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A dominant negative allele of Gs alpha blocks signaling through adenylyl cyclase
Author(s) -
Rylaarsdam Robin Pals,
Sultan Dahlia,
Ghouse Amrien,
Patel Pooja
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.962.4
Subject(s) - adenylyl cyclase , mutation , gs alpha subunit , microbiology and biotechnology , hek 293 cells , g alpha subunit , mutant , g protein , biology , gnas complex locus , suppressor mutation , protein subunit , genetics , receptor , gene
McCune‐Albright Syndrome (MAS) is a human genetic disorder caused by a mutation that constitutively activates the G s 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 G s 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.