To provide new insights into the regions of the human A adenosine receptor (AAR) involved in ligand binding, a series of chimeric human A and rat A adenosine receptors (A/A) were studied. Binding studies were initially performed on acutely transfected COS cells using fixed doses of the AAR agonist [H]CGS-21680, the AAR agonist [H]2-chloro-N-cyclopentyladenosine (CCPA), and the AAR antagonist [H]8-cyclopentyl-1,3-dipropylxanthine (DPCPX). When the region of the AAR from the amino terminus to the end of transmembrane (TM) 1 was replaced by the corresponding region of the AAR (ATM1/A), [H]CGS-21680 and [H]CCPA binding was detectable. When an ATM1-2/A construct was studied, [H]CGS-21680 binding was lost and [H] DPCPX binding appeared. Saturation studies using [H]CCPA revealed that the ATM1/A construct had low affinity. However, with the subsequent addition of AAR TMs 2-4 receptor affinity improved markedly. Saturation studies using [H]DPCPX also revealed that the TMs 1-4 of the AAR conferred wild-type receptor affinity. When the ligand binding properties of ATM1-4/A, ATM1-6/A, and wild type AAR constructs were directly compared, no differences were found using 10 different compounds. When truncated AARs that extended 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 suggest that the important determinants for AAR agonist and antagonist binding and ligand specificity are present in TMs 1-4.

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