Interactions of the bovine brain A1-adenosine receptor with recombinant G protein alpha-subunits. Selectivity for rGi alpha-3

• Published in: The Journal of biological chemistry 1991-09, Vol.266 (27), p.17778-17783
• Main Authors: Freissmuth, M., Schütz, W., Linder, M.E.
• Format: Article
• Language: English
• Subjects: Animals; Binding, Competitive; Biological and medical sciences; Brain Chemistry - drug effects; Cattle; Ethylmaleimide - pharmacology; Fundamental and applied biological sciences. Psychology; GTP-Binding Proteins - metabolism; Guanosine Diphosphate - metabolism; Interactions. Associations; Intermolecular phenomena; Iodine Radioisotopes; Molecular biophysics; Phenylisopropyladenosine - analogs & derivatives; Radioligand Assay; Receptors, Purinergic - metabolism; Recombinant Proteins - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)55194-2

The ability of the bovine brain A1-adenosine receptor to discriminate between different G protein subtypes was tested using G protein alpha-subunits synthesized in Escherichia coli (rG alpha-subunits). When combined with a 3-fold molar excess of beta gamma-subunit purified from bovine brain and used at high concentrations, all three subtypes of rGi alpha (rGi alpha-1, rGi alpha-2, and rGi alpha-3) and rGo alpha were capable of reconstituting guanine nucleotide-sensitive high-affinity binding of the agonist radioligand (-)-N6-3-[125I] (iodo-4-hydroxyphenylisopropyl) adenosine ([125I]HPIA) to the purified A1-adenosine receptor (Kd approximately 1.2 nM). Titration of the A1-adenosine receptor with increasing amounts of rG alpha revealed a approximately 10-fold higher affinity for rGi alpha-3 compared with rGi alpha-1, rGi alpha-2, and rGo alpha. This selectivity was also observed in the absence of beta gamma. Other alpha-subunits (rGs alpha-s, rGs alpha-L, rGs alpha PT, and rGz alpha) did not promote [125I]HPIA binding to the purified receptor. In N-ethylmaleimide-treated bovine brain membranes, rGi alpha-3 was the only rG alpha-subunit capable of reconstituting high-affinity agonist binding. Similarly, rGi alpha-3 competed potently with rGo alpha for activation by the agonist-liganded A1-adenosine receptor, whereas a approximately 50-fold molar excess of rGo alpha was required to quench the receptor-mediated release of [alpha-32P]GDP from rGi alpha-3. Hence, in spite of the extensive homology between alpha-subunits belonging to the Gi/Go group, the A1-adenosine receptor appears to discriminate between the subtypes. This specificity is likely to govern transmembrane signaling pathways in vivo.