Constitutive activity of Gs α R201H is suppressed by disruption of either of two hydrophobic pockets within the protein

Abstract

McCune‐Albright Syndrome (MAS) is a disorder caused by mutations in Gs at R201 that inhibit GTP hydrolysis. We developed a yeast model for MAS using a strain engineered to undergo cell cycle arrest in the presence of constitutively active Gα. We constructed and screened a library of 32,000 additional mutations in a constitutively active Gα gene, and identified 13 potential intragenic suppressor mutations at sites homologous to human Gs. When expressed in HEK cells, three Gs isoforms carrying the MAS (R201H) mutation with one of the 13 substitutions suppressed the MAS mutation's constitutive activity. F142 and L266 participate in hydrophobic packing in the crystal structure. Mutations that reduced the hydrophobicity of either amino acid suppressed the R201H mutation. R231 also suppressed R201H, possibly by altering hydrogen bonds between Switch II and water molecules near the γ phosphate of GTP. The suppressor mutations alone did not alter cellular responses to hormone or constitutively activate the protein. Intragenic suppressor mutations can be used to model potential sites to which small molecule drugs may be targeted to reduce or block the constitutive activity of Gs.Supported by NIH 1R15ED020190–01