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Upon ligand binding to a G protein–coupled receptor, extracellular signals are transmitted into a cell through sets of residue interactions that translate ligand binding into structural rearrangements. These interactions needed for functions impose evolutionary constraints so that, on occasion, mutations in one position may be compensated by other mutations at functionally coupled positions. To quantify the impact of amino acid substitutions in the context of major evolutionary divergence in the G protein–coupled receptor subfamily of metabotropic glutamate receptors (mGluRs), we combined two phylogenetic-based algorithms, Evolutionary Trace and covariation Evolutionary Trace, to infer potential structure–function couplings and roles in mGluRs. We found a subset of evolutionarily important residues at known functional sites and evidence of coupling among distinct structural clusters in mGluR. In addition, experimental mutagenesis and functional assays confirmed that some highly covariant residues are coupled, revealing their synergy. Collectively, these findings inform a critical step toward understanding the molecular and structural basis of amino acid variation patterns within mGluRs and provide insight for drug development, protein engineering, and analysis of naturally occurring variants.

The glucagon-like peptide 1 receptor (GLP1R) is a major drug target with several agonists being prescribed in patients with type 2 diabetes (T2D) and obesity. The impact of genetic variability of the GLP1R gene on receptor function and its association with metabolic traits are unclear. Here, functional profiling of 59 rare and one common GLP1R variant across four signaling pathways reveals an unexpected diversity of phenotypes ranging from defective cell surface expression to complete or pathway-specific gain- and loss-of-functions. Defective insulin secretion of loss-of-function GLP1R variants was rescued by allosteric GLP1R ligands or high exendin-4 concentrations in INS-1 823/3 cells. Genetic association studies in 200K participants from UK Biobank show that impaired GLP1R cell surface expression contributes to impaired glucose control and increased adiposity with increased HbA1c, BMI and diastolic blood pressure. This study defines impaired GLP-1R cell surface expression as a risk factor for T2D- and obesity-associated traits.

Genetic variants drive the evolution of traits and diseases. We previously modeled these variants as small displacements in fitness landscapes and estimated their functional impact by differentiating the evolutionary relationship between genotype and phenotype. Conversely, here we integrate these derivatives to identify genes steering specific traits. Over cancer cohorts, integration identified 460 likely tumor-driving genes. Many have literature and experimental support but had eluded prior genomic searches for positive selection in tumors. Beyond providing cancer insights, these results introduce a general calculus of evolution to quantify the genotype–phenotype relationship and discover genes associated with complex traits and diseases.

Significance GPCRs and GPCR pathways are increasingly being implicated in human malignancies, placing them among the most promising cancer drug candidates. Our results reveal enrichment of highly impactful, recurrent GPCR mutations within cancers. We found that cognate mutations in selected class A GPCRs have deleterious effects on signaling function. The results also suggest that olfactory receptors, often considered inconsequential, display a nonrandom mutation pattern in tumors in which they are expressed. These findings support the idea that protein paralogs can act in parallel as members of an onco-group., 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 β-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.

International audience; Melatonin is a hormone mainly produced by the pineal gland and MT1 is one of the two G protein-coupled receptors (GPCRs) mediating its action. Despite an increasing number of available GPCR crystal structures, the molecular mechanism of activation of a large number of receptors, including MT1, remains poorly understood. The purpose of this study is to elucidate the structural elements involved in the process of MT1’s activation using naturally occurring variants affecting its function. Thirty-six nonsynonymous variants, including 34 rare ones, were identified in MTNR1A (encoding MT1) from a cohort of 8687 individuals and their signaling profiles were characterized using Bioluminescence Resonance Energy Transfer-based sensors probing 11 different signaling pathways. Computational analysis of the experimental data allowed us to group the variants in clusters according to their signaling profiles and to analyze the position of each variant in the context of the three-dimensional structure of MT1 to link functional selectivity to structure. MT1 variant signaling profiles revealed three clusters characterized by (1) wild-type-like variants, (2) variants with selective defect of βarrestin-2 recruitment, and (3) severely defective variants on all pathways. Our structural analysis allows us to identify important regions for βarrestin-2 recruitment as well as for Gα12 and Gα15 activation. In addition to identifying MT1 domains differentially controlling the activation of the various signaling effectors, this study illustrates how natural variants can be used as tools to study the molecular mechanisms of receptor activation.

Background A variety of anticancer chemotherapeutics induce adverse side effects including myelotoxicity. Dried roots of Phragmites communis Trinius, Phragmitis rhizoma, have been clinically used in traditional folk medicine to relieve various symptoms like fever. In this study, we evaluated the protective effect of the aqueous extract of Phragmitis rhizoma (EPR) against docetaxel-induced myelotoxicity in vitro and in vivo. Methods The in vitro myelo-protective effect of EPR was evaluated using the colony forming unit (CFU) assay with hematopoietic progenitor cells. The in vivo efficacy of EPR was evaluated in myelosuppressed C57BL/6 male mice which were induced by repeated intraperitoneal injections of 30 mg/kg docetaxel for 3 times. EPR was orally administered for 4 days to docetaxel-induced myelosuppressed C57BL/6 male mice which were induced by intraperitoneal injection of 30 mg/kg docetaxel for 3 times: Group 1 (vehicle control, n = 10), Group 2 (docetaxel plus vehicle, n = 10), Group 3 (docetaxel plus EPR 30 mg/kg, n = 10), Group 4 (docetaxel plus EPR 100 mg/kg, n = 10) and Group 5 (docetaxel plus EPR 300 mg/kg, n = 10). Whole blood counts were measured automatically, and immune organs were histologically examined. Expression of immunomodulatory cytokines was measured by quantitative real-time polymerase chain reaction or enzyme-linked immunosorbent assay. The toxicity of EPR itself was evaluated in normal human cell lines including IMR-90, foreskin fibroblast and human umbilical vein endothelial cells. The hepatotoxicity of EPR was predicted by multi-parametric assays involving cell viability, caspase 3/7 activity, GSH contents and LDH leakage using the HepaRG hepatic cell line. Results Co-treatment of EPR or its major component, p-hydroxycinnamic acid, increased the numbers of hematopoietic CFU counts in the docetaxel-induced in vitro myelotoxicity assay system. The in vitro protective effect of EPR against docetaxel toxicity was replicated in a myelosuppressed animal model: white blood cells, neutrophils, lymphocytes and red blood cells rebounded; bone marrow niche and structural integrity of the thymus were preserved; and the expression of immune-stimulating cytokines including IL3, IL6, SCF and GM-CSF was enhanced. Furthermore, EPR and p-hydroxycinnamic acid promoted the proliferation of primary splenocytes and thymocytes. In the toxicity assays, no remarkable signs related with toxicity were observed in all tested normal human cells and HepaRG. Conclusions EPR has the potential to ameliorate docetaxel-mediated myelotoxicity in both in vitro and in vivo models. However, the identification of the responsible active components and the precise underlying myelo-protective mechanism of EPR need to be elucidated before novel drug development using EPR can precede. Electronic supplementary material The online version of this article (doi:10.1186/s12906-017-1890-1) contains supplementary material, which is available to authorized users.

In vitro myelo-protective effects of Phragmitis rhizoma extract. (PPTX 43 kb)