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Frank Ivey

Advisor: Katherine Borkovich, Ph.D.

Heterotrimeric GTP-binding proteins, G proteins, are integral components of eukaryotic signaling systems linking extracellular signals to intracellular responses. Through coupling to seven-transmembrane helix receptors, G proteins convey primary signaling events into multi-leveled cascades of intracellular activity by regulating downstream enzymes, collectively called effectors. The effector enzymes regulated by G proteins include adenylyl cyclase, cGMP phosphodiesterase, phospholipase C-beta, mitogen-activated protein kinases, and ion channels. Neurospora crassa is a multicellular, filamentous fungus that is capable of both asexual and sexual reproduction by elaboration of specialized, developmentally controlled structures that give rise to either asexual or sexual spores, respectively. N. crassa possesses at least three heterotrimeric G-alpha proteins (GNA-1-3) and one G-beta subunit (GNB-1). GNA-1 was the first microbial G-alpha protein that could be classified in the G-alpha-i superfamily based on its amino acid identity and demonstration that it is a substrate for ADP-ribosylation by pertussis toxin. Experiments were designed to identify the signal transduction pathways and the effector enzymes regulated by GNA-1. Targeted gene-replacement of gna-1 revealed that GNA-1 controls multiple developmental pathways including both asexual and sexual reproduction, maintenance of growth, and resistance to osmotic stress. The G-alpha-i and G-alpha-z members of the G-alpha-i superfamily negatively regulate adenylyl cyclase activity in mammalian cells therefore, adenylyl cyclase and cAMP levels were measured in Delta-gna-1 strains and also in strains that were deleted for both gna-1 and gna-2, a second G-alpha in N. crassa shown to have overlapping functions with GNA-1. Direct measurements of adenylyl cyclase activity revealed that GNA-1, but not GNA-2, was responsible for GTP-stimulated adenylyl cyclase activity in N. crassa. Furthermore, anti-GNA-1 IgG could specifically inhibit GTP-stimulated adenylyl cyclase activity in wild-type strain extracts. These studies also provided evidence that N. crassa possesses feedback mechanisms that control steady-state cAMP levels through indirect regulation of cAMP-phosphodiesterase activity; mutations in gna-1 and gna-2 were additive in their effect on lowering cAMP-phosphodiesterase activity under growth conditions where steady-state cAMP levels were normal but GTP-stimulated adenylyl cyclase activity was reduced 90% in comparison to control strains. Genetic and biochemical epistasis experiments utilizing a Delta-gna-1 cr-1 mutant suggest that GNA-1 is essential for female fertility in a cAMP-independent pathway. Furthermore, deletion of gna-1 in a cr-1 background exacerbated many of the defects already observed in the cr-1 strain including more severe growth restriction and developmental defects. However, deletion of gna-1 had no effect on the increased thermotolerance of cr-1, which has been attributed to loss of cAMP. cr-1 possesses GNA-1 protein, and plasma membrane fractions from this strain reconstituted GTP-stimulated adenylyl cyclase activity in Delta-gna-1 membrane fractions. These studies provide direct evidence for the involvement of G-alpha proteins in the regulation of adenylyl cyclase activity in eukaryotic microbes.

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Research Info

Functional Analysis of GNA-1, a G-alpha-i Superfamily Member Found in the Filamentous Fungus Neurospora crassa