RESUMO
Gain-of-function mutations in heterotrimeric G-protein α 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.
Assuntos
Displasia Fibrosa Poliostótica/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Substituição de Aminoácidos , Linhagem Celular Tumoral , Cromograninas , AMP Cíclico/antagonistas & inibidores , AMP Cíclico/biossíntese , Ossos Faciais/anormalidades , Biblioteca Gênica , Células HEK293 , Humanos , Mutação , Crânio/anormalidades , Supressão GenéticaRESUMO
McCune-Albright syndrome (MAS) is a human genetic disorder caused by a mutation that constitutively activates the G(s)α 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 EC(50) 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 G(s)α. Interestingly, F222P/D223V behaved similar to R201H in causing increases in basal cAMP production, thus demonstrating constitutive activity. Substitution of another acidic (E) or polar (N, T, and 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.
Assuntos
Alelos , Displasia Fibrosa Poliostótica/genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Supressão Genética , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular , AMP Cíclico , Subunidades alfa Gs de Proteínas de Ligação ao GTP/química , Regulação da Expressão Gênica , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Alinhamento de SequênciaRESUMO
McCune-Albright syndrome (MAS) causes a variety of bone and endocrine abnormalities due to the post-zygotic mutation of the alpha subunit of the stimulatory G-protein Gsalpha. This mutation causes signal-independent activity of the G-protein in the affected cells. We report the development of a system to study the effects of MAS mutations using Saccharomyces cerevisiae, wherein activation of the yeast G-protein pathway results in growth arrest in a genetically recessive fashion. We introduced the MAS mutation into the analogous site in the yeast Galpha gene, GPA1 and randomly mutated the gene to produce intragenic suppressors. Yeast with normal and mutated G-protein genes were induced to lose the normal gene, and mutations able to intragenically suppress the constitutive activity of the MAS mutation were identified based on their ability to form colonies. We report one mutation in GPA1, also in the active site, that is an intragenic suppressor of the MAS defect.
Assuntos
Displasia Fibrosa Poliostótica/metabolismo , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Supressão Genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Displasia Fibrosa Poliostótica/genética , Subunidades alfa de Proteínas de Ligação ao GTP/química , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Transdução de SinaisRESUMO
We report a novel mutant of the luteinizing hormone receptor (LHR) in a case of familial Leydig cell hypoplasia and pseudohermaphrotidism. The proband was homozygous for two missense mutations, T1121C and C1175T, causing substitutions I374T and T3921. The molecular effects of the mutations were investigated by heterologous expression of the WT LHR, the double mutant LHR, or receptors with either the I374T or the T392I mutation, and measuring hormone binding and cAMP signaling. All mutant LHRs exhibited severe defects, including loss of ligand binding and cAMP production. Immunoblots showed little difference in protein levels between the WT and mutant receptors.