Heterotrimeric guanine nucleotide-binding proteins (G proteins), consisting of alpha, beta and gamma subunits, are involved in the response of eukaryotic cells to light, growth factors, hormones and neurotransmitters. Mammalian alpha proteins are grouped into four classes, Gi, Gs, Gq and G12, based on amino acid sequence identity. Constitutively-activated Gi-alpha family members have been implicated in cell proliferation and mitogenesis in mammals, possibly through regulation of cAMP levels and/or ion channel activity. There is also mounting evidence that Gi-alpha proteins may control blue light responses important for cell differentiation, phototropism, and the circadian clock in plants and vertebrates. We have isolated a Gi-alpha family gene (gna- 1) from the multicellular fungus Neurospora crassa. The sequence of the encoded Gna-1 protein is 55% identical to that of other members of the Gi family, and possesses the myristoylation and pertussis toxin modification sites which are the sequence hallmarks of this group. the gna-1 gene contains 3 introns, 2 of which are in positions conserved in the Gi family. In addition, a Neurospora membrane protein of the approximate size of Gna-1 is labeled by pertussis toxin in vitro. These results indicate that Neurospora crassa Gna-1 is the first microbial alpha subunit to belong to any mammalian class. Analogous to the situation in higher organisms, both [cAMP] and [Ca2+] are implicated in cell proliferation and morphogenesis in Neurospora, while blue light responses, such as morphogenesis and resetting the circadian clock, may involve changes in [cAMP]. Therefore, we hypothesize that Gna-1 will regulate cAMP and/or Ca2+ levels, and thus cell proliferation, morphogenesis and the circadian clock in Neurospora crassa.
The Specific Aims are: 1) To screen for other genes related to gna-1 in Neurospora crassa, in order to determine whether gna-1 is part of a gene family. 2) To overexpress wild type gna-1, and to create both null and constitutively-activating mutations in the gna-1 gene in Neurospora in vivo. We will also test the ability of the cloned gna-1 gene to complement morphological mutations which map near gna-1, to determine if they are allelic. The overexpressing, null and """"""""oncogenic"""""""" mutant strains will be assessed for changed morphologies and alterations in cAMP and/or Ca2+ levels. 3) To overexpress and purify the Gna-1 protein for both biochemical analysis (Aim 4) and antibody generation. the antisera will be used for quantitation and immunolocalization of the Gna-1 protein, and for immunoinhibition experiments (Aim 4). To set up reconstitution assays coupling Gna-1 to regulation of downstream effectors such as adenylyl cyclase, cAMP phosphodiesterase or Ca2+ channels. Gna-1 antibody will be used to immunoinhibit these activities, and human Gi family proteins will be tested as substitutes for Gna-1 in the assays. Investigation of the function of the Neurospora Gi homologue Gna-1 will elucidate the roles of human Gi-family alpha proteins in development, mitogenesis and the circadian clock.
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