@article {Ciruela788, author = {Francisco Ciruela and Carles Saura and Enric I. Canela and Josefa Mallol and Carmen Llu{\'\i}s and Rafael Franco}, title = {Ligand-Induced Phosphorylation, Clustering, and Desensitization of A1 Adenosine Receptors}, volume = {52}, number = {5}, pages = {788--797}, year = {1997}, doi = {10.1124/mol.52.5.788}, publisher = {American Society for Pharmacology and Experimental Therapeutics}, abstract = {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{\textendash}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.}, issn = {0026-895X}, URL = {https://molpharm.aspetjournals.org/content/52/5/788}, eprint = {https://molpharm.aspetjournals.org/content/52/5/788.full.pdf}, journal = {Molecular Pharmacology} }