Intracellular calcium and alpha(1b)-adrenoceptor phosphorylation

Background. Desensitization of G protein-coupled receptors is associated wi th receptor phosphorylation. Two groups of kinases seem to participate in s uch receptor phosphorylation, i. e., second messenger-activated protein kin ases and G protein-coupled receptor kinases. Calcium seems to play a role i n the phosphorylation of some G protein-coupled receptors. The role of calc ium in alpha(1b)-adrenoceptor phosphorylation has not been critically asses sed, Method's. Rat-1 fibroblasts stably expressing the hamster alpha(1b)-adrener gic receptor were used. To study receptor phosphorylation cells metabolical ly labeled with [P-32]Pi were lysed and the receptor immunoprecipitated usi ng a polyclonal antibody generated against the receptor carboxyl terminal d ecapeptide. Intracellular calcium was determined by using Fura-2 fluorescen ce, Results, Norepinepkrine, endothelin-1, and lysophospharidic acid increased intracellular calcium concentration. All these agents and phorbol myristate acetate (PMA) induce alpha(1b)-adrenoceptor phosphorylation, The intracell ular chelator, BAPTA, abolished the increase in intracellular calcium induc ed by the previously mentioned agents but did not affect the receptor phosp horylation induced by norepinephrine, PMA, or lysophosphatidic acid. Under these conditions, receptor phosphorylation induced by endothelin was slight ly but consistently decreased, Thapsigargin increased intracellular calcium concentration but was unable to induce alpha(1b)-adrenoceptor phosphorylat ion and decreased PMA-induced receptor phosphorylation. No increase in rece ptor phosphorylation was observed when calcium ionophores were used. Conclusions. Our data indicate that an increase in [Ca2+]i is not sufficien t to induce alpha(1b)-adrenoceptor phosphorylation and that buffering of [C a2+]i does not alter the receptor phosphorylation induced by norepinephrine , lysophosphatidic acid, and PMA. A marginal role of calcium in the alpha(1 b)-adrenoceptor phosphorylation induced by endothelin-l cannot be discarded . (C) 1999 IMSS. Published by Elsevier Science Inc.