Purification of A1 adenosine receptor–G-protein complexes: effects of receptor down-regulation and phosphorylation on coupling

摘要

pWe examined the effects of exposing Asub1/sub adenosine receptors (Asub1/subARs) to an agonist on the stability and phosphorylation state of receptor–guanine nucleotide-binding regulatory protein (R–G-protein) complexes. Non-denatured recombinant human Asub1/subARs extended on the N-terminus with hexahistidine (Hissub6/sub) and the FLAG (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) epitope (H/F) were purified to near homogeneity from stably transfected Chinese-hamster ovary (CHO)-K1 cells. Purified receptors have pharmacological properties similar to receptors in membranes. G-proteins were co-purified with 15±2% of H/F-Asub1/subAR unless receptor–G-protein (R–G) complexes were uncoupled by pre-treating cell membranes with GTP. By silver staining, purified Asub1/subAR–G-protein complexes contain receptors, G-protein α and β subunits and an unidentified 97 kDa protein. Pretreating intact cells with iN/isup6/sup-cyclopentyladenosine (CPA) for 24 h decreased both the total number of receptors measured in membranes and the number of purified Asub1/subARs by about 50%. In contrast, pretreating cells with CPA decreased the number of R–G complexes measured in membranes (54±6%) significantly less than it decreased the number of purified R–G complexes (78±3%) as detected by sup125/supI-iN/isup6/sup-(4-aminobenzyl)adenosine binding or by Western blotting Gsubi/subα2. The effect of CPA to decrease the fraction of receptors purified as R–G complexes was not associated with any change in low-level Asub1/subAR phosphorylation (found on serine), or low-level phosphorylation of G-protein α or β subunits or the 97 kDa protein. These experiments reveal a novel aspect of agonist-induced down-regulation, namely a diminished stability of receptor–G-protein complexes that is manifested as uncoupling during receptor purification./p