Ligand-Induced Phosphorylation, Clustering, and Desensitization of A1 Adenosine Receptors
- Departament de Bioquı́mica i Biologia Molecular, Facultat de Quı́mica, Universitat de Barcelona 08028, Barcelona, Catalunya, Spain
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
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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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↵2 R. Franco, unpublished observations.
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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.
- Abbreviations:
- A1R
- A1 adenosine receptor
- ADA
- adenosine deaminase
- PKA
- protein kinase A
- PKC
- protein kinase C
- HBSS
- Hanks’ balanced salt solution
- DMEM
- Dulbecco’s modified Eagle’s medium
- PBS
- phosphate-buffered saline
- CHO
- Chinese hamster ovary
- HEPES
- 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
- AM
- acetoxymethyl ester
- (R)-PIA
- (R)-phenylisopropyladenosine
- DPCPX
- 1,3-dipropyl-8-cyclopentylxanthine
- PMA
- phorbol-12-myristate-13-acetate
- Clφ
- chlorophenyl
- SDS
- sodium dodecyl sulfate
- PAGE
- polyacrylamide gel electrophoresis
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- Received February 18, 1997.
- Accepted July 23, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
