Activated alleles of the Schizosaccharomyces pombe gpa2+ Galpha gene identify residues involved in GDP-GTP exchange.
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.
Biology Department, Boston College, Chestnut Hill, MA 02467, USA.
F Douglas
FD
Ivey
Francis X
FX
Taglia
Fan
F
Yang
Matthew M
MM
Lander
David A
DA
Kelly
Charles S
CS
Hoffman
D000483:Alleles; D000595:Amino Acid Sequence; D000242:Cyclic AMP / Q000378:metabolism; D044385:GTP-Binding Protein alpha Subunits / Q000235:genetics* / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D005947:Glucose / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D020962:Heterotrimeric GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D017434:Protein Structure, Tertiary; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism; D012568:Schizosaccharomyces / Q000235:genetics / Q000378:metabolism; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics* / Q000378:metabolism*; D016415:Sequence Alignment; D015398:Signal Transduction / Q000502:physiology; D020798:Two-Hybrid System Techniques
D044385:GTP-Binding Protein alpha Subunits; D043562:Receptors, G-Protein-Coupled; D029702:Schizosaccharomyces pombe Proteins; C416142:git3 protein, S pombe; C101525:git5 protein, S pombe; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D000242:Cyclic AMP; D020962:Heterotrimeric GTP-Binding Proteins; C540695:gpa2 protein, S pombe; D005947:Glucose