A dominant negative allele of Gs alpha blocks signaling through adenylyl cyclase

Abstract

McCune‐Albright Syndrome (MAS) is a human genetic disorder caused by a mutation that constitutively activates the Gs alpha subunit by abolishing GTP hydrolysis. Previous work in this laboratory modeled the MAS mutation in a yeast system, and identified an intragenic suppressor of the MAS mutation, which substituted two residues in the GTP‐binding site: L318P and D319V. To extend these studies, the human GNAS1 gene, encoding Gs alpha, was subjected to site‐directed mutagenesis. Constructs expressing the MAS mutation (R201H), the MAS mutation plus the mutations homologous to the yeast suppressors (R201H/F699P/D700V), the MAS mutation plus each of the amino acid changes in the yeast suppressor (R201H/F699P), (R201H/D700V), or the yeast suppressor mutation alone (F699P/D700V) were transfected into HEK293 cells, and basal and receptor‐stimulated cAMP levels were measured. R201H/F699P/D700V abolished the constitutive activity of the MAS mutation. Interestingly, F699P/D700V blocked the response to hCG. Cotransfection of F699P/D700V with R201H blocked the rise in basal cAMP. Thus, F669P/D700V acts as a dominant negative allele by competing with other G‐proteins for adenylyl cyclase, not the GPCR.