Abstract

I analyzed in this work the effect of agonists and unlabeled guanyl nucleotides on [³⁵S]GTPγS and [³H]NMS binding to transfected CHO cells expressing hM₁ muscarinic receptors. I was unable to explain my kinetic results by “traditional” (one-site, two-site, or two-step) bimolecular binding models. I therefore examined the equations that describe catalytic G protein activation. My results were fully consistent with the following interpretation: G protein-coupled receptors either interacted with GDP-bound G proteins and facilitated the GDP release or recognized empty G proteins, depending on the incubation conditions. The receptor-coupled empty G proteins (RG) then recognized GTPγS, and the occupied G protein (G GTPγS* ) dissociated reversibly from the receptor. Agonists accelerated the GDP release from receptor-coupled G proteins and accelerated theG GTPγS* dissociation: both effects accelerated synergically the G protein-GTPγS association reaction in the presence of GDP. GTPγS-bound G proteins,G GTPγS* , competed efficiently with inactive (empty or GDP-bound) G proteins for receptor recognition, and were able, therefore, at low concentrations, to quench the [³⁵S]GTPγS binding reaction.