RIC8 Is a Guanine-Nucleotide Exchange Factor for Gα Subunits That Regulates Growth and Development in Neurospora crassa

Abstract

Heterotrimeric (abg) G proteins are crucial components of eukaryotic signal transduction pathways. G-protein-coupled receptors (GPCRs) act as guanine nucleotide exchange factors (GEFs) for Ga subunits. Recently, facilitated GDP/GTP exchange by non-GPCR GEFs, such as RIC8, has emerged as an important mechanism for Ga 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 Ga genes gna-1 and gna-3. In addition, constitutively activated alleles of gna-1 and gna-3 rescue many defects of Dric8 mutants. Similar to reports in Drosophila, Neurospora Dric8 strains have greatly reduced levels of G-protein subunits. Effects on cAMP signaling are suggested by low levels of adenylyl cyclase protein in Dric8 mutants and suppression of Dric8 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.E UKARYOTIC cells sense many hormones, growth factors, neurotransmitters, and the presence of light via G-proteinsignaling pathways. The G-protein heterotrimer consists of a Ga subunit, which binds and hydrolyzes GTP, and of tightly associated Gb 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 andWilkie andKinch 2005). GDP-bound Ga subunits are associated with the Gbg dimer and the GPCR (Wilkie and Kinch 2005). Ligand binding to the receptor leads to exchange of GDP for GTP on the Ga, leading to dissociation of the heterotrimer into Ga-GTP and Gbg 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 Ga proteins. The activation cycle is terminated by hydrolysis of GTP to GDP by the Ga subunit and reassociation with Gbg and the GPCR.Recently a non-GPCR protein, RIC8, has been implicated as a positive regulator of Ga 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 Ga and Gb protein in Drosophila (H...

Global Gene Expression and Focused Knockout Analysis Reveals Genes Associated with Fungal Fruiting Body Development in Neurospora crassa

“…Expression of some GPCRs and G-protein subunits, especially gpr-1 and gna-3, increased after 48 h. A perithecial defect was observed for both ⌬bek-1 and ⌬gpr-1 strains (66), and abnormal perithecia with a large propotion of nonviable ascospores were found in ⌬gna-3 strains (67). Genetic tests suggest that RIC8 activates GNA-1 and GNA-3, but not GNA-2, and positively regulates the cAMP pathway in vegetative growth of N. crassa (68). In our study, expression of ric8, cr-1, gna-2, and gpr-6 was highly correlated.…”

Genetic and Physical Interactions between Gα Subunits and Components of the Gβγ Dimer of Heterotrimeric G Proteins in Neurospora crassa

“…In this scenario, the recently discovered cytosolic guanine nucleotide exchange factor (GEF) RIC8 may function to load GTP on the remaining free G␣ proteins. We have shown that RIC8 binds to and regulates GDP/GTP exchange on GNA-1 and GNA-3 in N. crassa (57).…”

“…The membranes for Western analysis were probed using previously described primary antibodies for GNA-1, GNA-2, and GNB-1 at a final dilution of 1:1,000 (3,22,34,57,60). A previously described GNA-3 antibody (see Fig.…”

The Genomes of Three Uneven Siblings: Footprints of the Lifestyles of Three Trichoderma Species

“…In addition to the membrane-localized GPCRs, cytoplasmic nonreceptor GEFs, such as N. crassa RIC-8 (635), have recently been identified which bind to the G-protein and accelerate guaninenucleotide exchange. In M. oryzae, MoRic8 was shown to interact with and positively regulate MagB (636), and in N. crassa RIC-8 exhibited GEF activity toward the GNA-1 and GNA-3 G␣ proteins (635). Another protein with GEF activity, Arr4p/Get3p, has been described in S. cerevisiae.…”