The effects of saponin on the binding and functional properties of the human adenosine A(1) receptor

1 Experiments with adenosine deaminase suggest that adenosine is present in membrane preparations from CHO cells bearing adenosine A(1) receptors. 2 Pretreatment of the membranes (ca 0.6 mg protein ml(-1)) with the permeabilizing agent saponin (100 mu g ml(-1)) or addition of saponin (10 mu g ml(-1)) to the membranes (0.02-0.08 mg protein ml(-1)) in the assay, generates homogeneous low affinity agonist binding curves in the presence of GTP and an increased function, assessed by agonist stimulation of [S-35]-GTP gamma S binding. The affinity constants for the binding of an agonist and an antagonist are not affected by this saponin treatment. Saponin facilitates the interaction of guanine nucleotides with receptor G-protein complexes, possibly by removing a permeability barrier to access of G-proteins by GTP. However, adenosine is still present in the binding assays after saponin treatment. 3 The agonist binding properties of the human A(1) receptor have been characterized. In saponin pretreated membranes, 80-90% of the A(1) receptors are capable of forming agonist-receptor-G protein complexes in the absence of GTP. These complexes have a 300-600 fold higher affinity than uncoupled receptors for N-6-cyclohexyladenosine. 4 A very slow component is observed in the association and dissociation kinetics of the agonist [H-3]-N-6-cyclohexyladenosine ([H-3]-CHA) and in the association but not dissociation kinetics of the antagonist [H-3]-8-cyclopentyl-1,3-dipropylxanthine ([H-3]-DPCPX). The slow association component of [H-3]-DPCPX is essentially absent when incubations are carried out in the presence of GTP. The slow dissociation component of [H-3]-CHA binding is rapidly disrupted by GTP. 5 It is hypothesized that long-lasting adenosine-receptor-G protein complexes are present in the CHO membrane preparations. The existence of these complexes, resistant to the action of adenosine deaminase but sensitive to GTP, may rationalize the observed kinetics and the increase in H-3-antagonist binding produced by GTP which has been observed in essentially all studies of A(1) receptors and has been ascribed previo usly to precoupling of A(1) receptors to G-proteins in the absence of agonists.