McCune-Albright Syndrome (MAS) is a human genetic disorder caused by a mutation that constitutively activates the Gsα subunit by abolishing GTP hydrolysis. MAS patients suffer from a range of endocrinopathies as well as polyostotic fibrous dysplasia of bone. We previously identified an intragenic suppressor of the MAS mutation in a yeast system, which substituted two residues in the GTP-binding site of Gpa1: L318P and D319V to suppress the constitutive activity of an R297H mutation, corresponding to the human F222P, D223V, and R201H mutations, respectively. To extend these studies, the human GNAS gene was subjected to site-directed mutagenesis. Constructs expressing the MAS mutation (R201H), the MAS mutation plus the mutations homologous to the yeast suppressors (R201H, F222P/D223V), or the yeast suppressor mutation alone (F222P/D223V) were transfected into HEK293 cells, and basal and receptor-stimulated cAMP levels were measured. Expression of R201H increased the basal cAMP levels and decreased the EC50 for hormone-stimulated cAMP production. These effects were dependent on the amount of R201H protein expressed. R201H, F222P/D223V abolished the constitutive activity of the MAS mutation, and caused responses to hormone that were not different from those measured in cells expressing WT Gsα. Interestingly, F222P/D223V behaved similarly to R201H in causing increases in basal cAMP production, thus demonstrating constitutive activity. Substitution of another acidic (E) or polar (N, T, G) amino acid at position 223 caused no suppression of R201H activity, while substitution of a second nonpolar amino acid (A) at this position partially suppressed, and the larger polar I residue completely suppressed the effects of R201H.

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.

Inappropriate activation of the Gs alpha subunit by mutation of modification of Arg201 is associated with the human diseases of McCune‐Albright Syndrome and cholera. Previous work in our laboratory identified Asp223 as a site where substitution to valine could reverse the constitutive activity of an Arg201His mutation. In this study, Asp223 was substituted with a variety of other amino acids, including glutamic acid, asparagine, glutamine, alanine, leucine, and glycine. HEK cells transfected with plasmids carrying the different Gs alpha subunit alleles were used to measure basal levels of cAMP. The Arg201His allele significantly elevates basal cAMP. Substitution of Asp223 with nonpolar residues suppressed the elevation in basal cAMP, while substitution with acidic or polar residues had no effect on the constitutive activity of the Arg201His mutation. This characterization of which amino acids at residue 223 can suppress the Arg201His mutation may provide a foundation for identifying small molecules that can be used as effective therapies against McCune‐Albirght Syndrome.Supported by NIH grant1R15DE02109‐01

Heterotrimeric G‐proteins cycle between an active/GTP‐bound conformation and an inactive/GDP‐bound conformation. Reduction in GTP hydrolysis by the G‐protein alpha subunit results in constitutive activity of the protein. McCune‐Albright Syndrome (MAS) is a human genetic disorder caused by a mutation that constitutively activates the Gs alpha subunit by inhibiting GTP hydrolysis. Arginine 201 substitutions with histidine, serine, or cysteine have been reported in biopsies from MAS patients. 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. The homologous mutations (F222P, D223V) in the human Gs alpha subunit also suppressed the constitutive activity of the MAS mutation when the protein was expressed in human cells. To extend these studies, F222 and D223 were mutated to a variety of other amino acids to determine the structural requirements for suppression of the MAS mutation. Basal and receptor‐stimulated cAMP levels were measured in HEK293 cells transfected with the different alleles of Gs alpha. While some single mutations at either F222 or D223 were able to suppress the MAS R201H mutation, a range of levels of suppression were observed.