Coupling of g proteins to reconstituted monomers and tetramers of the M2 muscarinic receptor.

Dar'ya S Redka, Takefumi Morizumi, Gwendolynne Elmslie, Pranavan Paranthaman, Rabindra V Shivnaraine, John Ellis, Oliver P Ernst, James W Wells

The Journal of biological chemistry 2014 Aug 29

filter terms:

G protein-coupled receptors can be reconstituted as monomers in nanodiscs and as tetramers in liposomes. When reconstituted with G proteins, both forms enable an allosteric interaction between agonists and guanylyl nucleotides. Both forms, therefore, are candidates for the complex that controls signaling at the level of the receptor. To identify the biologically relevant form, reconstituted monomers and tetramers of the purified M2 muscarinic receptor were compared with muscarinic receptors in sarcolemmal membranes for the effect of guanosine 5'-[β,γ-imido]triphosphate (GMP-PNP) on the inhibition of N-[(3)H]methylscopolamine by the agonist oxotremorine-M. With monomers, a stepwise increase in the concentration of GMP-PNP effected a lateral, rightward shift in the semilogarithmic binding profile (i.e. a progressive decrease in the apparent affinity of oxotremorine-M). With tetramers and receptors in sarcolemmal membranes, GMP-PNP effected a vertical, upward shift (i.e. an apparent redistribution of sites from a state of high affinity to one of low affinity with no change in affinity per se). The data were analyzed in terms of a mechanistic scheme based on a ligand-regulated equilibrium between uncoupled and G protein-coupled receptors (the "ternary complex model"). The model predicts a rightward shift in the presence of GMP-PNP and could not account for the effects at tetramers in vesicles or receptors in sarcolemmal membranes. Monomers present a special case of the model in which agonists and guanylyl nucleotides interact within a complex that is both constitutive and stable. The results favor oligomers of the M2 receptor over monomers as the biologically relevant state for coupling to G proteins. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Citation

Dar'ya S Redka, Takefumi Morizumi, Gwendolynne Elmslie, Pranavan Paranthaman, Rabindra V Shivnaraine, John Ellis, Oliver P Ernst, James W Wells. Coupling of g proteins to reconstituted monomers and tetramers of the M2 muscarinic receptor. The Journal of biological chemistry. 2014 Aug 29;289(35):24347-65