Activation of guanosine 5'-[gamma-(35)S]thio-triphosphate binding through M(1) muscarinic receptors in transfected Chinese hamster ovary cell membranes; 1. Mathematical analysis of catalytic G protein activation.
I analyzed in this work the effect of agonists and unlabeled guanyl nucleotides on [(35)S]GTP gamma S and [(3)H]NMS binding to transfected CHO cells expressing hM(1) 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 GTP gamma S, and the occupied G protein (G) dissociated reversibly from the receptor. Agonists accelerated the GDP release from receptor-coupled G proteins and accelerated the G dissociation: both effects accelerated synergically the G protein-GTP gamma S association reaction in the presence of GDP. GTP gamma S-bound G proteins, G, competed efficiently with inactive (empty or GDP-bound) G proteins for receptor recognition, and were able, therefore, at low concentrations, to quench the [(35)S]GTP gamma S binding reaction.
PubMed Abstracts
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