LIGAND-INDUCED PHOSPHORYLATION, CLUSTERING, AND DESENSITIZATION OF A(1) ADENOSINE RECEPTORS

Through immunocytochemistry with the use of antibodies against A(1) ad enosine receptors (A(1)Rs) and confocal microscopy, we show that stimu lation of A(1)Rs by the agonist (R)-phenylisopropyladenosine [(R)-PIA] caused a rapid (5-15 min) aggregation (clustering) of receptor molecu les on the surface of DDT1MF-2 cells. Internalization of the chronical ly stimulated receptor was slower and occurred concomitantly, with a t ime-dependent decrease (50%) in the number of cell surface [H-3](R)-PI A binding sites. The reduction of binding sites was due partly (30%) t o internalization and partly (20%) to the presence of desensitized cel l surface receptor molecules that were unable to bind the ligand. Chro nic exposure of DDT1MF-2 cells to 50 nM (R)-PIA produced functional de sensitization, as deduced from second messenger production assays. Qua ntification of the content of A(1)Rs by immunoblotting and flow cytome try in cells pretreated with 50 nM (R)-PIA indicates a time-dependent slow down-regulation of the receptor. Receptor clustering and agonist- induced receptor phosphorylation, which occurred in serine and tyrosin e, were simultaneous. The finding that activators of protein kinase A or C were able to induce functional desensitization of A(1)Rs, phospho rylate A(1)Rs in serine and threonine, and trigger clustering of the r eceptor suggests that phosphorylation of A(1)Rs in serine/threonine is involved in desensitization-related events.