Evidence for receptor-mediated inhibition of intrinsic activity of GTP-binding protein, Gi1 and Gi2, but not G0in 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 G₀, 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 G₀. These findings indicate that ϰ-opioid receptor is coupled to inhibition of intrinsic activities of G_i1 and G_i2, but not G₀, 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.