Human G Protein-coupled Receptors Research Articles

Homologous G Protein-Coupled Receptors Boost the Modeling and Interpretation of Bioactivities of Ligand Molecules.

G protein-coupled receptors (GPCRs) are one of the most important drug targets, accounting for ∼34% of drugs on the market. For drug discovery, accurate modeling and explanation of bioactivities of ligands is critical for the screening and optimization of hit compounds. Homologous GPCRs are more likely to interact with chemically similar ligands, and they tend to share common binding modes with ligand molecules. The inclusion of homologous GPCRs in learning bioactivities of ligands potentially enhances the accuracy and interpretability of models due to utilizing increased training sample size and the existence of common ligand substructures that control bioactivities. Accurate modeling and interpretation of bioactivities of ligands by combining homologous GPCRs can be formulated as multitask learning with joint feature learning problem and naturally matched with the group lasso learning algorithm. Thus, we proposed a multitask regression learning with group lasso (MTR-GL) implemented by l2,1-norm regularization to model bioactivities of ligand molecules and then tested the algorithm on a series of thirty-five representative GPCRs datasets that cover nine subfamilies of human GPCRs. The results show that MTR-GL is overall superior to single-task learning methods and classic multitask learning with joint feature learning methods. Moreover, MTR-GL achieves better performance than state-of-the-art deep multitask learning based methods of predicting ligand bioactivities on most datasets (31/35), where MTR-GL obtained an average improvement of 38% on correlation coefficient (r2) and 29% on root-mean-square error over the DeepNeuralNet-QSAR predictors.

Extended Human G-Protein Coupled Receptor Network: Cell-Type-Specific Analysis of G-Protein Coupled Receptor Signaling Pathways.

G-protein coupled receptors (GPCRs) mediate crucial physiological functions in humans, have been implicated in an array of diseases, and are therefore prime drug targets. GPCRs signal via a multitude of pathways, mainly through G-proteins and β-arrestins, to regulate effectors responsible for cellular responses. The limited number of transducers results in different GPCRs exerting control on the same pathway, while the availability of signaling proteins in a cell defines the result of GPCR activation. The aim of this study was to construct the extended human GPCR network (hGPCRnet) and examine the effect that cell-type specificity has on GPCR signaling pathways. To achieve this, protein-protein interaction data between GPCRs, G-protein coupled receptor kinases (GRKs), Gα subunits, β-arrestins, and effectors were combined with protein expression data in cell types. This resulted in the hGPCRnet, a very large interconnected network, and similar cell-type-specific networks in which, distinct GPCR signaling pathways were formed. Finally, a user friendly web application, hGPCRnet ( http://bioinformatics.biol.uoa.gr/hGPCRnet ), was created to allow for the visualization and exploration of these networks and of GPCR signaling pathways. This work, and the resulting application, can be useful in further studies of GPCR function and pharmacology.

Precise modelling and interpretation of bioactivities of ligands targeting G protein-coupled receptors.

MotivationAccurate prediction and interpretation of ligand bioactivities are essential for virtual screening and drug discovery. Unfortunately, many important drug targets lack experimental data about the ligand bioactivities; this is particularly true for G protein-coupled receptors (GPCRs), which account for the targets of about a third of drugs currently on the market. Computational approaches with the potential of precise assessment of ligand bioactivities and determination of key substructural features which determine ligand bioactivities are needed to address this issue.ResultsA new method, SED, was proposed to predict ligand bioactivities and to recognize key substructures associated with GPCRs through the coupling of screening for Lasso of long extended-connectivity fingerprints (ECFPs) with deep neural network training. The SED pipeline contains three successive steps: (i) representation of long ECFPs for ligand molecules, (ii) feature selection by screening for Lasso of ECFPs and (iii) bioactivity prediction through a deep neural network regression model. The method was examined on a set of 16 representative GPCRs that cover most subfamilies of human GPCRs, where each has 300–5000 ligand associations. The results show that SED achieves excellent performance in modelling ligand bioactivities, especially for those in the GPCR datasets without sufficient ligand associations, where SED improved the baseline predictors by 12% in correlation coefficient (r2) and 19% in root mean square error. Detail data analyses suggest that the major advantage of SED lies on its ability to detect substructures from long ECFPs which significantly improves the predictive performance.Availability and implementationThe source code and datasets of SED are freely available at https://zhanglab.ccmb.med.umich.edu/SED/.Supplementary information Supplementary data are available at Bioinformatics online.

Development and application of a high-content virion display human GPCR array

Human G protein-coupled receptors (GPCRs) respond to various ligands and stimuli. However, GPCRs rely on membrane for proper folding, making their biochemical properties difficult to study. By displaying GPCRs in viral envelopes, we fabricated a Virion Display (VirD) array containing 315 non-olfactory human GPCRs for functional characterization. Using this array, we found that 10 of 20 anti-GPCR mAbs were ultra-specific. We further demonstrated that those failed in the mAb assays could recognize their canonical ligands, suggesting proper folding. Next, using two peptide ligands on the VirD-GPCR array, we identified expected interactions and novel interactions. Finally, we screened the array with group B Streptococcus, a major cause of neonatal meningitis, and demonstrated that inhibition of a newly identified target, CysLTR1, reduced bacterial penetration both in vitro and in vivo. We believe that the VirD-GPCR array holds great potential for high-throughput screening for small molecule drugs, affinity reagents, and ligand deorphanization.

SAT-LB072 Pharmacological Chaperone Rescue of Function in Human HPG Axis GPCRs with Inactivating Mutations

Inactivating mutations in G-protein coupled receptors (GPCRs) at all levels of the HPG axis give rise to incomplete reproductive development and adult infertility. The majority of the mutations in GPCRs cause misfolding of the receptor and a failure to traffic to the cell surface. We have identified cell permeant small molecule agonists and antagonists which can bind orthosterically or allosterically to stabilize the nascent GPCR in the endoplasmic reticulum and chaperone the mutant GPCR to the cell membrane. We have successfully 'rescued' many GnRH mutant receptors using small molecule antagonists which bind orthosterically. After removal of the antagonists the mutant GnRH receptors demonstrate good cell surface expression. Michael Conn's laboratory has demonstrated that such receptor rescue can restore reproductive competence in mice harboring an inactivating GnRH receptor mutation. We have also demonstrated rescue of cell surface expression and signaling in a substantial number of LH receptor mutations causing infertility in humans using a cell permeant allosteric agonist. This molecule is also able to rescue function in LHR binding and signalling deficient human mutations. Similarly we have rescued cell surface expression and signalling in human neurokinin B receptor mutations with an NKB antagonist and are exploring rescue of GPR54 utilising cell penetrating peptides. These discoveries represent an advance towards personalized medicine for GPCR deficiencies in the human HPG axis. Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. s presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.

SAT-043 A Trafficking Defective Mutant PROKR2, Identified In Patients With Hypogonadotropic Hypogonadism, Cycles Between Endoplasmic Reticulum (ER) And Golgi To Attenuate ER Stress

G protein-coupled receptors (GPCRs) are the largest family of transmembrane proteins and are a major class of therapeutic drug targets. Mutations in GPCRs resulting in loss of function or alteration of signaling can cause inherited or acquired diseases. Such mutations frequently cause impaired cell surface expression, resulting from protein misfolding and intracellular trapping due to careful monitoring by the cellular protein surveillance system. However, protein quality control mechanisms and intracellular trafficking of misfolded GPCRs remain incompletely understood. Here, we investigated the effects of mutations in prokineticin receptor 2 (PROKR2), identified in patients with hypogonadotropic hypogonadism or Kallmann syndrome, characterized by GnRH deficiency, as a model of the GPCR trafficking process. We first confirmed the impairment in cellular localization and downstream signaling of these PROKR2 mutants. To better understand the mechanisms underlying differences in cellular pathway targeting between wild type (WT) PROKR2 and P290S PROKR2, a trafficking defective mutant in which the mutation is in one of the most highly conserved amino acids among GPCRs, located in the 6th transmembrane domain, we performed a comparative interactome study using immunoprecipitation followed by mass spectrometry analysis. This revealed that P290S PROKR2 interacts strongly with proteins involved in the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway, transport to the Golgi, and retrograde transport from the Golgi to the ER. Guided by these interactome findings, we demonstrated that both the trafficking defective mutant, P290S PROKR2, as well as WT PROKR2 were degraded by the ERAD pathway, with the P290S mutant entering the ERAD pathway to a greater extent instead of being transported to the plasma membrane. Surprisingly, we found that P290S PROKR2 enters the Golgi, even though it is degraded by the ERAD pathway from the ER. When misfolded proteins accumulate in the ER, they evoke ER stress, which activates the unfolded protein response and can cause cell death. We found that misfolded P290S PROKR2 cycles to the ER-Golgi interface to reduce ER stress and to promote efficient degradation via the ERAD pathway. Our studies demonstrate that misfolded GPCRs are controlled in the early secretory pathway to maintain cellular homeostasis, thereby providing a fundamental strategy for restoration of defective cell surface transport caused by GPCR mutations as well as protection from the initiation and progression of human disease caused by ER stress. Our findings offer insights which could lead to development of potential therapeutic targets for diseases caused by human GPCR mutations.

Conserved 2nd Residue of Helix 8 of GPCR May Confer the Subclass-Characteristic and Distinct Roles through a Rapid Initial Interaction with Specific G Proteins.

To obtain a systematic view of the helix-8-second residue responsible for G protein-coupled receptor (GPCR)–G protein initial specific interactions, 786 human GPCRs were subclassified based on the pairs of agonist groups and target G proteins and compared with their conserved second residue of helix 8. Of 314 non-olfactory and deorphanized GPCRs, 273 (87%) conserved single amino acids in the subclasses, while 93 (58%) of the 160 subclasses possessed only a single GPCR member. Class B, C, Frizzled, and trace amine-associated GPCRs demonstrated 100% conservation, whereas class I and II olfactory and vomeronasal 1 receptors demonstrated much lower rates of conservation (20–47%). These conserved residues are characteristic of GPCR classes and G protein subtypes and confer their functionally-distinct roles.

High throughput screening at novel striatum orphan GPCRs identified by human transcriptomic profiling

The striatum performs essential brain functions including movement control, regulation of attention and is a critical component of reward systems. Current druggable targets have displayed limited efficacy in treating striatal diseases including addiction, schizophrenia and Parkinson's disease. G protein‐coupled receptors (GPCRs) are essential drug targets with ~35% of FDA approved medicines targeting these receptors. Of the 350 non‐sensory GPCRs, nearly 120 remain orphan GPCRs, whose endogenous ligands and G protein signaling are unknown. A previous challenge with orphan receptors has been the design of reliable drug screens, but a recent advance in the PRESTO‐Tango assay now allows for a measurement of receptor activation for over 300 GPCRs. Here we pursue the objective of identifying new druggable GPCRs, selectively expressed in the human striatum, using human tissue transcriptomics and the PRESTO‐Tango platform. We conducted a comprehensive genome‐wide survey of human tissue‐selective gene expression using 1640 high‐quality RNA‐seq samples from the Genotype Tissue Expression (GTEx) project. To identify receptor function and screen ligands, human clones of receptors were expressed in HEK293 cells and signaling was assessed using the Glo‐sensor cAMP and Tango beta‐arrestin assay. As a ligand discovery proof‐of‐concept, the Tango assay was used to screen the LOPAC library against five striatal orphan GPCRs. Human gene expression analyses identified expression of 18 striatal‐selective GPCRs. 11 of these receptors have known ligands including established therapeutic drug targets (e.g. dopamine D1, D2 receptors). The remaining 7 human striatal GPCRs, namely, GPR6, 52, 55, 88, 101, 139 and 149 were identified as class A orphan receptors. Biochemical and CRISPR/Cas9 knockout studies confirmed GPR6, GPR52 and GPR101 increased cAMP via Gs/olf G proteins, while GPR88 decreased cAMP levels via Gi/o G proteins. Screening the LOPAC library in parallel against five orphans (6, 52, 88, 101, 149) using the Tango assay revealed 60 compounds that selectively induced a 2‐fold activity above or below vehicle treated cells for one of the five receptors. Dose response assays confirmed two compounds with agonist activity for GPR88 (clozapine and carbachol) and one with agonist activity for GPR149 (PDTC). Interestingly, carbachol and clozapine are FDA approved drugs. Clozapine is a classic atypical antipsychotic medication and inverse agonist at serotonin and DA receptors; and carbachol is a cholinergic agonist currently used for the treatment of glaucoma. PDTC agonist activity for GPR149 represents the first described agonist compound for this orphan receptor. This research provides a template for identifying brain region specific targets using human tissue RNAseq transcriptomic datasets. Our findings also suggest the seven identified human orphan GPCRs are potential drug targets amenable to screening, whose pharmacological modulation may be therapeutic for treating striatum‐related neurological diseases.Support or Funding InformationRising Star Award, UT System (JAA); UTMB institutional funding (JAA); T32 DA07287 (DEF)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Structural Plasticity of a Human G Protein‐Coupled Receptor Studied by NMR Spectroscopy

The human genome encodes for 826 G protein‐coupled receptors (GPCRs), which are involved in nearly every physiological process and comprise the largest class of “druggable” proteins. Using nuclear magnetic resonance (NMR) in solution, we provide a global view of structural plasticity underlying the function of the human A2Aadenosine receptor (A2AAR), a representative class A human GPCR. To apply NMR to study a human GPCR we developed a robust molecular biology pipeline for production and sample preparation of stable‐isotope labeled receptors, yielding reproducible high‐quality NMR data. From these data, assignment of endogenous residues provided numerous probes distributed over the receptor intracellular surface, extracellular surface, and transmembrane domain, enabling a global characterization of A2AAR responses to variable efficacy of bound drugs. The NMR probes also permitted novel insights into changes in the local conformation of conserved GPCR activation motifs. In addition, we observed multiple local efficacy‐related conformers at the intracellular surface, which we relate directly to G protein signaling. The distributed NMR probes provide novel information on the role of a highly conserved GPCR allosteric center, permitting us to study the effect of this center on signaling pathways in the receptor. Amino acid replacement of the allosteric center caused striking changes in conformational polymorphisms at the intracellular surface but did not alter response to drug efficacy at the extracellular surface. We correlated changes at the intracellular surface to structural rearrangements at the toggle switch tryptophan and a conserved activation motif, providing novel insights into a widespread signaling mechanism in class A GPCRs. The presented work opens the door to exciting future studies of GPCR function, including studies of GPCRs in native environments.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Beyond the Flavour: The Potential Druggability of Chemosensory G Protein-Coupled Receptors.

G protein-coupled receptors (GPCRs) belong to the largest class of drug targets. Approximately half of the members of the human GPCR superfamily are chemosensory receptors, including odorant receptors (ORs), trace amine-associated receptors (TAARs), bitter taste receptors (TAS2Rs), sweet and umami taste receptors (TAS1Rs). Interestingly, these chemosensory GPCRs (csGPCRs) are expressed in several tissues of the body where they are supposed to play a role in biological functions other than chemosensation. Despite their abundance and physiological/pathological relevance, the druggability of csGPCRs has been suggested but not fully characterized. Here, we aim to explore the potential of targeting csGPCRs to treat diseases by reviewing the current knowledge of csGPCRs expressed throughout the body and by analysing the chemical space and the drug-likeness of flavour molecules.

Studying Structural Plasticity Underlying GPCR Function

The human genome encodes for 826 G protein-coupled receptors (GPCRs), which are involved in nearly every physiological process and comprise the largest class of “druggable” proteins. Using nuclear magnetic resonance (NMR) in solution, we provide a global view of structural plasticity underlying the function of the human A2A adenosine receptor (A2AAR), a representative class A human GPCR. To apply NMR to study a human GPCR we developed a robust molecular biology pipeline for production and sample preparation of stable-isotope labeled receptors, yielding reproducible high-quality NMR data. From these data, assignment of endogenous residues provided numerous probes distributed over the receptor intracellular surface, extracellular surface, and transmembrane domain, enabling a global characterization of A2AAR responses to variable efficacy of bound drugs. The NMR probes also permitted novel insights into changes in the local conformation of conserved GPCR activation motifs. In addition, we observed multiple local efficacy-related conformers at the intracellular surface, which we relate directly to G protein signaling. The distributed NMR probes provide novel information on the role of a highly conserved GPCR allosteric center, permitting us to study the effect of this center on signaling pathways in the receptor. Amino acid replacement of the allosteric center caused striking changes in conformational polymorphisms at the intracellular surface but did not alter response to drug efficacy at the extracellular surface. We correlated changes at the intracellular surface to structural rearrangements at the toggle switch tryptophan and a conserved activation motif, providing novel insights into a widespread signaling mechanism in class A GPCRs. The presented work opens the door to exciting future studies of GPCR function, including studies of GPCRs in native environments.

Coupling of Human Rhodopsin to a Yeast Signaling Pathway Enables Characterization of Mutations Associated with Retinal Disease.

G protein-coupled receptors (GPCRs) are crucial sensors of extracellular signals in eukaryotes, with multiple GPCR mutations linked to human diseases. With the growing number of sequenced human genomes, determining the pathogenicity of a mutation is challenging, but can be aided by a direct measurement of GPCR-mediated signaling. This is particularly difficult for the visual pigment rhodopsin-a GPCR activated by light-for which hundreds of mutations have been linked to inherited degenerative retinal diseases such as retinitis pigmentosa. In this study, we successfully engineered, for the first time, activation by human rhodopsin of the yeast mating pathway, resulting in signaling via a fluorescent reporter. We combine this novel assay for rhodopsin light-dependent activation with studies of subcellular localization, and the upregulation of the unfolded protein response in response to misfolded rhodopsin protein. We use these assays to characterize a panel of rhodopsin mutations with known molecular phenotypes, finding that rhodopsin maintains a similar molecular phenotype in yeast, with some interesting differences. Furthermore, we compare our assays in yeast with clinical phenotypes from patients with novel disease-linked mutations. We demonstrate that our engineered yeast strain can be useful in rhodopsin mutant classification, and in helping to determine the molecular mechanisms underlying their pathogenicity. This approach may also be applied to better understand the clinical relevance of other human GPCR mutations, furthering the use of yeast as a tool for investigating molecular mechanisms relevant to human disease.

GPCR drug discovery: integrating solution NMR data with crystal and cryo-EM structures.

The 826 G protein-coupled receptors (GPCRs) in the human proteome regulate key physiological processes and thus have long been attractive drug targets. With the crystal structures of more than 50 different human GPCRs determined over the past decade, an initial platform for structure-based rational design has been established for drugs that target GPCRs, which is currently being augmented with cryo-electron microscopy (cryo-EM) structures of higher-order GPCR complexes. Nuclear magnetic resonance (NMR) spectroscopy in solution is one of the key approaches for expanding this platform with dynamic features, which can be accessed at physiological temperature and with minimal modification of the wild-type GPCR covalent structures. Here, we review strategies for the use of advanced biochemistry and NMR techniques with GPCRs, survey projects in which crystal or cryo-EM structures have been complemented with NMR investigations and discuss the impact of this integrative approach on GPCR biology and drug discovery.

A benchmarking study on virtual ligand screening against homology models of human GPCRs.

G-protein-coupled receptor (GPCR) is an important target class of proteins for drug discovery, with over 27% of FDA-approved drugs targeting GPCRs. However, being a membrane protein, it is difficult to obtain the 3D crystal structures of GPCRs for virtual screening of ligands by molecular docking. Thus, we evaluated the virtual screening performance of homology models of human GPCRs with respect to the corresponding crystal structures. Among the 19 GPCRs involved in this study, we observed that 10 GPCRs have homology models that have better or comparable performance with respect to the corresponding X-ray structures, making homology models a viable choice for virtual screening. For a small subset of GPCRs, we also explored how certain methods like consensus enrichment and sidechain perturbation affect the utility of homology models in virtual screening, as well as the selectivity between agonists and antagonists. Most notably, consensus enrichment across multiple homology models often yields results comparable to the best performing model, suggesting that ligand candidates predicted with consensus scores from multiple models can be the optimal option in practical applications where the performance of each model cannot be estimated.

On the use of Pichia pastoris for isotopic labeling of human GPCRs for NMR studies.

NMR studies of human integral membrane proteins provide unique opportunities to probe structure and dynamics at specific locations and on multiple timescales, often with significant implications for disease mechanism and drug development. Since membrane proteins such as G protein-coupled receptors (GPCRs) are highly dynamic and regulated by ligands or other perturbations, NMR methods are potentially well suited to answer basic functional questions (such as addressing the biophysical basis of ligand efficacy) as well as guiding applications (such as novel ligand design). However, such studies on eukaryotic membrane proteins have often been limited by the inability to incorporate optimal isotopic labels for NMR methods developed for large protein/lipid complexes, including methyl TROSY. We review the different expression systems for production of isotopically labeled membrane proteins and highlight the use of the yeast Pichia pastoris to achieve perdeuteration and 13C methyl probe incorporation within isoleucine sidechains. We further illustrate the use of this method for labeling of several biomedically significant GPCRs.

The Listeriolysin O PEST-like Sequence Co-opts AP-2-Mediated Endocytosis to Prevent Plasma Membrane Damage during Listeria Infection

Listeriolysin O (LLO) is a cholesterol-dependent cytolysin that mediates escape of Listeria monocytogenes from a phagosome, enabling growth of the bacteria in the host cell cytosol. LLO contains a PEST-like sequence that prevents it from killing infected cells, but the mechanism involved is unknown. We found that the LLO PEST-like sequence was necessary to mediate removal of LLO from the interior face of the plasma membrane, where it coalesces into discrete puncta. LLO interacts with Ap2a2, an adaptor protein involved in endocytosis, via its PEST-like sequence, and Ap2a2-dependent endocytosis is required to prevent LLO-induced cytotoxicity. An unrelated PEST-like sequence from a human Gprotein-coupled receptor (GPCR), which also interacts with Ap2a2, could functionally complement the PEST-like sequence in L.monocytogenes LLO. These data revealed that LLO co-opts the host endocytosis machinery to protect the integrity of the host plasma membrane during L.monocytogenes infection.

The role of disordered regions in GPCR‐G protein signaling

G-protein-coupled receptors (GPCRs) are physiologically important membrane proteins that sense signaling molecules such as hormones and neurotransmitters and are the targets of several prescribed drugs. The ~800 human GPCRs allosterically activate 16 G proteins and trigger GDP release to regulate intracellular signaling. In this presentation, I will present our findings on the allosteric mechanism governing Gα activation and the determinants of selective coupling between receptors and G proteins. In the first part of the talk, I will present evidence, which suggests that different GPCRs interact with and activate Gα proteins through a highly conserved mechanism. Through comparison of Gα with the small G protein Ras, we found that the evolution of short segments that undergo disorder-to-order transitions can decouple regions important for allosteric activation from receptor binding specificity [1]. In the second part of the talk, I will present the existence of a selectivity barcode (i.e., patterns of amino acids) within the disordered tail of each of the 16 human G proteins that is recognized by distinct regions on the ~800 human receptors. Although universally conserved positions in the barcode allow the receptors to bind and activate G proteins in a similar manner, different receptors recognize the unique positions of the G-protein barcode through distinct residues, like multiple keys (receptors) opening the same lock (G protein) using non-identical cuts. Considering the evolutionary history of GPCRs, specifically, the disordered intracellular loops allows the identification of these selectivity-determining residues [2]. These observations might explain how the GPCR-Gα system diversified rapidly while conserving the allosteric activation mechanism and provides a framework for understanding the molecular basis of coupling selectivity within individual receptors and G proteins. Support or Funding Information Medical Research Council (MC_U105185859) GPCR-G protein interface This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Fungal G-protein-coupled receptors: mediators of pathogenesis and targets for disease control

G-protein signalling pathways are involved in sensing the environment, enabling fungi to coordinate cell function, metabolism and development with their surroundings, thereby promoting their survival, propagation and virulence. G-protein-coupled receptors (GPCRs) are the largest class of cell surface receptors in fungi. Despite the apparent importance of GPCR signalling to fungal biology and virulence, relatively few GPCR-G-protein interactions, and even fewer receptor-binding ligands, have been identified. Approximately 40% of current pharmaceuticals target human GPCRs, due to their cell surface location and central role in cell signalling. Fungal GPCRs do not belong to any of the mammalian receptor classes, making them druggable targets for antifungal development. This Review Article evaluates developments in our understanding of fungal GPCR-mediated signalling, while substantiating the rationale for considering these receptors as potential antifungal targets. The need for insights into the structure-function relationship of receptor-ligand interactions is highlighted, which could facilitate the development of receptor-interfering compounds that could be used in disease control.

Defining G protein-coupled receptor peptide ligand expressomes and signalomes in human and mouse islets

IntroductionIslets synthesise and secrete numerous peptides, some of which are known to be important regulators of islet function and glucose homeostasis. In this study, we quantified mRNAs encoding all peptide ligands of islet G protein-coupled receptors (GPCRs) in isolated human and mouse islets and carried out in vitro islet hormone secretion studies to provide functional confirmation for the species-specific role of peptide YY (PYY) in mouse islets.Materials and methodsGPCR peptide ligand mRNAs in human and mouse islets were quantified by quantitative real-time PCR relative to the reference genes ACTB, GAPDH, PPIA, TBP and TFRC. The pathways connecting GPCR peptide ligands with their receptors were identified by manual searches in the PubMed, IUPHAR and Ingenuity databases. Distribution of PYY protein in mouse and human islets was determined by immunohistochemistry. Insulin, glucagon and somatostatin secretion from islets was measured by radioimmunoassay.ResultsWe have quantified GPCR peptide ligand mRNA expression in human and mouse islets and created specific signalomes mapping the pathways by which islet peptide ligands regulate human and mouse GPCR signalling. We also identified species-specific islet expression of several GPCR ligands. In particular, PYY mRNA levels were ~ 40,000-fold higher in mouse than human islets, suggesting a more important role of locally secreted Pyy in mouse islets. This was confirmed by IHC and functional experiments measuring insulin, glucagon and somatostatin secretion.DiscussionThe detailed human and mouse islet GPCR peptide ligand atlases will allow accurate translation of mouse islet functional studies for the identification of GPCR/peptide signalling pathways relevant for human physiology, which may lead to novel treatment modalities of diabetes and metabolic disease.

Yeast surface display platform for rapid discovery of conformationally selective nanobodies.

Camelid single-domain antibody fragments (“nanobodies”) provide the remarkable specificity of antibodies within a single 15 kDa immunoglobulin VHH domain. This unique feature has enabled applications ranging from use as biochemical tools to therapeutic agents. Nanobodies have emerged as especially useful tools in protein structural biology, facilitating studies of conformationally dynamic proteins such as G protein-coupled receptors (GPCRs). Nearly all nanobodies available to date have been obtained by animal immunization, a bottleneck restricting many applications of this technology. To solve this problem, we report a fully in vitro platform for nanobody discovery based on yeast surface display. We provide a blueprint for identifying nanobodies, demonstrate the utility of the library by crystallizing a nanobody with its antigen, and most importantly, we utilize the platform to discover conformationally-selective nanobodies to two distinct human GPCRs. To facilitate broad deployment of this platform, the library and associated protocols are freely available for non-profit research.