Dominance of G(s) in doubly G(s)/G(i)-coupled chimaeric A(1)/A(2A) adenosine receptors in HEK-293 cells.

A(1) adenosine receptors inhibit adenylate cyclase by activating G(i)/G(o), whereas A(2A) receptors activate G(s). We examined how regions of A(1) and A(2A) receptors regulate coupling to G-proteins by constructing chimaeras in which the third intracellular loops (3ICL or L) and/or the C-termini (or T) were switched. Pertussis toxin (PTX) was used in membrane radioligand binding assays to calculate the fraction of recombinant receptors coupled to G(i)/G(o) and in whole cells to differentially influence agonist-stimulated cAMP accumulation. Switching A(1)/A(2A) 3ICL domains results in receptors that maintain binding selectivity for ligands but are doubly coupled. Receptor chimaeras with an A(1) 3ICL sequence (A(2A)/A(1)L or A(2A)/A(1)LT) respond to agonist stimulation with elevated cAMP despite being coupled predominantly to G(i)/G(o). These chimaeras have basal cAMP levels lower than those of wild-type A(2A) receptors, similar to wild-type A(1) receptors. The A(1) C-terminus modulates the coupling of receptors with A(1) 3ICL such that A(2A)/A(1)LT is better coupled to G(i)/G(o) than A(2A)/A(1)L. The C-terminus has little impact on coupling to receptors containing A(2A) 3ICL sequence. Our results show that the C-terminus sequence selectively facilitates coupling to G(i)/G(o) mediated by A(1) 3ICL and not by other intracellular domains that favour G(i) coupling. The C-terminus sequence has little or no effect on coupling to G(s). For doubly G(s)/G(i)-coupled adenosine receptors in HEK-293 cells, G(s)-mediated stimulation predominates over G(i)/G(o)-mediated inhibition of adenylate cyclase. We discuss the signalling consequences of simultaneously activating opposing G-proteins within single cells.

A1 adenosine receptors. Two amino acids are responsible for species differences in ligand recognition.

Species differences in ligand binding to A1 adenosine receptors were localized to the seventh transmembrane (TM7) region based on the binding of [8-3H]cyclopentyl-1, 3-dipropylxanthine and three other ligands to wild type and six bovine/canine interspecies receptor chimeras expressed in COS-1 cells. Subsequent site-directed mutagenesis experiments identified amino acid 270 (isoleucine/methionine, bovine/canine) as being primarily responsible for species differences in the binding of N6-adenine-substituted compounds, R-N6-phenylisopropyladenosine (R-PIA) and (S)-N6-endonorbornan-2-yl-9-methyladenine, and the C-8-substituted xanthine, [3H]cyclopentyl-1,3-dipropylxanthine. These data are consistent with the hypothesis that the N6 region of adenines and the C-8-region of xanthines bind to the same region of the receptor. A second 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 nature of amino acid 270. We hypothesize that amino acid 270 of the A1 receptor interacts with the N6 region of adenosine, while amino acid 277 is important, especially in the absence of an N6 substitution, for interactions with a distinct nucleoside region, possibly on the ribose.