title: Ligand-induced phosphorylation, clustering, and desensitization of A1 adenosine receptors.
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.
authors.affiliation: Departament de Bioquímica i Biologia Molecular, Facultat de Química, Universitat de Barcelona 08028, Barcelona, Catalunya, Spain.
authors.forename: F
authors.initials: F
authors.lastname: Ciruela
authors.forename: C
authors.initials: C
authors.lastname: Saura
authors.forename: E I
authors.initials: EI
authors.lastname: Canela
authors.forename: J
authors.initials: J
authors.lastname: Mallol
authors.forename: C
authors.initials: C
authors.lastname: Lluís
authors.forename: R
authors.initials: R
authors.lastname: Franco
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D001667:Binding, Competitive; D006224:Cricetinae; D015536:Down-Regulation; D005455:Fluorescent Antibody Technique; D008647:Mesocricetus; D018613:Microscopy, Confocal; D009130:Muscle, Smooth / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010766:Phosphorylation; D018047:Receptors, Purinergic P1 / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D014407:Tumor Cells, Cultured / Q000187:drug effects; D014665:Vasodilator Agents / Q000494:pharmacology*
chemical_list: D018047:Receptors, Purinergic P1; D014665:Vasodilator Agents; C047235:N-(1-methyl-2-phenylethyl)adenosine; D000241:Adenosine