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

Abstract

We examined the effects of exposing A1 adenosine receptors (A1ARs) to an agonist on the stability and phosphorylation state of receptor–guanine nucleotide-binding regulatory protein (R–G-protein) complexes. Non-denatured recombinant human A1ARs extended on the N-terminus with hexahistidine (His6) 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-A1AR unless receptor–G-protein (R–G) complexes were uncoupled by pre-treating cell membranes with GTP. By silver staining, purified A1AR–G-protein complexes contain receptors, G-protein α and β subunits and an unidentified 97 kDa protein. Pretreating intact cells with N6-cyclopentyladenosine (CPA) for 24 h decreased both the total number of receptors measured in membranes and the number of purified A1ARs 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 125I-N6-(4-aminobenzyl)adenosine binding or by Western blotting Giα2. The effect of CPA to decrease the fraction of receptors purified as R–G complexes was not associated with any change in low-level A1AR 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.

A Chemical Biological Approach to Study G Protein-Coupled Receptors: Labeling the Adenosine A1 Receptor Using an Electrophilic Covalent Probe

“…The slightly higher mass can be explained by N-glycosylation of the A 1 AR, as has been seen in early purification studies of endogenous A 1 AR. , Indeed, upon incubation with PNGase, a strong reduction is seen in the molecular weight of the corresponding band (Figure C). Preincubation with the reversible antagonist DPCPX resulted in full disappearance of both bands, thereby confirming that this pattern represents the A 1 AR.…”

Purification and characterization of the human adenosine A2a receptor functionally expressed in Escherichia coli

“…Puri®cation of a hA2aR fusion protein (M-A2aTr316-H10), expressed in E. coli, resulted in 1.5 mg of homogenous, fully functional protein from 100 g of wet cells. The amount of puri®ed receptor protein is at least 100-fold greater than that for adenosine A 1 receptor obtained from different tissues or Table 2. for tagged A 1 receptor puri®ed from stably transfected CHO cells [17,31]. To date, no quanti®ed puri®cation of the adenosine A 2a receptor has been reported.…”

Acute down‐regulation of adenosine A1 receptor activity in status epilepticus

“…A possible explanation is that adenosine concentrations in the DG following stimulus-bound SE are too low to be detected by A 1 Rs. Alternative explanations are that there is a down-regulation of A 1 Rs (Hettinger et al, 1998;Rebola et al, 2005;Roman et al, 2008;Mundell & Kelly, 2011) or that there has been an interruption in the intracellular signaling cascade (Green et al, 1990;Ciruela et al, 1997;Gao et al, 1999;Jajoo et al, 2010). We explored these possibilities further.…”