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.

Gain-of-function mutations in heterotrimeric G-protein a subunits are associated with a variety of human diseases. McCune-Albright syndrome (MAS) is caused by mutations in GNAS, the gene encoding Gs. Alterations at Arg201 significantly reduce the GTPase activity of the protein, rendering it constitutively active. In this study, we have constructed a library of random mutations in a constitutively active yeast GPA1 gene carrying a mutation homologous to the McCune-Albright allele (Arg297His). Intragenic suppressors found at sites with homology to the human Gs protein were tested for their ability to suppress the constitutive activity of an Arg201His mutation in Gs. Three intragenic suppressors, at Phe142, Arg231, and Leu266, were able to suppress elevated basal cAMP responses caused by Arg201His when expressed in HEK293 cells. A range of amino acid substitutions was introduced at each of these sites to investigate the chemical requirements for intragenic suppression. The ability of Gs proteins carrying the suppressor mutations alone to mediate receptor-induced cAMP production was measured. These results offer potential sites on Gs that could serve as drug targets for MAS therapies.

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

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

Siglecs are type I transmembrane proteins expressed primarily on leukocytes. Among them is Siglec-8, a CD33 subfamily member that is selectively expressed on the cell surface of human eosinophils. Siglec-8 has an intracellular immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM), putatively responsible for signal transduction. In cytokine-activated eosinophils, Siglec-8 binding causes apoptosis with increased mitochondrial damage and ROS production, but exact signaling mechanisms are unknown. Using a mAb (2C4) against Siglec-8 in combination with small molecule inhibitors, we first examined Siglec-8-mediated apoptosis and ROS production by flow cytometry after 24 hr IL-5 priming (30 ng/mL) of human eosinophils. We observed that 2C4-mediated eosinophil apoptosis was inhibited by PP1 and SU6656 (Src kinase inhibitors), ibrutinib (Btk inhibitor), LY294002 (PI3K inhibitor), GF109203x (PKC inhibitor), and sodium orthovanadate (a protein phosphatase inhibitor) at IC50’s of 1.7 μM, 1.8 μM, 0.9 nM, 1.2 μM, 2.4 μM, and 22 μM respectively. Complete inhibition of ROS production occurred at expected IC90’s. Western blot analysis following Siglec-8 cross-linking with 2C4 showed increased phosphorylation of Src530, Csk, PI3Kδ, and Blk that was detectable within 15 min, and c-Abl phosphorylation that was detectable within 60 min. Additionally, co-immunoprecipitation data shows that Siglec-8 associates with SHP-2, a protein tyrosine phosphatase. While the sequence of signaling events is yet to be determined, Siglec-8 mediated apoptosis in eosinophils involves the unexpected recruitment of molecules normally associated with cell survival.