A threonine residue in the seventh transmembrane domain of the human A1 adenosine receptor mediates specific agonist binding.
The A1 adenosine receptor is a member of the seven-transmembrane G protein-coupled, receptor superfamily. This receptor binds the purine nucleoside adenosine with high affinity and inhibits the activity of adenylate cyclase. We have used site-directed mutagenesis and functional expression studies to examine the role of the threonine residue, located at position 277 in transmembrane domain VII of the human A1 receptor. Mutation of Thr-277 to either serine or alanine resulted in the expression of receptors that had essentially no change in binding affinity for the A1 selective antagonist 8-cyclo-pentyl-1,3-dipropylxanthine. Mutation of Thr-277 to serine resulted in modest (4.4-8.6-fold) but significant increases in the observed Ki values for three adenosine agonists, namely N-(1-methyl-2-phenethyl)adenosine (R-PIA or S-PIA) and 1-(6-amino-9H-purin-9-yl)-1-deoxy-N-ethyl-beta-L- ribofuranuronamide) (NECA). However, mutation of Thr-277 to alanine resulted in no significant changes in the Ki for R-PIA or S-PIA but did result in a highly significant 437-fold increase in the Ki for NECA. This demonstrates that the hydroxyl moiety of Thr-277 mediates agonist but not antagonist binding and, more specifically, that this residue forms a probable molecular contact site with the 5' substitution found in NECA.
Townsend-Nicholson
A
A
Garvan Institute of Medical Research, Darlinghurst, New South Wales, Sydney, Australia.
Schofield
P R
PR
D000241:Adenosine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D019830:Adenosine-5'-(N-ethylcarboxamide); D000595:Amino Acid Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011869:Radioligand Assay; D018047:Receptors, Purinergic P1 / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013237:Stereoisomerism; D013806:Theophylline / Q000031:analogs & derivatives / Q000378:metabolism; D013912:Threonine; D014316:Tritium
D018047:Receptors, Purinergic P1; D011994:Recombinant Proteins; D014316:Tritium; C047235:N-(1-methyl-2-phenylethyl)adenosine; D013912:Threonine; C053907:8-cyclopentyl-1,3-dimethylxanthine; D019830:Adenosine-5'-(N-ethylcarboxamide); D013806:Theophylline; D000241:Adenosine