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.
Constitutive activity of Gs alpha is associated with several human diseases, including McCune‐Albright Syndrome (MAS). In this genetic disorder, Arg201 is mutated to His, Cys, or Ser, resulting in inhibition of GTP hydrolysis and subsequent overactivation of adenylyl cyclase and elevated basal cAMP levels. Previous studies identified three intragenic suppressor sites that blocked the constitutive activity caused by R201H: F142, R231, and L266. These mutations did not by themselves alter the activity of Gs alpha. The intragenic suppressors were examined for their ability to block the other MAS alleles. F142S and R231C suppressed the constitutive activity of both of MAS alleles. L266N suppressed R201S, and is currently being tested for the ability to block the constitutive activity of R201C. Thus, the intragenic suppressors may be useful drug targeting sites for MAS mutations generally, not just for R201H‐caused cases of the disease. Grant Funding Source: Supported by NIH grant 1R15ED020190‐01
Constitutive activation of Gsα is seen in the genetic disorder McCune‐Albright Syndrome, as well as some endocrine adenomas and in tissues affected by cholera toxin. Random mutagenesis of constitutively active Gα subunits in yeast identified three sites that, when mutated, suppressed the constitutive activity caused by R201H: F142, R231, and L266. Because R231 is on the surface of the protein, near the GTP‐binding site it was selected as a candidate site for drug targeting, with the goal of developing inhibitors of the constitutively active Gs alleles. Docking molecules in silico to the R231 binding pocket on the surface of Gs identified commercially available molecules with high potential to bind to the suppressor site. Eighteen molecules with high predicted binding affinity were tested for the ability to decrease the elevation in basal cAMP observed in HEK cells overexpressing the constitutively active Gs‐R201H allele. Two molecules, 6‐oxo‐N‐{[2‐(pyrrolidin‐1‐yl)pyridin‐4‐yl]methyl}‐1,6‐dihydropyridazine‐3‐carboxamide and N‐{[3‐(3‐methylphenyl)phenyl]methyl}‐6‐oxo‐1,6‐dihydropyridazine‐3‐carboxamide were able to significantly reduce cAMP levels by at least 10% from baseline. Neither of these molecules significantly reduced basal cAMP levels in cells transfected with wild‐type Gs constructs. These molecules will form the basis for further studies to develop antagonists of mutant Gs proteins associated with human disease.This work was Supported by NIH grant 1R15ED020190‐01
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2025 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.