Delineation of molecular determinants for FR900359 inhibition of G_q/11 unlocks inhibition of Ga_s

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 Ga_s inhibitor, FR900359-sensitive Ga_s mutants may prove useful in studies where delicate control over Ga_s signaling would be of the essence.