The density of G-protein coupled receptors at the cell surface can be increased by treatment with cell-permeable orthosteric ligands, called pharmacological chaperones. These compounds act in the endoplasmic reticulum, where they facilitate folding and stabilize the correct conformation of maturating proteins. In this work, we wanted to explore if endogenously generated adenosine can chaperone its cognate receptor. To test this hypothesis: (i) we used an ER-retained, folding-defective mutant of the human A1-adenosine receptor as a sensor and (ii) we raised the cellular levels of endogenous adenosine by simultaneous inhibition of adenosine kinase, adenosine deaminase and equilibrative nucleoside transporters or by hypoxia. The applied inhibitors restored the surface expression of the A1-Y288A-receptor in stably transfected HEK 293 cells, similarly to the A1-antagonist DPCPX.
The upregulated receptor had mature glycosylation pattern and was binding-competent, with a radioligand affinity indistinguishable form the wild type A1-receptor. The effect of the inhibitors was specific, as it did not enhance the cell surface expression of the folding-deficient V2-vasopressin receptor, which in contrast responded to pharmacochaperoning by their cognate antagonist. Application of hypoxia phenocopied the effect of the inhibitors; endogenous adenosine increased the number of the A1-Y288A-receptors at the plasma membrane within 1 hour. These results were recapitulated for the wild type A1-receptor.
Taken together, our work documents that endogenous adenosine may act as a ligand chaperone and thus counteract the damaging effects of hypoxia via increased A1-receptor signaling. This mechanism can be seen as a ramification of the retaliatory metabolite concept of adenosine and a new twist in understanding the role of the endoplasmic reticulum.