Recruitment of a cytoplasmic chaperone relay by the A(2A) adenosine receptor

The adenosine A(2A) receptor is a prototypical rhodopsin-like G protein-coupled receptor but has several unique structural features, in particular a long C terminus (of >120 residues) devoid of a palmitoylation site. It is known to interact with several accessory proteins other than those canonically involved in signaling. However, it is evident that many more proteins must interact with the A(2A) receptor, if the trafficking trajectory of the receptor is taken into account from its site of synthesis in the endoplasmic reticulum (ER) to its disposal by the lysosome. Affinity-tagged versions of the A(2A) receptor were expressed in HEK293 cells to identify interacting partners residing in the ER by a proteomics approach based on tandem affinity purification. The receptor-protein complexes were purified in quantities sufficient for analysis by mass spectrometry. We identified molecular chaperones (heat-shock proteins HSP90 and HSP70-1A) that interact with and retain partially folded A(2A) receptor prior to ER exit. Complex formation between the A(2A) receptor and HSP90 (but not HSP90) and HSP70-1A was confirmed by co-affinity precipitation. HSP90 inhibitors also enhanced surface expression of the receptor in PC12 cells, which endogenously express the A(2A) receptor. Finally, proteins of the HSP relay machinery (e.g. HOP/HSC70-HSP90 organizing protein and P23/HSP90 co-chaperone) were recovered in complexes with the A(2A) receptor. These observations are consistent with the proposed chaperone/coat protein complex II exchange model. This posits that cytosolic HSP proteins are sequentially recruited to folding intermediates of the A(2A) receptor. Release of HSP90 is required prior to recruitment of coat protein complex II components. This prevents premature ER export of partially folded receptors

Chaperoning of the A(1)-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