Abstract
The Schizosaccharomyces pombe glucose/cyclic AMP (cAMP) signaling pathway includes the Gpa2-Git5-Git11 heterotrimeric G protein, whose Gpa2 Galpha subunit directly binds to and activates adenylate cyclase in response to signaling from the Git3 G protein-coupled receptor. To study intrinsic and extrinsic regulation of Gpa2, we developed a plasmid-based screen to identify mutationally activated gpa2 alleles that bypass the loss of the Git5-Git11 Gbetagamma dimer to repress transcription of the glucose-regulated fbp1(+) gene. Fifteen independently isolated mutations alter 11 different Gpa2 residues, with all but one conferring a receptor-independent activated phenotype upon integration into the gpa2(+) chromosomal locus. Biochemical characterization of three activated Gpa2 proteins demonstrated an increased GDP-GTP exchange rate that would explain the mechanism of activation. Interestingly, the amino acid altered in the Gpa2(V90A) exchange rate mutant protein is in a region of Gpa2 with no obvious role in Galpha function, thus extending our understanding of Galpha protein structure-function relationships.
Publication types
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Research Support, N.I.H., Extramural
MeSH terms
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Alleles*
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Amino Acid Sequence
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Cyclic AMP / metabolism
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GTP-Binding Protein alpha Subunits / genetics*
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GTP-Binding Protein alpha Subunits / metabolism*
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Gene Expression Regulation, Fungal*
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Glucose / metabolism
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Guanosine Diphosphate / metabolism*
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Guanosine Triphosphate / metabolism*
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Heterotrimeric GTP-Binding Proteins / genetics
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Heterotrimeric GTP-Binding Proteins / metabolism
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Models, Molecular
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Molecular Sequence Data
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Mutation
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Protein Structure, Tertiary
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Receptors, G-Protein-Coupled / genetics
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Receptors, G-Protein-Coupled / metabolism
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Schizosaccharomyces pombe Proteins / genetics*
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Schizosaccharomyces pombe Proteins / metabolism*
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Schizosaccharomyces* / genetics
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Schizosaccharomyces* / metabolism
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Sequence Alignment
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Signal Transduction / physiology
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Two-Hybrid System Techniques
Substances
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GTP-Binding Protein alpha Subunits
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Receptors, G-Protein-Coupled
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Schizosaccharomyces pombe Proteins
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git3 protein, S pombe
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git5 protein, S pombe
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Guanosine Diphosphate
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Guanosine Triphosphate
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Cyclic AMP
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Heterotrimeric GTP-Binding Proteins
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gpa2 protein, S pombe
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Glucose