The Schizosaccharomyces pombe glucose/cyclic AMP (cAMP) signaling pathway includes the Gpa2-Git5-Git11 heterotrimeric G protein, whose Gpa2 G␣ 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 G␤␥ 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 G␣ function, thus extending our understanding of G␣ protein structure-function relationships.Heterotrimeric guanine-nucleotide binding proteins (G proteins) relay extracellular signals from surface receptors to intracellular effectors. G proteins are composed of G␣ subunits and G␤␥ dimers whose interaction depends upon whether G␣ is bound to GDP or GTP. The inactive heterotrimer contains a G␣ subunit bound to GDP. Ligand binding by seven transmembrane G protein-coupled receptors (GPCRs) leads to GDP release and GTP binding by G␣, resulting in a conformational change that decreases G␣ affinity for G␤␥ and, depending on the system, promotes effector binding (32). Either or both G␣-GTP and G␤␥ are then able to regulate downstream effectors (28). Restoration of the basal state is achieved through hydrolysis of the bound GTP by the G␣ and G protein heterotrimer reassociation (28). GTP hydrolysis can be accelerated by protein-protein interactions of G␣ with regulator of G protein signaling (40, 50) proteins or through G␣-effector interactions (4).A variety of approaches, including mutational studies, biochemical analyses of purified G protein subunits, and highresolution crystallography, inform our current understanding of the mechanisms behind guanine nucleotide binding and release, as well as GTP hydrolysis reactions that govern G␣ activation and inactivation cycles. Crystal structure data are available for G␣ subunits in the GDP-bound (20, 31) or GTPbound (5, 29, 42) conformation. G␣ subunits possess two domains, a helical domain and a GTPase domain that resembles small GTPases, such as Ras, Rho, and Rheb. Bound guanine nucleotides are buried in a cleft between these domains, which are connected by two linker sequences. Current models suggest that interdomain interactions regulate both receptor-activated and basal GDP release rates (8,9,34).Mutagenesis studies have been used to identify and characterize G␣ residues critical for guanine nucleotide binding (1,33,38,39), basal GDP release rates (34, 43), and GTP catalysis (19,43). O...