations did not affect the plasma membrane expression and G q/11 activations by the receptors. However, the S373A mutant almost abolished the receptor mediated cAMP formation, in contrast to the other mutant receptors never effected the G s activations. These results indicate that the ET‐A receptor mediated G s activations are induced via S373 phosphorylation mediated by PKC. Supported by the Intramural Research Program of the NICHD, NIH.