Phosphorylation and desensitization of α1d-adrenergic receptors

In rat-1 fibroblasts stably expressing rat alpha (1d)-adrenoceptors, noradrenaline and PMA markedly decreased alpha (1d)-adrenoceptor function (noradrenaline-elicited increases in calcium in whole cells and [S-35]guanosine 5'-[gamma -thio]triphosphate binding in membranes), suggesting homologous and heterologous desensitizations. Photoaffinity labelling, Western blotting and immunoprecipitation identified alpha (1d)-adrenoceptors as a broad band of 70-80 kDa. alpha (1d)-Adrenoceptors were phosphorylated in the basal state and noradrenaline and PMA increased it. The effect of noradrenaline was concentration-dependent (EC50 75 nM), rapid (maximum at 1 min) and transient. Phorbol ester-induced phosphorylation was concentration-dependent (EC50 25 nM), slightly slower (maximum at 5 min) and stable for at least 60 min. Inhibitors of protein kinase C decreased the effect of phorbol esters but not that of noradrenaline. Evidence of crosstalk of alpha (1d)-adrenoceptors with receptors endogenously expressed in rat-1 fibroblasts was given by the ability of endothelin, lysophosphatidic acid and bradykinin to induce alpha (1d)-adrenoceptor phosphorylation. In summary, it is shown for the first time here that alpha (1d)-adrenoceptors are phosphoproteins and that receptor phosphorylation is increased by the natural ligand, noradrenaline, by direct activation of protein kinase C and via cross-talk with other receptors endogenously expressed in rat-1 fibroblasts. Receptor phosphorylation has functional repercussions.