IDENTIFICATION OF DOMAINS OF THE HUMAN A(1) ADENOSINE RECEPTOR THAT ARE IMPORTANT FOR BINDING-RECEPTOR SUBTYPE-SELECTIVE LIGANDS USING CHIMERIC A(1) A(2A) ADENOSINE RECEPTORS/

To provide new insights into the regions of the human A(1) adenosine r eceptor (A(1)AR) involved in ligand binding, a series of chimeric huma n A(1) and rat A(2a) adenosine receptors (A(1)/A(2a)) were studied. Bi nding studies were initially performed on acutely transfected COS cell s using fixed doses of the A(2a)AR agonist [H-3]CGS-21680, the A(1)AR agonist [H-3]2-chloro-N-6-cyclopentyladenosine (CCPA), and the A(1)AR antagonist [H-3]8-cyclopentyl-1,3-dipropylxanthine (DPCPX). When the r egion of the A(2a)AR from the amino terminus to the end of transmembra ne (TM) 1 was replaced by the corresponding region of the A(1)AR (A(1) TM1/A(2a)), [H-3]CGS-21680 and [H-3]CCPA binding was detectable. When an A(1)TM1-2/A(2a) construct was studied, [H-3]CGS-21680 binding was l ost and [H-3] DPCPX binding appeared. Saturation studies using [H-3]CC PA revealed that the A(1)TM1/A(2a) construct had low affinity. However , with the subsequent addition of A(1)AR TMs 2-4 receptor affinity imp roved markedly. Saturation studies using [H-3]DPCPX also revealed that the TMs 1-4 of the A(1)AR conferred wild-type receptor affinity. When the ligand binding properties of A(1)TM1-4/A(2a), A(1)TM1-6/A(2a), an d wild type A(1)AR constructs were directly compared, no differences w ere found using 10 different compounds. When truncated A(1)ARs that ex tended from the amino terminus to shortly after TM4 were examined, no binding was detectable suggesting that the amino half of the receptor alone is not sufficient for ligand binding. Collectively, these data s uggest that the important determinants for A(1)AR agonist and antagoni st binding and ligand specificity are present in TMs 1-4.