Chaperoning of the A1-adenosine receptor by endogenous adenosine - an extension of the retaliatory metabolite concept.

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 A(1)-adenosine receptor as a sensor to explore whether endogenously produced adenosine can exert a chaperoning effect. This A(1)-receptor-Y(288)A was retained in the ER of stably transfected human embryonic kidney 293 cells but rapidly reached the plasma membrane in cells incubated with an A(1) 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 A(1)-selective antagonist with an affinity indistinguishable from the wild-type A(1) receptor. The effect of the inhibitor combination was specific, because it did not result in enhanced surface levels of two folding-defective human V-2-vasopressin receptor mutants, which were susceptible to pharmacochaperoning by their cognate antagonist. Raising cellular adenosine levels by subjecting cells to hypoxia (5% O-2) reproduced chaperoning by the inhibitor combination and enhanced surface expression of A(1)-receptor-Y(288)A within 1 hour. These findings were recapitulated for the wild-type A(1) receptor. Taken together, our observations document that endogenously formed adenosine can chaperone its cognate A(1) 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 A(1) receptors, these cells will be more susceptible to extracellular adenosine and thus more likely to cope with metabolic distress.