Identification of Binding Sites for Small Molecule Modulators of Gβγ Signaling

G‐protein coupled receptors (GPCRs) transduce extracellular stimuli to intracellular signaling events via G‐proteins and β‐arrestins and are targeted by up to 40% of FDA approved drugs. Targeting proteins downstream of the GPCR, such as G‐protein β, allows for selective modulation of specific pathways downstream of GPCRs. Inhibition of interactions between Gβγ and specific effectors has shown to have therapeutic potential in diseases such as heart failure and inflammation (Lehmann et al., 2008. and Casey et al., 2010.) Previously, our laboratory identified small molecules that bind to a common site for protein‐protein interactions on Gβ. These compounds selectively modulate Gβγ‐dependent activity of various downstream effectors. We hypothesize that these compounds differentially modulate Gβγ‐mediated signals due to binding to specific subsets of amino acids on Gβ. To find the binding site for these compounds, we used a random mutagenesis screen in yeast. The yeast mating response to α‐factor is mediated by the Gβγ homolog, Ste4p. This pathway when activated can inhibit yeast growth making it an attractive tool for examining the inhibition of βγ and homologs using small molecules. Gallein, a well‐characterized Gβγ inhibitor, inhibited the α‐factor/Ste4P mediated mating response pathway and it suppressed α‐factor induced growth arrest. To screen for residues on Ste4p that bind gallein, we set up a yeast strain where α‐factor stimulates growth on media lacking histidine and showed that gallein inhibits growth of this strain in the presence of α‐factor. We randomly mutagenized ste4p and 500,000 variants were screened for colonies that grew in the presence of gallein. Two classes of mutations were found. Class I mutants had high basal activity suggesting that the mutations decreased the affinity of Ste4P for Gpa1p. Indeed, these mutations were located at positions known to be important for mammalian Gα binding to Gβγ. Class II mutants did not exhibit high basal activity. To test whether these mutants were resistant to gallein inhibition because they lack a gallein binding site, we examined the concentration dependence for gallein inhibition in a reporter assay. The IC 50 's for the Class I mutants were not different from wild‐type suggesting that resistance was not conferred by a loss of gallein binding. We are in the course of examining class II mutants. One double mutant, G182R, C282E, showed an increased IC 50 for gallein while other remain to be examined. All of these mutations map to a region on a critical Gβγ protein‐protein interaction surface. Finding gallein binding specific residues will allow us to define an inhibitor binding site on Gβ, and may allow for rational design of novel therapeutic inhibitors. In addition, it will help us understand the mechanism for selective inhibition of signaling downstream of Gβγ by small molecules.