Computational prediction yields efficient and scalable initial assessments of how variants of unknown significance may affect human health. However, when discrepancies between these predictions and ...direct experimental measurements of functional impact arise, inaccurate computational predictions are frequently assumed as the source. Here, we present a methodological analysis indicating that shortcomings in both computational and biological data can contribute to these disagreements. We demonstrate that incomplete assaying of multifunctional proteins can affect the strength of correlations between prediction and experiments; a variant's full impact on function is better quantified by considering multiple assays that probe an ensemble of protein functions. Additionally, many variants predictions are sensitive to protein alignment construction and can be customized to maximize relevance of predictions to a specific experimental question. We conclude that inconsistencies between computation and experiment can often be attributed to the fact that they do not test identical hypotheses. Aligning the design of the computational input with the design of the experimental output will require cooperation between computational and biological scientists, but will also lead to improved estimations of computational prediction accuracy and a better understanding of the genotype–phenotype relationship.
When discrepancies between SNV impact predictions and direct experimental measurements of functional impact arise, inaccurate computational predictions are frequently attributed as the source. Here, we present a methodological analysis examining potential causes of disagreement between computational prediction and experimental assessments of variant impact, including robustness of experimental design, protein multifunctionality, and alignment selection. Understanding and addressing these factors led to improved agreement between prediction and experimental validation and a more consistent translation of the genotype‐phenotype relationship.
G‐protein‐coupled receptors (GPCRs) are the largest family of integral membrane receptors with key roles in regulating signaling pathways targeted by therapeutics, but are difficult to study using ...existing proteomics technologies due to their complex biochemical features. To obtain a global view of GPCR‐mediated signaling and to identify novel components of their pathways, we used a modified membrane yeast two‐hybrid (MYTH) approach and identified interacting partners for 48 selected full‐length human ligand‐unoccupied GPCRs in their native membrane environment. The resulting GPCR interactome connects 686 proteins by 987 unique interactions, including 299 membrane proteins involved in a diverse range of cellular functions. To demonstrate the biological relevance of the GPCR interactome, we validated novel interactions of the GPR37, serotonin 5‐HT4d, and adenosine ADORA2A receptors. Our data represent the first large‐scale interactome mapping for human GPCRs and provide a valuable resource for the analysis of signaling pathways involving this druggable family of integral membrane proteins.
Synopsis
The complete interactome of 48 disease‐associated, full‐length human G‐coupled protein receptors (GPCRs) is mapped in the native environment of the cellular membrane, using the membrane yeast two‐hybrid (MYTH) technology.
MYTH is used to identify interacting partners for 48 human GPCRS, resulting in an interactome containing 686 proteins and 987 unique interactions.
Bioinformatics analyses of the GPCR interactome indicate enrichment for diverse pathways, diseases, molecular functions, biological processes, domains, and drug targets.
Orthogonal analyses using co‐immunoprecipitation and BRET validate a subset of the identified interactions.
Functional characterization of novel GPCR interactions identifies potential roles in neurobiological processes.
The complete interactome of 48 disease‐associated, full‐length human G‐coupled protein receptors (GPCRs) is mapped in the native environment of the cellular membrane, using the membrane yeast two‐hybrid (MYTH) technology.
Activation of heterotrimeric G proteins by their cognate seven transmembrane domain receptors is believed to involve conformational changes propagated from the receptor to the G proteins. However, ...the nature of these changes remains unknown. We monitored the conformational rearrangements at the interfaces between receptors and G proteins and between G protein subunits by measuring bioluminescence resonance energy transfer between probes inserted at multiple sites in receptor-G protein complexes. Using the data obtained for the alpha(2A)AR-G alpha(i1) beta1gamma2 complex and the available crystal structures of G alpha(i1) beta1gamma2, we propose a model wherein agonist binding induces conformational reorganization of a preexisting receptor-G protein complex, leading the G alpha-G betagamma interface to open but not dissociate. This conformational change may represent the movement required to allow nucleotide exit from the G alpha subunit, thus reflecting the initial activation event.
Receptor heteromers constitute a new area of research that is reshaping our thinking about biochemistry, cell biology, pharmacology and drug discovery. In this commentary, we recommend clear ...definitions that should facilitate both information exchange and research on this growing class of transmembrane signal transduction units and their complex properties. We also consider research questions underlying the proposed nomenclature, with recommendations for receptor heteromer identification in native tissues and their use as targets for drug development.
Signaling diversity of G protein-coupled (GPCR) ligands provides novel opportunities to develop more effective, better-tolerated therapeutics. Taking advantage of these opportunities requires ...identifying which effectors should be specifically activated or avoided so as to promote desired clinical responses and avoid side effects. However, identifying signaling profiles that support desired clinical outcomes remains challenging. This study describes signaling diversity of mu opioid receptor (MOR) ligands in terms of logistic and operational parameters for ten different in vitro readouts. It then uses unsupervised clustering of curve parameters to: classify MOR ligands according to similarities in type and magnitude of response, associate resulting ligand categories with frequency of undesired events reported to the pharmacovigilance program of the Food and Drug Administration and associate signals to side effects. The ability of the classification method to associate specific in vitro signaling profiles to clinically relevant responses was corroborated using β2-adrenergic receptor ligands.
Skeletal muscle is populated with a reservoir of quiescent muscle stem cells (MuSCs), which regenerate the tissue after injury. Here, we show that the adhesion G-protein-coupled receptor Gpr116 is ...essential for long-term maintenance of the MuSC pool. Quiescent MuSCs express high levels of Gpr116, which is rapidly downregulated upon MuSC activation. MuSCs deficient for Gpr116 exhibit progressive depletion over time and are defective in self-renewal. Adhesion G-protein-coupled receptors contain an agonistic peptide sequence, called the “Stachel” sequence, within their long N-terminal ectodomains. Stimulation of MuSCs with the GPR116 Stachel peptide delays MuSC activation and differentiation. Stachel peptide stimulation of GPR116 leads to strong interaction with β-arrestins. Stimulation of GPR116 increases the nuclear localization of β-arrestin1, where it interacts with cAMP response element binding protein to regulate gene expression. Altogether, we propose a model by which GPR116 maintains the MuSC pool via nuclear functions of β-arrestin1.
Display omitted
•Quiescent muscle stem cells express the adhesion G-protein-coupled receptor Gpr116•Gpr116-deficient muscle stem cells are incapable of maintaining quiescence•The GPR116 Stachel agonist peptide prevents MuSC activation•GPR116 signaling is mediated in part by nuclear activity of β-arrestin1
Sénéchal et al. show the adhesion G-protein-coupled receptor GPR116 regulates muscle stem cell quiescence. Gpr116-deficient muscle stem cells activate, with consequent depletion over time. The GPR116 N-terminal Stachel peptide agonist prevents muscle stem cell activation, mediated in part by downstream signaling leading to nuclear accumulation and β-arrestin1 activity.
G protein-coupled receptors (GPCRs) represent the largest family of proteins involved in signal transduction. Here we present a bioluminescence resonance energy transfer (BRET) assay that directly ...monitors in real time the early interactions between human GPCRs and their cognate G-protein subunits in living human cells. In addition to detecting basal precoupling of the receptors to Galpha-, Gbeta- and Ggamma-subunits, BRET measured very rapid ligand-induced increases in the interaction between receptor and Galphabetagamma-complexes (t(1/2) approximately 300 ms) followed by a slower (several minutes) decrease, reflecting receptor desensitization. The agonist-promoted increase in GPCR-Gbetagamma interaction was highly dependent on the identity of the Galpha-subunit present in the complex. Therefore, this G protein-activity biosensor provides a novel tool to directly probe the dynamics and selectivity of receptor-mediated, G-protein activation-deactivation cycles that could be advantageously used to identify ligands for orphan GPCRs.
PDF
