The effect of endothelin-1 on the phosphorylation of ␣ 1b -adrenoreceptors, transfected into rat-1 fibroblasts, was studied. Basal ␣ 1b -adrenoreceptor phosphorylation was markedly increased by endothelin-1, norepinephrine, and phorbol esters. The effect of endothelin-1 was dose dependent (EC 50 Ϸ 1 nM), reached its maximum 5 min after stimulation, and was inhibited by BQ-123, an antagonist selective for ET A receptors. Endothelin-1-induced ␣ 1b -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 ␣ 1b -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 [ 35 S]GTP␥S 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 ␣ 1b -adrenoreceptor internalization. Our data indicate that activation of ET A receptors by endothelin-1 induces ␣ 1b -adrenoreceptor phosphorylation and alters G protein coupling.

No abstract

In rat hepatic C9 cells, angiotensin II (Ang II)-induced activation of angiotensin type 1 (AT 1 ) receptors (AT 1 -Rs) stimulates extracellular signal-regulated kinase (ERK) 1/2 phosphorylation via transactivation of the endogenous epidermal growth factor (EGF) receptor (EGF-R) by a protein kinase C (PKC) ␦/Src/Pyk2-dependent pathway. This leads to phosphorylation of the EGF-R as well as its subsequent internalization. On the other hand, EGF-induced activation of the EGF-R in C9 cells was found to cause phosphorylation of the AT 1 -R. This was prevented by selective inhibition of the intrinsic tyrosine kinase activity of the EGF-R by AG1478 [4-(3Ј-chloroanilino)-6,7-dimethoxy-quinazoline] and was reduced by inhibition of PKC and phosphoinositide 3-kinase. EGF-induced AT 1 -R phosphorylation was associated with a decrease in membrane-associated AT 1 -Rs and a reduced inositol phosphate response to Ang II. Agonist activation of endogenous AT 1 -Rs and EGF-Rs induced the formation of a multireceptor complex containing both the AT 1 -R and the transactivated EGF-R. The dependence of these responses on caveolin was indicated by the finding that cholesterol depletion of C9 cells abolished Ang II-induced inositol phosphate production, activation of Akt/PKB and ERK1/2, and AT 1 -R internalization. Confocal microscopy demonstrated that caveolin-1 was endogenously phosphorylated and was distributed on the plasma membrane in patches that undergo redistribution during Ang II stimulation. Agonist-induced phosphorylation and association of caveolin 1 with the AT 1 -R was observed, consistent with a scaffolding role of caveolin during transactivation of the EGF-R by Ang II. The EGF-induced AT 1 -R/caveolin association was abolished by AG1478, suggesting that activation of the EGF-R promotes the association of caveolin and the AT 1 -R.

No abstract