Purification of A1 adenosine receptor-G-protein complexes: effects of receptor down-regulation and phosphorylation on coupling.
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 alpha and beta 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 Gialpha2. 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 alpha or beta 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.
Department of Molecular Physiology and Biological Physics, Health Sciences Center Box 449, University of Virginia, Charlottesville, VA 22908, USA.
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Gao
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Robeva
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Linden
D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D002846:Chromatography, Affinity; D006224:Cricetinae; D015536:Down-Regulation; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D010766:Phosphorylation; D058906:Purinergic P1 Receptor Agonists; D018047:Receptors, Purinergic P1 / Q000302:isolation & purification* / Q000378:metabolism
D058906:Purinergic P1 Receptor Agonists; D018047:Receptors, Purinergic P1; D019204:GTP-Binding Proteins