Interaction of purified bovine brain A1-adenosine receptors with guanine nucleotide-binding proteins of human platelet membranes following reconstitution.
A1-adenosine receptors and associated guanine nucleotide-binding proteins (G proteins) have been co-purified from bovine cerebral cortex by agonist affinity chromatography [J. Biol. Chem. 264:14853-14859 (1989)]. In this study we have reconstituted purified bovine brain A1 receptors into human platelet membranes that contain A2- but no detectable A1-adenosine receptors. The recovery of reconstituted receptors was assessed from the binding of the antagonist radioligand [125I]3-(4-amino-3-iodo)phenethyl-1-propyl-8-cyclopentyl-xanthine and ranged from 32 to 84%. Coupling of reconstituted A1 receptors to platelet G proteins was evaluated by measurement of the high affinity binding of an agonist radioligand, 125I-aminobenzyladenosine, to receptor-G protein complexes and by stereospecific photoaffinity labeling of a 35,000-Da receptor polypeptide with the agonist photoaffinity label 125I-azidobenzyladenosine. Fifty percent of receptors reconstituted into platelet membranes bound agonists with high affinity, indicative of coupling to platelet G proteins. Reconstituted A1 receptors bound various ligands with affinities characteristic of A1 receptors of bovine brain. Although platelets contain both pertussis toxin-sensitive and -insensitive G proteins, reconstituted high affinity agonist binding was almost completely abolished by treatment of platelet membranes with guanosine 5'-3-O-(thio)triphosphate, pertussis toxin, N-ethylmaleimide, or heparin. Following reconstitution, A1 receptors could be resolubilized in complexes with platelet G proteins. The data suggest that marked species differences in the binding affinity of ligands to adenosine receptors result from differences in the receptors rather than membrane structure or G proteins and, further, that A1 receptors couple selectively and tightly to pertussis toxin-sensitive G proteins.
Munshi
R
R
Department of Internal Medicine, Cardiology, University of Virginia, Charlottesville 22908.
Linden
J
J
D000241:Adenosine / Q000031:analogs & derivatives; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000345:Affinity Labels; D000818:Animals; D001667:Binding, Competitive; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D002417:Cattle; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002540:Cerebral Cortex / Q000378:metabolism*; D002793:Cholic Acids; D003902:Detergents; D005033:Ethylmaleimide / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D037342:Pertussis Toxin; D011869:Radioligand Assay; D011983:Receptors, Purinergic / Q000302:isolation & purification / Q000378:metabolism*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D000345:Affinity Labels; D002793:Cholic Acids; D003902:Detergents; D011983:Receptors, Purinergic; D010566:Virulence Factors, Bordetella; D000246:Adenosine Diphosphate Ribose; D006493:Heparin; C060191:N(6)-(4-aminobenzyl)adenosine; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D000241:Adenosine; D005033:Ethylmaleimide; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate