A "locked-on," constitutively active mutant of the adenosine A1 receptor.

Abstract We studied the wild-type human adenosine A 1 receptor and three mutant receptors, in which the glycine at position 14 had been changed into an alanine, a leucine, or a threonine residue. All receptors were characterized in radioligand binding experiments, the wild-type and the Gly 14 Thr mutant receptor in greater detail. Both receptors were allosterically modulated by sodium ions and PD81,723 (2-amino-4,5-dimethyl-3-thienyl-[3(trifluoromethyl)-phenyl]methanone), although in a different way. All mutant receptors appeared to be spontaneously or “constitutively” active in a [ 35 S]GTPγS binding assay, the first demonstration of the existence of such CAM (constitutively active mutant) receptors for the adenosine A 1 receptor. The Gly 14 Thr mutant receptor was also constitutively active in another functional assay, i.e., the inhibition of forskolin-induced cAMP production in intact cells. Importantly, this mutant displayed a peculiar “locked-on” phenotype, i.e., neither agonist nor inverse agonist was capable of modulating the basal activity in both the GTPγS and the cAMP assay, unlike the wild-type and the two other mutant receptors.