INTERACTIONS OF PURIFIED BOVINE BRAIN ADENOSINE-A1-RECEPTORS WITH G-PROTEINS - RECIPROCAL MODULATION OF AGONIST AND ANTAGONIST BINDING

The bovine brain A1-adenosine receptor was purified 8000-fold by affinity chromatography on xanthine-amine-congener (XAC)-Sepharose. Addition of a 120-fold molar excess of a purified bovine brain G-protein preparation (G(o,i) a mixture of G(o) and G(i), containing predominantly G(o)) decreases the B(max.) of the binding of the antagonist radioligand [H-3]XAC to the receptor. This decease is observed not only after insertion into phospholipid vesicles but also in detergent solution, and is reversed by GTP analogues. In the presence of G(o,i) about 20 and 40% of the receptors display guanine-nucleotide-sensitive high-affinity binding of the agonist radioligand (-)-N6-3-([I-125]iodo-4-hydroxyphenylisopropyl)adenosine after reconstitution into lipid vesicles and in detergent solution, respectively. The ability of G(o,i) to enhance agonist binding and decrease antagonist binding is concentration-dependent, with a half-maximal effect occurring at approximately 10-fold molar excess of G-proteins over A1-adenosine receptors. In the presence of the receptor, the rate of guanosine 5'-[gamma-[S-35]thio]triphosphate (GTP[S-35]) binding to G(o,i) is accelerated. This rate is further enhanced if the receptor is activated by the agonist (-)(R)-N6-phenylisopropyladenosine, whereas the antagonist XAC decreases the association rate of GTP[S-35] to levels observed in the absence of receptor. These results show (1) that detergent removal is not a prerequisite for the observation of coupling between the A1-adenosine receptor and G(o,i) and (2) that the regulatory effect of G-proteins on antagonist binding to the A1-adenosine receptor can be reconstituted by using purified components.