The Schizosaccharomyces pombe G alpha protein Gpa2 regulates glucose-induced cAMP production through a direct interaction with adenylate cyclase.
F. Douglas Ivey, Charles S. Hoffman
Biology Department, Boston College, 140 Commonwealth Ave, Chestnut Hill, MA, 02467, USA
In Schizosaccharomyces pombe, a heterotrimeric GTP-binding protein couples receptor-mediated detection of extracellular glucose to the stimulation of cAMP production through adenylate cyclase activation. We have identified a domain of adenylate cyclase that binds to Gpa2 using a yeast two-hybrid screen with a Gpa2 bait and a library of random fragments of the git2+/cyr1+ gene preys. This interaction appears to be valid as this domain of adenylate cyclase binds a mutationally-activated Gpa2 bait better the wild type Gpa2 bait. Deletion analysis of a representative prey plasmid allowed the localization of a short stretch of amino acids that are required for the Gpa2 interaction. Site-directed mutagenesis of codons within this domain identified critical amino acids required for the interaction between Gpa2 and adenylate cyclase. Overexpression of this putative Gpa2-binding domain of adenylate cyclase results in a dominant negative phenotype characterized by improper transcriptional regulation of the fbp1+ fructose-1,6-bisphosphatase gene. Studies are currently underway to characterize the in vivo glucose-stimulated cAMP response in strains carrying specific, site-directed 'knock-in' mutant alleles of adenylate cyclase. Some of these mutants initiate a cAMP response, but then fail to sustain the response relative to wild type cells, suggesting that adenylate cyclase activation may involve both Gpa2-independent and Gpa2-mediated activation mechanisms.
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