Academic journal article Genetics

RIC8 Is a Guanine-Nucleotide Exchange Factor for G[alpha] Subunits That Regulates Growth and Development in Neurospora Crassa

Academic journal article Genetics

RIC8 Is a Guanine-Nucleotide Exchange Factor for G[alpha] Subunits That Regulates Growth and Development in Neurospora Crassa

Article excerpt

ABSTRACT Heterotrimeric (αβγ) G proteins are crucial components of eukaryotic signal transduction pathways. G-protein-coupled receptors (GPCRs) act as guanine nucleotide exchange factors (GEFs) for Gα subunits. Recently, facilitated GDP/GTP exchange by non- GPCR GEFs, such as RIC8, has emerged as an important mechanism for Gα regulation in animals. RIC8 is present in animals and filamentous fungi, such as the model eukaryote Neurospora crassa, but is absent from the genomes of baker's yeast and plants. In Neurospora, deletion of ric8 leads to profound defects in growth and asexual and sexual development, similar to those observed for a mutant lacking the Gα genes gna-1 and gna-3. In addition, constitutively activated alleles of gna-1 and gna-3 rescue many defects of Δric8 mutants. Similar to reports in Drosophila, Neurospora Δric8 strains have greatly reduced levels of G-protein subunits. Effects on cAMP signaling are suggested by low levels of adenylyl cyclase protein in Δric8 mutants and suppression of Δric8 by a mutation in the protein kinase A regulatory subunit. RIC8 acts as a GEF for GNA-1 and GNA-3 in vitro, with the strongest effect on GNA-3. Our results support a role for RIC8 in regulating GNA-1 and GNA-3 in Neurospora.

EUKARYOTIC cells sense many hormones, growth factors, neurotransmitters, and the presence of light via G-proteinsignaling pathways. The G-protein heterotrimer consists of a Gα subunit, which binds and hydrolyzes GTP, and of tightly associated Gβ and Gg subunits. G proteins interact with seven-transmembrane helix G-protein-coupled receptors (GPCRs) to regulate downstream signaling pathways (reviewed in Neves et al. 2002 and Wilkie and Kinch 2005). GDP-bound Gα subunits are associated with the Gβγ dimer and the GPCR (Wilkie and Kinch 2005). Ligand binding to the receptor leads to exchange of GDP for GTP on the Gα, leading to dissociation of the heterotrimer into Gα-GTP and Gβγ units, which can both interact with effector proteins to generate changes in cellular physiology (Neves et al. 2002). GPCRs thus act as guanine nucleotide-exchange factors (GEFs) for heterotrimeric Gα proteins. The activation cycle is terminated by hydrolysis of GTP to GDP by the Gα subunit and reassociation with Gβγ and the GPCR.

Recently a non-GPCR protein, RIC8, has been implicated as a positive regulator of Gα proteins in several animal species, including Caenorhabditis elegans, Drosophila melanogaster, and mammalian cells (reviewed in Wilkie and Kinch 2005). Invertebrates such as C. elegans and Drosophila contain one RIC8 protein, while vertebrates contain two homologs, Ric-8A and Ric-8B (Tall et al. 2003). RIC8 is required for asymmetric cell division in zygotes and priming of synaptic vesicles in C. elegans (Miller and Rand 2000; Wilkie and Kinch 2005). In Drosophila, RIC8 is essential for responses to extracellular ligands and for maintenance of polarity during asymmetric cell division in embryogenesis (Hampoelz et al. 2005). Furthermore, RIC8 is also required for stability of a Gα and Gβ protein in Drosophila (Hampoelz et al. 2005). Of the two mammalian RIC8 proteins, only Ric- 8A has demonstrated GEF activity toward the Gα proteins Gαq, Gαi, Gαo, and Gα13 (Tall et al. 2003). Ric-8A binds the GDP-bound Gα in the absence of Gβγ, and GDP is then released, forming a stable Ric-8A:Gα complex (Tall et al. 2003). Subsequently, GTP binds the Gα protein and Ric-8A is released. Similar to Drosophila RIC8, Ric-8B regulates the stability of a Gα subunit in mammalian cells (Nagai et al. 2010).

Neurospora crassa possesses three Gα subunits (GNA-1, GNA-2, and GNA-3), one Gβ (GNB-1), one Gg (GNG-1), and .25 putative GPCRs (reviewed in Li et al. 2007). We have previously demonstrated that loss of gna-1 and gna-3 or of all three Gα genes leads to a severe decrease in extension of both basal and aerial hyphae. The relatively subtle phenotypes of GPCR mutants in comparison to strains lacking one or more Gα genes (Kim and Borkovich 2004; Krystofova and Borkovich 2006; Li and Borkovich 2006; Bieszke et al. …

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