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

Abstract

Heterotrimeric G proteins are essential mediators of intracellular signaling of G protein-coupled receptors. The Gq/11subfamily 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 suggest 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 for targeting by other, novel inhibitor scaffolds. In lack of a selective Gαs inhibitor, FR900359 sensitive Gas mutants may prove useful in studies where delicate control over Gαs signaling would be of the essence.

An experimental strategy to probe Gq contribution to signal transduction in living cells

“…Earlier studies by us and others have introduced Gαq variants that are functional and either partially or entirely resistant to the inhibitory action of FR and YM ( 25 , 63 , 73 ). Yet, neither study intended to probe a causal relationship between FR action and Gq inhibition or to interrogate the occurrence of potential off-target effects, which may confound data analysis and interpretation.…”

“…This distinction ideally requires a mutant designed to achieve the greatest possible loss of FR sensitivity with the minimal number of amino acid replacements. Currently available FR-insensitive Gαq mutants were generated by swapping either five or three Gαq residues for those of Gα16 and Gαs, respectively, two proteins which are naturally not FR regulated ( 63 , 73 ). We reasoned that even fewer but more drastic amino acid changes may suffice to fully ablate inhibitor action, provided that they disrupt key hydrophobic forces between FR and its protein target.…”

“…We reasoned that even fewer but more drastic amino acid changes may suffice to fully ablate inhibitor action, provided that they disrupt key hydrophobic forces between FR and its protein target. Guided by our own and published knowledge of the FR binding site ( 25 , 63 , 73 ), we predicted replacement of phenylalanine 75 (CGN: H.HA.7) and isoleucine 190 (CGN: G.S2.2) to be particularly impactful. Both residues are key components of a small cluster of nonpolar amino acids and form crucial hydrophobic interactions with FR by engaging with the N-acetyl-hydroxyleucine and the phenyllactic acid building blocks of the inhibitor ( Fig.…”

“…2 B i ), or expression ( Fig. S3 ) and because FR inhibition occurred at exceedingly high concentrations only, which are not commonly used ( 24 , 25 , 73 ), we selected the double-mutant Gαq F75K I190W for further mechanistic characterization.

Figure 3 A Gαq triple mutant has no obvious comparative advantage over the double mutant.

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Hypermethylation of GNA14 and its tumor-suppressive role in hepatitis B virus-related hepatocellular carcinoma

“…Heterotrimeric G protein complex is a type of intracellular guanine nucleotide binding protein composed of Gα, Gβ, and Gγ subunits, which can mediate G protein coupled receptor (GPCR) signal transduction 30 . There are 16 Gα subunit proteins.…”