Abstract

Cell-permeable orthosteric ligands can assist folding of G protein–coupled receptors in the endoplasmic reticulum (ER); this pharmacochaperoning translates into increased cell surface levels of receptors. Here we used a folding-defective mutant of human A₁-adenosine receptor as a sensor to explore whether endogenously produced adenosine can exert a chaperoning effect. This A₁-receptor-Y²⁸⁸A was retained in the ER of stably transfected human embryonic kidney 293 cells but rapidly reached the plasma membrane in cells incubated with an A₁ antagonist. This was phenocopied by raising intracellular adenosine levels with a combination of inhibitors of adenosine kinase, adenosine deaminase, and the equilibrative nucleoside transporter: mature receptors with complex glycosylation accumulated at the cell surface and bound to an A₁-selective antagonist with an affinity indistinguishable from the wild-type A₁ receptor. The effect of the inhibitor combination was specific, because it did not result in enhanced surface levels of two folding-defective human V₂-vasopressin receptor mutants, which were susceptible to pharmacochaperoning by their cognate antagonist. Raising cellular adenosine levels by subjecting cells to hypoxia (5% O₂) reproduced chaperoning by the inhibitor combination and enhanced surface expression of A₁-receptor-Y²⁸⁸A within 1 hour. These findings were recapitulated for the wild-type A₁ receptor. Taken together, our observations document that endogenously formed adenosine can chaperone its cognate A₁ receptor. This results in a positive feedback loop that has implications for the retaliatory metabolite concept of adenosine action: if chaperoning by intracellular adenosine results in elevated cell surface levels of A₁ receptors, these cells will be more susceptible to extracellular adenosine and thus more likely to cope with metabolic distress.