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.