Delineation of molecular determinants for FR900359 inhibition of Gq/11 unlocks inhibition of Gαs

Michael W Boesgaard,Kasper Harpsøe,Michelle Malmberg,Christina R Underwood,Asuka Inoue,Jesper M Mathiesen,Gabriele M König,Evi Kostenis,David E Gloriam,Hans Bräuner-Osborne

doi:10.1074/jbc.ra120.013002

Michael W Boesgaard, Kasper Harpsøe + Show 8 more

Open Access

https://doi.org/10.1074/jbc.ra120.013002

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Abstract

Heterotrimeric G proteins are essential mediators of intracellular signaling of G protein-coupled receptors. The Gq/11 subfamily consists of Gq, G11, G14, and G16 proteins, of which all but G16 are inhibited by the structurally related natural products YM-254890 and FR900359. These inhibitors act by preventing the GDP/GTP exchange, which is necessary for activation of all G proteins. A homologous putative binding site for YM-254890/FR900359 can also be found in members of the other three G protein families, Gs, Gi/o, and G12/13, but none of the published analogs of YM-254890/FR900359 have shown any inhibitory activity for any of these. To explain why the YM-254890/FR900359 scaffold only inhibits Gq/11/14, the present study delineated the molecular selectivity determinants by exchanging amino acid residues in the YM-254890/FR900359-binding site in Gq and Gs We found that the activity of a Gs mutant with a Gq-like binding site for YM-254890/FR900359 can be inhibited by FR900359, and a minimum of three mutations are necessary to introduce inhibition in Gs In all, this suggests that although the YM-254890/FR900359 scaffold has proven unsuccessful to derive Gs, Gi/o, and G12/13 inhibitors, the mechanism of inhibition between families of G proteins is conserved, opening up the possibility of targeting by other, novel inhibitor scaffolds. In lack of a selective Gαs inhibitor, FR900359-sensitive Gαs mutants may prove useful in studies where delicate control over Gαs signaling would be of the essence.

Highlights

G protein–coupled receptors (GPCRs) comprise the largest family of membrane-bound receptors in the human genome, with ;800 genes [1]
To evaluate G protein conformational changes associated with depsipeptide inhibitor binding, we compared the structures of the inactive GDP-bound heterotrimeric YM-254890– bound Gq protein complex [23] and the GDP=AlF24 -activated monomeric Gaq [38]
The Switch I loop, which is in direct contact with YM254890 [23] and connects the two domains, displays different conformations, and both of these changes provide a tighter fit to the depsipeptide inhibitor (Fig. 1A)

Summary

Introduction

G protein–coupled receptors (GPCRs) comprise the largest family of membrane-bound receptors in the human genome, with ;800 genes (around half of which are olfactory) [1]. We showed that all G protein mutants generated in this study, both in Gaq and Gas, resulted in robust agonist-induced responses relative to baseline when activated by the endogenously expressed M3 or b2 receptors and displayed sufficient expression, making them suitable to investigate FR900359 inhibition

Results

Conclusion