TY - JOUR T1 - Ligand-Induced Phosphorylation, Clustering, and Desensitization of A<sub>1</sub> Adenosine Receptors JF - Molecular Pharmacology JO - Mol Pharmacol SP - 788 LP - 797 DO - 10.1124/mol.52.5.788 VL - 52 IS - 5 AU - Francisco Ciruela AU - Carles Saura AU - Enric I. Canela AU - Josefa Mallol AU - Carmen Lluís AU - Rafael Franco Y1 - 1997/11/01 UR - http://molpharm.aspetjournals.org/content/52/5/788.abstract N2 - Through immunocytochemistry with the use of antibodies against A1 adenosine receptors (A1Rs) and confocal microscopy, we show that stimulation of A1Rs 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 [3H](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 A1Rs 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 A1Rs, phosphorylate A1Rs in serine and threonine, and trigger clustering of the receptor suggests that phosphorylation of A1Rs in serine/threonine is involved in desensitization-related events. ER -