Adenosine Deaminase and A1 Adenosine Receptors Internalize Together following Agonist-induced Receptor Desensitization*
- From the Departament de Bioquı́mica i Biologia Molecular, Facultat de Quı́mica, Universitat de Barcelona, 08028 Barcelona, Catalunya, Spain
Abstract
A1 adenosine receptors (A1Rs) and adenosine deaminase (ADA; EC 3.5.4.4) interact on the cell surface of DDT1MF-2 smooth muscle cells. The interaction facilitates ligand binding and signaling via A1R, but it is not known whether it has a role in homologous desensitization of A1Rs. Here we show that chronic exposure of DDT1MF-2 cells to the A1R agonist,N 6-(R)-(phenylisopropyl)adenosine (R-PIA), caused a rapid aggregation or clustering of A1 receptor molecules on the cell membrane, which was enhanced by pretreatment with ADA. Colocalization between A1R and ADA occurred in the R-PIA-induced clusters. Interestingly, colocalization between A1R and ADA also occurred in intracellular vesicles after internalization of both protein molecules in response to R-PIA. Agonist-induced aggregation of A1Rs was mediated by phosphorylation of A1Rs, which was enhanced and accelerated in the presence of ADA. Ligand-induced second-messenger desensitization of A1Rs was also accelerated in the presence of exogenous ADA, and it correlated well with receptor phosphorylation. However, although phosphorylation of A1R returned to its basal state within minutes, desensitization continued for hours. The loss of cell-surface binding sites (sequestration) induced by the agonist was time-dependent (t½= 10 ± 1 h) and was accelerated by ADA. All of these results strongly suggest that ADA plays a key role in the regulation of A1Rs by accelerating ligand-induced desensitization and internalization and provide evidence that the two cell surface proteins internalize via the same endocytic pathway.
Footnotes
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↵* This work was supported by a joint (Echevarne Fundation and Spanish Ministry of Education) PETRI Grant (PTR92/0047) administered by Fundació Bosch i Gimpera and by Fondo de Investigaciones Sanitarias de la Seguridad Social Grant 91/0272, ComissióInterdepartamental de Recerca i InnovacióTecnológica-Comisión Interministerial de Ciencia y Tecnologı́a, Spain (CICYT) Grant QFN93/4423, and CICYT Grants PB94/0941 and SAF97/0066.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ To whom correspondence should be addressed: Dept. Bioquı́mica i Biologia Molecular, Universitat de Barcelona, Facultat de Quı́mica, Martı́ i Franquès 1, 08028 Barcelona, Spain. Tel.: 34-934021208; Fax: 34-934021219; E-mail:r.franco{at}sun.bq.ub.es.
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↵1 The abbreviations used are: A1R, A1 adenosine receptor; ADA, adenosine deaminase;R-PIA,N 6-(R)-(phenylisopropyl)adenosine; FITC, fluorescein isothiocyanate; TRITC, rhodamine isothiocyanate; β2-AR, β2-adrenergic receptor; DMEM, Dulbecco’s modified Eagle’s medium; PBS, phosphate-buffered saline; HBSS, Hanks’ balanced salt solution.
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↵2 A. Navarro, C. A. Saura, J. Mallol, E. I. Canela, C. Lluis, and R. Franco, manuscript in preparation.
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- Received November 12, 1997.
- Revision received March 10, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
