Filamentous fungi are multicellular eukaryotic organisms known for nutrient recycling as well as for antibiotic and food production. This group of organisms also contains the most devastating plant pathogens and several important human pathogens. Since the first report of heterotrimeric G proteins in filamentous fungi in 1993, it has been demonstrated that G proteins are essential for growth, asexual and sexual development, and virulence in both animal and plant pathogenic filamentous species. Numerous G protein subunit and G protein-coupled receptor genes have been identified, many from whole-genome sequences. Several regulatory pathways have now been delineated, including those for nutrient sensing, pheromone response and mating, and pathogenesis. This review provides a comparative analysis of G protein pathways in several filamentous species, with discussion of both unifying themes and important unique signaling paradigms.

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...

Here we characterize the relationship between the PRE-2 pheromone receptor and its ligand, CCG-4, and the general requirements for receptors, pheromones, G proteins, and mating type genes during fusion of opposite mating-type cells and sexual sporulation in the multicellular fungus Neurospora crassa. PRE-2 is highly expressed in mat a cells and is localized in male and female reproductive structures. Dpre-2 mat a females do not respond chemotropically to mat A males (conidia) or form mature fruiting bodies (perithecia) or meiotic progeny (ascospores). Strains with swapped identity due to heterologous expression of pre-2 or ccg-4 behave normally in crosses with opposite mating-type strains. Coexpression of pre-2 and ccg-4 in the mat A background leads to self-attraction and development of barren perithecia without ascospores. Further perithecial development is achieved by inactivation of Sad-1, a gene required for meiotic gene silencing. Findings from studies involving forced heterokaryons of opposite mating-type strains show that presence of one receptor and its compatible pheromone is necessary and sufficient for perithecial development and ascospore production. Taken together, the results demonstrate that although receptors and pheromones control sexual identity, the mating-type genes (mat A and mat a) must be in two different nuclei to allow meiosis and sexual sporulation to occur.

Beta adrenergic receptors (βARs) are G-protein-coupled receptors essential for physiological responses to the hormones/neurotransmitters epinephrine and norepinephrine which are found in the nervous system and throughout the body. They are the targets of numerous widely used drugs, especially in the case of the most extensively studied βAR, βAR, whose ligands are used for asthma and cardiovascular disease. βARs signal through Gα G-proteins and via activation of adenylyl cyclase and cAMP-dependent protein kinase, but some alternative downstream pathways have also been proposed that could be important for understanding normal physiological functioning of βAR signaling and its disruption in disease. Using fluorescence-based Ca flux assays combined with pharmacology and gene knock-out methods, we discovered a previously unrecognized endogenous pathway in HEK-293 cells whereby βAR activation leads to robust Ca mobilization from intracellular stores via activation of phospholipase C and opening of inositol trisphosphate (InsP) receptors. This pathway did not involve cAMP, Gα, or Gα or the participation of the other members of the canonical βAR signaling cascade and, therefore, constitutes a novel signaling mechanism for this receptor. This newly uncovered mechanism for Ca mobilization by βAR has broad implications for adrenergic signaling, cross-talk with other signaling pathways, and the effects of βAR-directed drugs.

Background: TRPM1 is essential for the light response of retinal depolarizing bipolar cells. Results: Recombinant purified TRPM1 is mostly dimeric, and a low resolution cryo-EM structure is presented. Conclusion: Because most TRP channels function as tetramers, active TRPM1 channels likely require additional partner subunits. Significance: The results suggest a novel paradigm for structure and regulation within the TRP channel family.