Benedictine University, Biology, Lisle, IL 60532
Turning a biological system off can be just as important as turning it on. In classical G-protein coupled receptor signaling pathways, the heterotrimeric G-proteins cycle between an active/GTP-bound conformation and an inactive/GDP-bound conformation. This cycling is controlled by activated receptors that turn on the pathway, along with an endogenous GTPase activity of the G-protein alpha subunit that turns the pathway off. McCune-Albright Syndrome (MAS) is a genetic disorder caused by a mutation (R201H) that inhibits GTP hydrolysis in Gs alpha, permanently activating the protein. We have developed a yeast model system for MAS in which mutating the homologous residue in the yeast G alpha subunit (R297H) prevents colony formation on media containing 5-floro-orotic acid (5-FOA). We constructed a library of 32,000 unique plasmids carrying additional mutations in the constitutively active G alpha gene, and used it to identify 13 mutations at sites homologous to the human Gs protein that were potential intragenic suppressor sites. In this presentation, we will describe our characterization of these mutant forms of Gs expressed in human cultured cells. Three sites successfully suppressed the McCune-Albright mutation’s constitutive activity. Extensive mutagenesis of each of these sites revealed biochemical requirements for suppression at each site. None of the three mutations by themselves caused constitutive activity of the protein or significantly changed cellular responsiveness to hormone. Intragenic suppressor mutations can be used to model potential sites to which small molecule drugs may be targeted, potentially interacting with the mutant protein in the same way that the mutation did, and reducing or blocking the constitutive activity of the protein. A complete map of the locations and chemical compositions of suppressor mutations for the MAS allele will provide important information to drive rational drug design for the treatment of this disease.
This work was supported by NIH grant 1R15ED020190-01 to R.P. Rylaarsdam
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