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 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.

Human A2AAdenosine Receptors: High-Affinity Agonist Binding to Receptor-G Protein Complexes Containing Gβ4

“…In contrast to recombinant hA 2A ARs, recombinant hA 1 ARs and hA 3 ARs do couple sufficiently well to G proteins to readily allow detection of the high-affinity agonist binding conformation characterized by agonist binding that is largely inhibited by GTP␥S (Gao et al, 1999). This may be related to the fact that G i/o is more abundant in mammalian cells than is G s .…”

Gα11 Signaling through ARF6 Regulates F-Actin Mobilization and GLUT4 Glucose Transporter Translocation to the Plasma Membrane

“…21,2001 G ␣ 11 SIGNALING AND ARF6 5269 contrast, similar expression of HA-ARF6(T27N) in the 3T3-L1 adipocytes had no inhibitory effect on basal hexose uptake or on the marked response of 2-deoxyglucose uptake to insulin (Fig. 10).…”

“…In rat epitrochlearis and soleus muscles, removing adenosine with adenosine deaminase or blocking its action with an adenosine receptor antagonist markedly reduces the responsiveness of glucose transport to stimulation by insulin (23). The A 1 adenosine receptor is tightly coupled to the heterotrimeric G protein G i (21). Agents which downregulate G i [prostaglandin E1 and N 6 -(2-phenylisopropyl)adenosine] have been shown to decrease the maximal response of the cells to insulin by ϳ30% (22,23).…”

A3Adenosine Receptors in Human Astrocytoma Cells: Agonist-Mediated Desensitization, Internalization, and Down-Regulation

“…These results are also supported by the known differences in desensitization properties between the A 1 and A 3 AR subtypes (Palmer et al, 1996), whereas A 3 AR undergoes a rapid desensitization (as also supported by the present results), signaling via the A 1 AR does not easily subside upon sustained agonist exposure. It takes about 12 h to down-regulate A 1 AR receptor number and to attenuate its ability to inhibit adenylyl cyclase (Ciruela et al, 1997;Gao et al, 1999). These desensitization pattern differences have been attributed to a different sensitivity of the two receptor subtypes to phosphorylation by G protein-coupled receptor kinase proteins (Ferguson et al, 2000).…”