Evidence for receptor-mediated inhibition of intrinsic activity of GTP-binding protein, G i1 and G i2, but not G 0 in reconstitution experiments

Abstract

The receptor-mediated inhibition of intrinsic activities of GTP-binding proteins (G-proteins) was studied. Pertussis toxin (IAP)-substrate G-protein, G i1, G i2 or G 0, was prelabeled with [α- 32P]GDP and reconstituted with synaptic membranes of the guinea pig cerebellum in the presence of 0.02% of Chaps. Intrinsic activities of G-proteins were evaluated by the release of [α- 32P]GDP in exchange for added GppNHp or GDP in reconstituted preparations. U-50,488H (1 nM-10 μM), a specific ϰ-subtype of opioid receptor agonist, inhibited the [α- 32P]GDP release in exchange for added 1 μM GppNHp in G i1-reconstituted preparations in a concentration-dependent manner. On the other hand, the ϰ-opioid agonist at 10 μM increases the K m values of GppNHp, but not GDP in exchange for [α- 32P]GDP release in preparations reconstituted with G i1 or G i2, but not with G 0. These findings indicate that ϰ-opioid receptor is coupled to inhibition of intrinsic activities of G i1 and G i2, but not G 0, in guinea pig cerebellar membranes. In addition, it was revealed that the mode of action is mediated by a decrease in affinity of GTP (or its analog) for G-proteins, but not by a change in affinity of GDP.