Abstract
Chronic exposure of A1 adenosine receptors (A1R) to A1R agonists leads to activation, phosphorylation, desensitization, and internalization to intracellular compartments of the receptor. Desensitization and internalization of A1R is modulated by adenosine deaminase (ADA), an enzyme that regulates the extracellular concentration of adenosine. ADA interacts with A1R on the cell surface of the smooth muscle cell line DDT1 MF-2, and both proteins are internalized following agonist stimulation of the receptor. The mechanism involved in A1R and ADA internalization upon agonist exposure is poorly understood in epithelial cells. In this report, we show that A1R and ADA interact in LLC-PK1 epithelial cells. Exposure of LLC-PK1 cells to A1R agonists induces aggregation of A1R and ADA on the cell surface and their translocation to intracellular compartments. Biochemical and cell biology assays were used to characterize the intracellular vesicles containing both proteins after agonist treatment. A1R and ADA colocalized together with the rafts marker protein caveolin. Filipin, a sterol-binding agent that disrupts rafts (small microdomains of the plasma membrane), was able to inhibit A1R internalization. In contrast, acid treatment of the cells, which disrupts internalization via clathrin-coated vesicles, did not inhibit agonist-stimulated A1R internalization. We demonstrated that A1R agonist N 6-(R)-phenylisopropyl adenosine promotes the translocation of A1R into low-density gradient fractions containing caveolin. Furthermore, a direct interaction of the C-terminal domain of A1R with caveolin-1 was demonstrated by pull down experiments. These results indicate that A1R and ADA form a stable complex in the cell surface of LLC-PK1 cells and that agonist-induced internalization of the A1 adenosine receptor and ADA is mediated by clathrin-independent endocytosis.
Footnotes
- Received June 14, 2000.
- Accepted January 29, 2001.
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Send reprint requests to: Rafael Franco, Dept. Bioquı́mica i Biologia Molecular, Universitat de Barcelona, Martı́ i Franquès 1, 08028 Barcelona, Spain. E-mail address: r.franco{at}sun.bq.ub.es
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This research was supported by Spanish Comisión Interministerial de Ciencia y Tecnologı́a R+D program (Grants PB97-0984 and BIO1999-0601-C02).
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1 Present address: Molecular Neurogenetics Unit, Massachusetts General Hospital, 149 13th St., Charlestown, MA 02129.
- The American Society for Pharmacology and Experimental Therapeutics
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