Recurrent high-impact mutations at cognate structural positions in class A G protein-coupled receptors expressed in tumors.

G protein-coupled receptors (GPCRs) are the largest family of human proteins. They have a common structure and, signaling through a much smaller set of G proteins, arrestins, and effectors, activate downstream pathways that often modulate hallmark mechanisms of cancer. Because there are many more GPCRs than effectors, mutations in different receptors could perturb signaling similarly so as to favor a tumor. We hypothesized that somatic mutations in tumor samples may not be enriched within a single gene but rather that cognate mutations with similar effects on GPCR function are distributed across many receptors. To test this possibility, we systematically aggregated somatic cancer mutations across class A GPCRs and found a nonrandom distribution of positions with variant amino acid residues. Individual cancer types were enriched for highly impactful, recurrent mutations at selected cognate positions of known functional motifs. We also discovered that no single receptor drives this pattern, but rather multiple receptors contain amino acid substitutions at a few cognate positions. Phenotypic characterization suggests these mutations induce perturbation of G protein activation and/or ß-arrestin recruitment. These data suggest that recurrent impactful oncogenic mutations perturb different GPCRs to subvert signaling and promote tumor growth or survival. The possibility that multiple different GPCRs could moonlight as drivers or enablers of a given cancer through mutations located at cognate positions across GPCR paralogs opens a window into cancer mechanisms and potential approaches to therapeutics.

Genetic trade-offs underlie divergent life history strategies for local adaptation in white clover.

Local adaptation is common in plants, yet characterization of its underlying genetic basis is rare in herbaceous perennials. Moreover, while many plant species exhibit intraspecific chemical defence polymorphisms, their importance for local adaptation remains poorly understood. We examined the genetic architecture of local adaptation in a perennial, obligately-outcrossing herbaceous legume, white clover (Trifolium repens). This widespread species displays a well-studied chemical defence polymorphism for cyanogenesis (HCN release following tissue damage) and has evolved climate-associated cyanogenesis clines throughout its range. Two biparental F2  mapping populations, derived from three parents collected in environments spanning the U.S. latitudinal species range (Duluth, MN, St. Louis, MO and Gainesville, FL), were grown in triplicate for two years in reciprocal common garden experiments in the parental environments (6,012 total plants). Vegetative growth and reproductive fitness traits displayed trade-offs across reciprocal environments, indicating local adaptation. Genetic mapping of fitness traits revealed a genetic architecture characterized by allelic trade-offs between environments, with 100% and 80% of fitness QTL in the two mapping populations showing significant QTL×E interactions, consistent with antagonistic pleiotropy. Across the genome there were three hotspots of QTL colocalization. Unexpectedly, we found little evidence that the cyanogenesis polymorphism contributes to local adaptation. Instead, divergent life history strategies in reciprocal environments were major fitness determinants: selection favoured early investment in flowering at the cost of multiyear survival in the southernmost site versus delayed flowering and multiyear persistence in the northern environments. Our findings demonstrate that multilocus genetic trade-offs contribute to contrasting life history characteristics that allow for local adaptation in this outcrossing herbaceous perennial.

Dual-species origin of an adaptive chemical defense polymorphism.

Allopolyploid speciation and chemical defense diversification are two of the most characteristic features of plant evolution; although the former has likely shaped the latter, this has rarely been documented. Here we document allopolyploidy-mediated chemical defense evolution in the origin of cyanogenesis (HCN release upon tissue damage) in white clover (Trifolium repens). We combined linkage mapping of the loci that control cyanogenesis (Ac, controlling production of cyanogenic glucosides; and Li, controlling production of their hydrolyzing enzyme linamarase) with genome sequence comparisons between white clover, a recently evolved allotetraploid, and its diploid progenitors (Trifolium pallescens, Trifolium occidentale). The Ac locus (a three-gene cluster comprising the cyanogenic glucoside pathway) is derived from T. occidentale; it maps to linkage group 2O (occidentale subgenome) and is orthologous to a highly similar cluster in the T. occidentale reference genome. By contrast, Li maps to linkage group 4P (pallescens subgenome), indicating an origin in the other progenitor species. These results indicate that cyanogenesis evolved in white clover as a product of the interspecific hybridization that created the species. This allopolyploidization-derived chemical defense, together with subsequent selection on intraspecific cyanogenesis variation, appears to have contributed to white clover's ecological success as a globally distributed weed species.

Residues and residue pairs of evolutionary importance differentially direct signaling bias of D2 dopamine receptors.

The D2 dopamine receptor and the serotonin 5-hydroxytryptamine 2A receptor (5-HT2A) are closely-related G-protein-coupled receptors (GPCRs) from the class A bioamine subfamily. Despite structural similarity, they respond to distinct ligands through distinct downstream pathways, whose dysregulation is linked to depression, bipolar disorder, addiction, and psychosis. They are important drug targets, and it is important to understand how their bias toward G-protein versus ß-arrestin signaling pathways is regulated. Previously, evolution-based computational approaches, difference Evolutionary Trace and Evolutionary Trace-Mutual information (ET-Mip), revealed residues and residue pairs that, when switched in the D2 receptor to the corresponding residues from 5-HT2A, altered ligand potency and G-protein activation efficiency. We have tested these residue swaps for their ability to trigger recruitment of ß-arrestin2 in response to dopamine or serotonin. The results reveal that the selected residues modulate agonist potency, maximal efficacy, and constitutive activity of ß-arrestin2 recruitment. Whereas dopamine potency for most variants was similar to that for WT and lower than for G-protein activation, potency in ß-arrestin2 recruitment for N124H3.42 was more than 5-fold higher. T205M5.54 displayed high constitutive activity, enhanced dopamine potency, and enhanced efficacy in ß-arrestin2 recruitment relative to WT, and L379F6.41 was virtually inactive. These striking differences from WT activity were largely reversed by a compensating mutation (T205M5.54/L379F6.41) at residues previously identified by ET-Mip as functionally coupled. The observation that the signs and relative magnitudes of the effects of mutations in several cases are at odds with their effects on G-protein activation suggests that they also modulate signaling bias.

ß2-Adrenergic receptor activation mobilizes intracellular calcium via a non-canonical cAMP-independent signaling pathway.

Beta adrenergic receptors (ßARs) 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 ßAR, ß2AR, whose ligands are used for asthma and cardiovascular disease. ßARs signal through Gαs 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 ßAR signaling and its disruption in disease. Using fluorescence-based Ca2+ flux assays combined with pharmacology and gene knock-out methods, we discovered a previously unrecognized endogenous pathway in HEK-293 cells whereby ß2AR activation leads to robust Ca2+ mobilization from intracellular stores via activation of phospholipase C and opening of inositol trisphosphate (InsP3) receptors. This pathway did not involve cAMP, Gαs, or Gαi or the participation of the other members of the canonical ß2AR signaling cascade and, therefore, constitutes a novel signaling mechanism for this receptor. This newly uncovered mechanism for Ca2+ mobilization by ß2AR has broad implications for adrenergic signaling, cross-talk with other signaling pathways, and the effects of ßAR-directed drugs.

Continent-Wide Climatic Variation Drives Local Adaptation in North American White Clover.

Climate-associated clines in adaptive polymorphisms are commonly cited as evidence of local adaptation within species. However, the contribution of the clinally varying trait to overall fitness is often unknown. To address this question, we examined survival, vegetative growth, and reproductive output in a central US common garden experiment using 161 genotypes of white clover (Trifolium repens L.) originating from 15 locations across North America. White clover is polymorphic for cyanogenesis (hydrogen cyanide release upon tissue damage), a chemical defense against generalist herbivores, and climate-associated cyanogenesis clines have repeatedly evolved across the species range. Over a 12-month experiment, we observed striking correlations between the population of origin and plant performance in the common garden, with climatic distance from the common garden site predicting fitness more accurately than geographic distance. Assessments of herbivore leaf damage over the 2015 growing season indicated marginally lower herbivory on cyanogenic plants; however, this effect did not result in increased fitness in the common garden location. Linear mixed modeling suggested that while cyanogenesis variation had little predictive value for vegetative growth, it is as important as climatic variation for predicting reproductive output in the central United States. Together, our findings suggest that knowledge of climate similarity, as well as knowledge of locally favored adaptive traits, will help to inform transplantation strategies for restoration ecology and other conservation efforts in the face of climate change.

Genome-Wide Association Study of Arabidopsis thaliana Identifies Determinants of Natural Variation in Seed Oil Composition.

The renewable source of highly reduced carbon provided by plant triacylglycerols (TAGs) fills an ever increasing demand for food, biodiesel, and industrial chemicals. Each of these uses requires different compositions of fatty acid proportions in seed oils. Identifying the genes responsible for variation in seed oil composition in nature provides targets for bioengineering fatty acid proportions optimized for various industrial and nutrition goals. Here, we characterized the seed oil composition of 391 world-wide, wild accessions of Arabidopsis thaliana, and performed a genome-wide association study (GWAS) of the 9 major fatty acids in the seed oil and 4 composite measures of the fatty acids. Four to 19 regions of interest were associated with the seed oil composition traits. Thirty-four of the genes in these regions are involved in lipid metabolism or transport, with 14 specific to fatty acid synthesis or breakdown. Eight of the genes encode transcription factors. We have identified genes significantly associated with variation in fatty acid proportions that can be used as a resource across the Brassicaceae. Two-thirds of the regions identified contain candidate genes that have never been implicated in lipid metabolism and represent potential new targets for bioengineering.

Genome-Wide Association Study in Arabidopsis thaliana of Natural Variation in Seed Oil Melting Point: A Widespread Adaptive Trait in Plants.

Seed oil melting point is an adaptive, quantitative trait determined by the relative proportions of the fatty acids that compose the oil. Micro- and macro-evolutionary evidence suggests selection has changed the melting point of seed oils to covary with germination temperatures because of a trade-off between total energy stores and the rate of energy acquisition during germination under competition. The seed oil compositions of 391 natural accessions of Arabidopsis thaliana, grown under common-garden conditions, were used to assess whether seed oil melting point within a species varied with germination temperature. In support of the adaptive explanation, long-term monthly spring and fall field temperatures of the accession collection sites significantly predicted their seed oil melting points. In addition, a genome-wide association study (GWAS) was performed to determine which genes were most likely responsible for the natural variation in seed oil melting point. The GWAS found a single highly significant association within the coding region of FAD2, which encodes a fatty acid desaturase central to the oil biosynthesis pathway. In a separate analysis of 15 a priori oil synthesis candidate genes, 2 (FAD2 and FATB) were located near significant SNPs associated with seed oil melting point. These results comport with others' molecular work showing that lines with alterations in these genes affect seed oil melting point as expected. Our results suggest natural selection has acted on a small number of loci to alter a quantitative trait in response to local environmental conditions.

Oligomeric state of purified transient receptor potential melastatin-1 (TRPM1), a protein essential for dim light vision.

Transient receptor potential melastatin-1 (TRPM1) is essential for the light-induced depolarization of retinal ON bipolar cells. TRPM1 likely forms a multimeric channel complex, although almost nothing is known about the structure or subunit composition of channels formed by TRPM1 or any of its close relatives. Recombinant TRPM1 was robustly expressed in insect cells, but only a small fraction was localized to the plasma membrane. Similar intracellular localization was observed when TRPM1 was heterologously expressed in mammalian cells. TRPM1 was affinity-purified from Sf9 cells and complexed with amphipol, followed by detergent removal. In blue native gels and size exclusion chromatography, TRPM1 migrated with a mobility consistent with detergent- or amphipol-bound dimers. Cross-linking experiments were also consistent with a dimeric subunit stoichiometry, and cryoelectron microscopy and single particle analysis without symmetry imposition yielded a model with approximate 2-fold symmetrical features. Finally, electron microscopy of TRPM1-antibody complexes revealed a large particle that can accommodate TRPM1 and two antibody molecules. Taken together, these data indicate that purified TRPM1 is mostly dimeric. The three-dimensional structure of TRPM1 dimers is characterized by a small putative transmembrane domain and a larger domain with a hollow cavity. Blue native gels of solubilized mouse retina indicate that TRPM1 is present in two distinct complexes: one similar in size to the recombinant protein and one much larger. Because dimers are likely not functional ion channels, these results suggest that additional partner subunits participate in forming the transduction channel required for dim light vision and the ON pathway.

Regulator of G Protein Signaling Proteins Control Growth, Development and Cellulase Production in Neurospora crassa.

Heterotrimeric (αßγ) G protein signaling pathways are critical environmental sensing systems found in eukaryotic cells. Exchange of GDP for GTP on the Gα subunit leads to its activation. In contrast, GTP hydrolysis on the Gα is accelerated by Regulator of G protein Signaling (RGS) proteins, resulting in a return to the GDP-bound, inactive state. Here, we analyzed growth, development and extracellular cellulase production in strains with knockout mutations in the seven identified RGS genes (rgs-1 to rgs-7) in the filamentous fungus, Neurospora crassa. We compared phenotypes to those of strains with either knockout mutations or expressing predicted constitutively activated, GTPase-deficient alleles for each of the three Gα subunit genes (gna-1Q204L, gna-2Q205L or gna-3Q208L). Our data revealed that six RGS mutants have taller aerial hyphae than wild type and all seven mutants exhibit reduced asexual sporulation, phenotypes shared with strains expressing the gna-1Q204L or gna-3Q208L allele. In contrast, Δrgs-1 and Δrgs-3 were the only RGS mutants with a slower growth rate phenotype, a defect in common with gna-1Q204L strains. With respect to female sexual development, Δrgs-1 possessed defects most similar to gna-3Q208L strains, while those of Δrgs-2 mutants resembled strains expressing the gna-1Q204L allele. Finally, we observed that four of the seven RGS mutants had significantly different extracellular cellulase levels relative to wild type. Of interest, the Δrgs-2 mutant had no detectable activity, similar to the gna-3Q208L strain. In contrast, the Δrgs-1 and Δrgs-4 mutants and gna-1Q204L and gna-2Q205L strains exhibited significantly higher cellulase activity than wild type. With the exception of sexual development, our results demonstrate the greatest number of genetic interactions between rgs-1 and gna-1 and rgs-2 and gna-3 in N. crassa.

Roles for receptors, pheromones, G proteins, and mating type genes during sexual reproduction in Neurospora crassa.

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. Δpre-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.

The guanine nucleotide exchange factor RIC8 regulates conidial germination through Gα proteins in Neurospora crassa.

Heterotrimeric G protein signaling is essential for normal hyphal growth in the filamentous fungus Neurospora crassa. We have previously demonstrated that the non-receptor guanine nucleotide exchange factor RIC8 acts upstream of the Gα proteins GNA-1 and GNA-3 to regulate hyphal extension. Here we demonstrate that regulation of hyphal extension results at least in part, from an important role in control of asexual spore (conidia) germination. Loss of GNA-3 leads to a drastic reduction in conidial germination, which is exacerbated in the absence of GNA-1. Mutation of RIC8 leads to a reduction in germination similar to that in the Δgna-1, Δgna-3 double mutant, suggesting that RIC8 regulates conidial germination through both GNA-1 and GNA-3. Support for a more significant role for GNA-3 is indicated by the observation that expression of a GTPase-deficient, constitutively active gna-3 allele in the Δric8 mutant leads to a significant increase in conidial germination. Localization of the three Gα proteins during conidial germination was probed through analysis of cells expressing fluorescently tagged proteins. Functional TagRFP fusions of each of the three Gα subunits were constructed through insertion of TagRFP in a conserved loop region of the Gα subunits. The results demonstrated that GNA-1 localizes to the plasma membrane and vacuoles, and also to septa throughout conidial germination. GNA-2 and GNA-3 localize to both the plasma membrane and vacuoles during early germination, but are then found in intracellular vacuoles later during hyphal outgrowth.

RIC8 is a guanine-nucleotide exchange factor for Galpha subunits that regulates growth and development in Neurospora crassa.

Heterotrimeric (αßγ) 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.

Heterotrimeric G protein signaling in filamentous fungi.

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.