Differences in the Allosteric Interaction between Agonists and GMP-PNP in Monomers and Oligomers of the M2 Muscarinic Cholinergic Receptor Fused to GαI1

Abstract

The nature of the signaling complex between G protein-coupled receptors (GPCRs) and G proteins remains unclear. GPCRs can exist as oligomers, yet monomers can activate G proteins when reconstituted in nanodiscs; the interaction with the G protein often is considered transient, yet some evidence suggests that the RG complex remains intact during signaling. Two classes of sites typically are observed in the binding of agonists to GPCRs in natural membranes, and guanylyl nucleotides such as GMP-PNP effect an apparent interconversion from higher (KH) to lower affinity (KL) without affecting affinity per se. We have examined three models of a stable RG complex in which the M2 muscarinic receptor is fused to Gαi1 via different linking sequences. Each fusion protein was expressed in Sf9 cells and characterized as an oligomer in digitonin-solubilized preparations, as a purified monomer in solution, and as an oligomer reconstituted in phospholipid vesicles. In the oligomeric state, the agonist oxotremorine-M recognized two classes of sites; GMP-PNP progressively increased the low-affinity fraction without affecting KH or KL. In the monomeric state, oxotremorine-M similarly recognized two classes of sites, most of which were of high affinity (FH=71%); GMP-PNP progressively increased KH, as expected for an allosteric interaction between two sites, without affecting KL or FH. The effect of GMP-PNP in monomers required the presence of DTT; the nucleotide-independent sites of low affinity may represent a subpopulation in which communication between the receptor- and αi1-domains has been compromised. Only the oligomeric form of the fusion protein mimics the behavior of GPCRs in natural membranes, suggesting that signaling in vivo proceeds via a stable complex comprising multiple equivalents of receptor and G protein. (Supported by HSFO and CIHR)