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Delineation of molecular determinants for FR900359 inhibition of G_q/11 unlocks inhibition of Gα_s.

ABSTRACT: Heterotrimeric G proteins are essential mediators of intracellular signaling of G protein-coupled receptors. The G_q/11 subfamily consists of G_q, 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, G_s, G_i/o, and G_12/13, 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 G_q/11/14, the present study delineated the molecular selectivity determinants by exchanging amino acid residues in the YM-254890/FR900359-binding site in G_q and G_s We found that the activity of a G_s mutant with a G_q-like binding site for YM-254890/FR900359 can be inhibited by FR900359, and a minimum of three mutations are necessary to introduce inhibition in G_s In all, this suggests that although the YM-254890/FR900359 scaffold has proven unsuccessful to derive G_s, G_i/o, and G_12/13 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 Gα_s inhibitor, FR900359-sensitive Gα_s mutants may prove useful in studies where delicate control over Gα_s signaling would be of the essence.

SUBMITTER: Boesgaard MW

PROVIDER: S-EPMC7535923 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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

Boesgaard Michael W MW Harpsøe Kasper K Malmberg Michelle M Underwood Christina R CR Inoue Asuka A Mathiesen Jesper M JM König Gabriele M GM Kostenis Evi E Gloriam David E DE Bräuner-Osborne Hans H

The Journal of biological chemistry 20200804 40

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-25489 ...[more]

PMID: 32753482

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Project description:Background & aimsProliferation, differentiation, and morphogenesis of the intestinal epithelium are tightly regulated by a number of molecular pathways. Coordinated action of intestine is achieved by gastrointestinal hormones, most of which exert these actions through G-protein-coupled receptors. We herein investigated the role of Gαq/11-mediated signaling in intestinal homeostasis.MethodsIntestinal tissues from control (Gnaqflox/floxGna11+/+ ), Int-Gq knock-out (KO) (VilCre+/-Gnaqflox/floxGna11+/+ ), G11 KO (Gnaqflox/floxGna11-/- ), and Int-Gq/G11 double knock-out (DKO) (VilCre+/-Gnaqflox/floxGna11-/- ) mice were examined by microscopy, transmission electron microscopy, and immunohistochemistry. The effect of Gαq/11-mediated signaling was studied in the cell lineage, proliferation, and apoptosis. Dextran sodium sulfate (DSS) colitis was induced to study the role of Gαq/11 in colon.ResultsPaneth cells were enlarged, increased in number, and mislocalized in Int-Gq/G11 DKO small intestine. Paneth cells also reacted with PAS and Muc2 antibody, indicating an intermediate character of Paneth and goblet cells. The nuclear β-catenin, T-cell factor 1, and Sox9 expression were reduced severely in the crypt base of Int-Gq/G11 DKO intestine. Proliferation was activated in the crypt base and apoptosis was enhanced along the crypt. Int-Gq/G11 DKO mice were susceptible to DSS colitis. Proliferation was inhibited in the crypt of unaffected and regenerative areas. Cystic crypts, periodic acid-Schiff-positive cells, and Muc2-positive cells were unusually observed in the ulcerative region.ConclusionsThe Gαq/11-mediated pathway plays a pivotal role in the preservation of intestinal homeostasis, especially in Paneth cell maturation and positioning. Wnt/β-catenin signaling was reduced significantly in the crypt base in Gαq/G11-deficient mice, resulting in the defective maturation of Paneth cells, induction of differentiation toward goblet cells, and susceptibility to DSS colitis.

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Delineation of molecular determinants for FR900359 inhibition of G_q/11 unlocks inhibition of Gα_s.

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Delineation of molecular determinants for FR900359 inhibition of G<sub>q/11</sub> unlocks inhibition of Gα<sub>s</sub>.

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