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 prese nt in membrane preparations from CHO cells bearing adenosine A(1) rece ptors. 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 sap onin (10 mu g ml(-1)) to the membranes (0.02-0.08 mg protein ml(-1)) i n 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 nucl eotides with receptor G-protein complexes, possibly by removing a perm eability barrier to access of G-proteins by GTP. However, adenosine is still present in the binding assays after saponin treatment. 3 The ag onist binding properties of the human A(1) receptor have been characte rized. In saponin pretreated membranes, 80-90% of the A(1) receptors a re capable of forming agonist-receptor-G protein complexes in the abse nce of GTP. These complexes have a 300-600 fold higher affinity than u ncoupled receptors for N-6-cyclohexyladenosine. 4 A very slow componen t is observed in the association and dissociation kinetics of the agon ist [H-3]-N-6-cyclohexyladenosine ([H-3]-CHA) and in the association b ut 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 a denosine-receptor-G protein complexes are present in the CHO membrane preparations. The existence of these complexes, resistant to the actio n of adenosine deaminase but sensitive to GTP, may rationalize the obs erved kinetics and the increase in H-3-antagonist binding produced by GTP which has been observed in essentially all studies of A(1) recepto rs and has been ascribed previo usly to precoupling of A(1) receptors to G-proteins in the absence of agonists.