Activation of endothelin ETA receptors induces phosphorylation of alpha1b-adrenoreceptors in Rat-1 fibroblasts.

The effect of endothelin-1 on the phosphorylation of alpha1b-adrenoreceptors, transfected into rat-1 fibroblasts, was studied. Basal alpha1b-adrenoreceptor phosphorylation was markedly increased by endothelin-1, norepinephrine, and phorbol esters. The effect of endothelin-1 was dose dependent (EC50 approximately 1 nM), reached its maximum 5 min after stimulation, and was inhibited by BQ-123, an antagonist selective for ETA receptors. Endothelin-1-induced alpha1b-adrenoreceptor phosphorylation was attenuated by staurosporine or genistein and essentially abolished when both inhibitors were used together. The effect of norepinephrine was not modified by either staurosporine or genistein alone, and it was only partially inhibited when both were used together. These data suggest the participation of protein kinase C and tyrosine kinase(s) in endothelin-1-induced receptor phosphorylation. However, phosphoaminoacid analysis revealed the presence of phosphoserine and traces of phosphothreonine, but not of phosphotyrosine, suggesting that the putative tyrosine kinase(s), activated by endothelin, could act in a step previous to receptor phosphorylation. The effect of endothelin-1 on alpha1b-adrenoreceptor phosphorylation was not mediated through pertussis toxin-sensitive G proteins. Calcium mobilization induced by norepinephrine was diminished by endothelin-1. Norepinephrine and endothelin-1 increased [35S]GTPgammaS binding to control membranes. The effect of norepinephrine was abolished in membranes obtained from cells pretreated with endothelin-1. Interestingly, genistein plus staurosporine inhibited this effect of the endothelial peptide. Endothelin-1 did not induce alpha1b-adrenoreceptor internalization. Our data indicate that activation of ETA receptors by endothelin-1 induces alpha1b-adrenoreceptor phosphorylation and alters G protein coupling.