Ligand-induced phosphorylation, clustering, and desensitization of A(1) adenosine receptors

Through immunocytochemistry with the use of antibodies against A(1) adenosine receptors (A(1)Rs) and confocal microscopy, we show that stimulation of A(1)Rs by the agonist (R)-phenylisopropyladenosine [(R)-PIA] caused a rapid (5-15 min) aggregation (clustering) of receptor molecules on the surface of DDT1MF-2 cells. Internalization of the chronically stimulated receptor was slower and occurred concomitantly, with a time-dependent decrease (50%) in the number of cell surface [H-3](R)-PIA binding sites. The reduction of binding sites was due partly (30%) to internalization and partly (20%) to the presence of desensitized cell surface receptor molecules that were unable to bind the ligand. Chronic exposure of DDT1MF-2 cells to 50 nM (R)-PIA produced functional desensitization, as deduced from second messenger production assays. Quantification of the content of A(1)Rs by immunoblotting and flow cytometry 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 tyrosine, were simultaneous. The finding that activators of protein kinase A or C were able to induce functional desensitization of A(1)Rs, phosphorylate A(1)Rs in serine and threonine, and trigger clustering of the receptor suggests that phosphorylation of A(1)Rs in serine/threonine is involved in desensitization-related events.