Interactions of the bovine brain A1-adenosine receptor with recombinant G protein alpha-subunits. Selectivity for rGi alpha-3.
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.
Institute of Pharmacology, University of Vienna, Austria.
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Freissmuth
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Schütz
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Linder
D000818:Animals; D001667:Binding, Competitive; D001923:Brain Chemistry / Q000187:drug effects; D002417:Cattle; D005033:Ethylmaleimide / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D007457:Iodine Radioisotopes; D010660:Phenylisopropyladenosine / Q000031:analogs & derivatives; D011869:Radioligand Assay; D011983:Receptors, Purinergic / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
D007457:Iodine Radioisotopes; D011983:Receptors, Purinergic; D011994:Recombinant Proteins; D006153:Guanosine Diphosphate; D010660:Phenylisopropyladenosine; C043459:N(6)-(3-iodo-4-hydroxyphenyl)-1-isopropyladenosine; D019204:GTP-Binding Proteins; D005033:Ethylmaleimide