Al. Tucker et al., A(1) ADENOSINE RECEPTORS - 2 AMINO-ACIDS ARE RESPONSIBLE FOR SPECIES-DIFFERENCES IN LIGAND RECOGNITION, The Journal of biological chemistry, 269(45), 1994, pp. 27900-27906
Species differences in ligand binding to A, adenosine receptors were l
ocalized to the seventh transmembrane (TM7) region based on the bindin
g of [8-S-3]cyclopentyl-1,3-dipropylxanthine and three other ligands t
o wild type and six bovine/canine interspecies receptor chimeras expre
ssed in COS-1 cells. Subsequent site-directed mutagenesis experiments
identified amino acid 270 (isoleucine/methionine, bovine/canine) as be
ing primarily responsible for species differences in the binding of N-
6-adenine-substituted compounds, R-N-6-phenylisopropyladenosine (R-PIA
) and (S)-N-6-endonorbornan-2-yl-9-methyladenine, and the C-8-substitu
ted xanthine, [H-3]cyclopentyl-1,3-dipropylxanthine. These data are co
nsistent with the hypothesis that the N-6 region of adenines and the C
-8-region of xanthines bind to the same region of the receptor. A seco
nd TM7 amino acid, 277 (serine/threonine, bovine/canine), selectively
influences the binding of the ribose-substituted adenosine analog, 5'-
N-ethylcarboxamidoadenosine to a variable extent, depending on the nat
ure of amino acid 270. We hypothesize that amino acid 270 of the A, re
ceptor interacts with the N-6 region of adenosine, while amino acid 27
7 is important, especially in the absence of an N-6 substitution, for
interactions with a distinct nucleoside region, possibly on the ribose
.