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 (5–15 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.
Footnotes
- Received February 18, 1997.
- Accepted July 23, 1997.
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Send reprint requests to: Prof. Rafael Franco, Departament de Bioquı́mica i Biologia Molecular, Facultat de Quı́mica, Martı́ i Franquès, 1, 08028 Barcelona, Spain. E-mail: r.franco{at}sun.bq.ub.es
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↵1 Current affiliation: MRC Anatomical Neuropharmacology Unit, University of Oxford, Oxford OX1 3TH, UK.
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This work was supported by Comisión Interministerial de Ciencia y Tecnologica Grants PB91/0263 and PB94/0941 and Comissió Interdepartamental de Recerca i Innovació Tecnológica/Comisión Interministerial de Ciencia y Tecnologica Grant QFN93/4423. F.C. and C.S. contributed equally to this work.
- The American Society for Pharmacology and Experimental Therapeutics
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