Mutations In G Protein-coupled Receptors Research Articles

Multi-omics integration analysis of GPCRs in pan-cancer to uncover inter-omics relationships and potential driver genes

G protein-coupled receptors (GPCRs) are the largest drug target family. Unfortunately, applications of GPCRs in cancer therapy are scarce due to very limited knowledge regarding their correlations with cancers. Multi-omics data enables systematic investigations of GPCRs, yet their effective integration remains a challenge due to the complexity of the data. Here, we adopt two types of integration strategies, multi-staged and meta-dimensional approaches, to fully characterize somatic mutations, somatic copy number alterations (SCNAs), DNA methylations, and mRNA expressions of GPCRs in 33 cancers. Results from the multi-staged integration reveal that GPCR mutations cannot well predict expression dysregulation. The correlations between expressions and SCNAs are primarily positive, while correlations of the methylations with expressions and SCNAs are bimodal with negative correlations predominating. Based on these correlations, 32 and 144 potential cancer-related GPCRs driven by aberrant SCNA and methylation are identified, respectively. In addition, the meta-dimensional integration analysis is carried out by using deep learning models, which predict more than one hundred GPCRs as potential oncogenes. When comparing results between the two integration strategies, 165 cancer-related GPCRs are common in both, suggesting that they should be prioritized in future studies. However, 172 GPCRs emerge in only one, indicating that the two integration strategies should be considered concurrently to complement the information missed by the other such that obtain a more comprehensive understanding. Finally, correlation analysis further reveals that GPCRs, in particular for the class A and adhesion receptors, are generally immune-related. In a whole, the work is for the first time to reveal the associations between different omics layers and highlight the necessity of combing the two strategies in identifying cancer-related GPCRs.

Single nucleotide variations encoding missense mutations in G protein-coupled receptors may contribute to autism.

Autism is a neurodevelopmental condition with a range of symptoms that vary in intensity and severity from person to person. Genetic sequencing has identified thousands of genes containing mutations in autistic individuals, which may contribute to the development of autistic symptoms. Several of these genes encode G protein-coupled receptors (GPCRs), which are cell surface expressed proteins that transduce extracellular messages to the intracellular space. Mutations in GPCRs can impact their function, resulting in aberrant signalling within cells and across neurotransmitter systems in the brain. This review summarises the current knowledge on autism-associated single nucleotide variations encoding missense mutations in GPCRs and the impact of these genetic mutations on GPCR function. For some autism-associated mutations, changes in GPCR expression levels, ligand affinity, potency and efficacy have been observed. However, for many the functional consequences remain unknown. Thus, further work to characterise the functional impacts of the genetically identified mutations is required.

MUG: A mutation overview of GPCR subfamily A17 receptors

G protein-coupled receptors (GPCRs) mediate several signaling pathways through a general mechanism that involves their activation, upholding a chain of events that lead to the release of molecules responsible for cytoplasmic action and further regulation. These physiological functions can be severely altered by mutations in GPCR genes. GPCRs subfamily A17 (dopamine, serotonin, adrenergic and trace amine receptors) are directly related with neurodegenerative diseases, and as such it is crucial to explore known mutations on these systems and their impact in structure and function. A comprehensive and detailed computational framework - MUG (Mutations Understanding GPCRs) - was constructed, illustrating key reported mutations and their effect on receptors of the subfamily A17 of GPCRs. We explored the type of mutations occurring overall and in the different families of subfamily A17, as well their localization within the receptor and potential effects on receptor functionality. The mutated residues were further analyzed considering their pathogenicity. The results reveal a high diversity of mutations in the GPCR subfamily A17 structures, drawing attention to the considerable number of mutations in conserved residues and domains. Mutated residues were typically hydrophobic residues enriched at the ligand binding pocket and known activating microdomains, which may lead to disruption of receptor function. MUG as an interactive web application is available for the management and visualization of this dataset. We expect that this interactive database helps the exploration of GPCR mutations, their influence, and their familywise and receptor-specific effects, constituting the first step in elucidating their structures and molecules at the atomic level.

Targeting trafficking as a therapeutic avenue for misfolded GPCRs leading to endocrine diseases.

G protein-coupled receptors (GPCRs) are plasma membrane proteins associated with an array of functions. Mutations in these receptors lead to a number of genetic diseases, including diseases involving the endocrine system. A particular subset of loss-of-function mutant GPCRs are misfolded receptors unable to traffic to their site of function (i.e. the cell surface plasma membrane). Endocrine disorders in humans caused by GPCR misfolding include, among others, hypo- and hyper-gonadotropic hypogonadism, morbid obesity, familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism, X-linked nephrogenic diabetes insipidus, congenital hypothyroidism, and familial glucocorticoid resistance. Several in vitro and in vivo experimental approaches have been employed to restore function of some misfolded GPCRs linked to endocrine disfunction. The most promising approach is by employing pharmacological chaperones or pharmacoperones, which assist abnormally and incompletely folded proteins to refold correctly and adopt a more stable configuration to pass the scrutiny of the cell’s quality control system, thereby correcting misrouting. This review covers the most important aspects that regulate folding and traffic of newly synthesized proteins, as well as the experimental approaches targeted to overcome protein misfolding, with special focus on GPCRs involved in endocrine diseases.

Hear the sounds: the role of G protein-coupled receptors in the cochlea.

Sound is converted by hair cells in the cochlea into electrical signals, which are transmitted by spiral ganglion neurons (SGNs) and heard by the auditory cortex. G protein-coupled receptors (GPCRs) are crucial receptors that regulate a wide range of physiological functions in different organ and tissues. The research of GPCRs in the cochlea is essential for the understanding of the cochlea development, hearing disorders, and the treatment for hearing loss. Recently, several GPCRs have been found to play important roles in the cochlea. Frizzleds and Lgrs are dominant GPCRs that regulate stem cell self-renew abilities. Moreover, Frizzleds and Celsrs have been demonstrated to play core roles in the modulation of cochlear planar cell polarity (PCP). In addition, hearing loss can be caused by mutations of certain GPCRs, such as Vlgr1, Gpr156, S1P2, and Gpr126. And A1, A2A, and CB2 activation by agonists has protective functions on noise- or drug-induced hearing loss. Here, we review the key findings of GPCR in the cochlea and discuss the role of GPCR in the cochlea, such as stem cell fate, PCP, hearing loss, and hearing protection.

Functional Rescue of Inactivating Mutations of the Human Neurokinin 3 Receptor Using Pharmacological Chaperones.

G protein-coupled receptors (GPCRs) facilitate the majority of signal transductions across cell membranes in humans, with numerous diseases attributed to inactivating GPCR mutations. Many of these mutations result in misfolding during nascent receptor synthesis in the endoplasmic reticulum (ER), resulting in intracellular retention and degradation. Pharmacological chaperones (PCs) are cell-permeant small molecules that can interact with misfolded receptors in the ER and stabilise/rescue their folding to promote ER exit and trafficking to the cell membrane. The neurokinin 3 receptor (NK3R) plays a pivotal role in the hypothalamic–pituitary–gonadal reproductive axis. We sought to determine whether NK3R missense mutations result in a loss of cell surface receptor expression and, if so, whether a cell-permeant small molecule NK3R antagonist could be repurposed as a PC to restore function to these mutants. Quantitation of cell surface expression levels of seven mutant NK3Rs identified in hypogonadal patients indicated that five had severely impaired cell surface expression. A small molecule NK3R antagonist, M8, increased cell surface expression in four of these five and resulted in post-translational receptor processing in a manner analogous to the wild type. Importantly, there was a significant improvement in receptor activation in response to neurokinin B (NKB) for all four receptors following their rescue with M8. This demonstrates that M8 may have potential for therapeutic development in the treatment of hypogonadal patients harbouring NK3R mutations. The repurposing of existing small molecule GPCR modulators as PCs represents a novel and therapeutically viable option for the treatment of disorders attributed to mutations in GPCRs that cause intracellular retention.

Phenotypic evaluation of constitutive GPCR/G-protein signaling in zebrafish embryos and larvae

Signal transduction initiation by G-protein-coupled receptors (GPCRs) normally begins upon extracellular ligand binding. Some oncogenic GPCR mutants are capable of inducing G-protein signaling without ligand stimulation, thus behaving as constitutively active receptors. Evaluation of disease-causing capacity of constitutively active mutations in animal models requires months of time-consuming experiments, which hampers research progress. Here, using zebrafish embryos transiently expressing with constitutively active mutations via mRNA microinjection, we describe G-protein-subtype-specific phenotypes that can be evaluated over several days. Exogenous expression of the cysteinyl leukotriene receptor type II (CysLT2R) with an oncogenic L1293.43Q mutation by mRNA injection into a fertilized embryo induced developmental arrest during epiboly and eventual embryonic lethality, which were suppressed by treatment with the Gq inhibitor, YM-254890. Embryos with a constitutively active Gαq mutant exhibited an analogous phenotype. Interestingly, expression of constitutively active Gαs, Gαi, and Gα13 mutants induced distinct phenotypes. These phenotypes may thus serve as useful indicators for rapid in vivo evaluation of signaling activity of GPCR and G-protein mutants.

Trafficking-defective mutant PROKR2 cycles between endoplasmic reticulum and Golgi to attenuate endoplasmic reticulum stress

G protein-coupled receptors (GPCRs) play crucial roles in numerous physiological and pathological processes. Mutations in GPCRs that result in loss of function or alterations in signaling can lead to inherited or acquired diseases. Herein, studying prokineticin receptor 2 (PROKR2), we initially identify distinct interactomes for wild-type (WT) versus a mutant (P290S) PROKR2 that causes hypogonadotropic hypogonadism. We then find that both the WT and mutant PROKR2 are targeted for endoplasmic reticulum (ER)-associated degradation, but the mutant is degraded to a greater extent. Further analysis revealed that both forms can also leave the ER to reach the Golgi. However, whereas most of the WT is further transported to the cell surface, most of the mutant is retrieved to the ER. Thus, the post-ER itinerary plays an important role in distinguishing the ultimate fate of the WT versus the mutant. We have further discovered that this post-ER itinerary reduces ER stress induced by the mutant PROKR2. Moreover, we extend the core findings to another model GPCR. Our findings advance the understanding of disease pathogenesis induced by a mutation at a key residue that is conserved across many GPCRs and thus contributes to a fundamental understanding of the diverse mechanisms used by cellular quality control to accommodate misfolded proteins.

Misfolded G Protein-Coupled Receptors and Endocrine Disease. Molecular Mechanisms and Therapeutic Prospects.

Misfolding of G protein-coupled receptors (GPCRs) caused by mutations frequently leads to disease due to intracellular trapping of the conformationally abnormal receptor. Several endocrine diseases due to inactivating mutations in GPCRs have been described, including X-linked nephrogenic diabetes insipidus, thyroid disorders, familial hypocalciuric hypercalcemia, obesity, familial glucocorticoid deficiency [melanocortin-2 receptor, MC2R (also known as adrenocorticotropin receptor, ACTHR), and reproductive disorders. In these mutant receptors, misfolding leads to endoplasmic reticulum retention, increased intracellular degradation, and deficient trafficking of the abnormal receptor to the cell surface plasma membrane, causing inability of the receptor to interact with agonists and trigger intracellular signaling. In this review, we discuss the mechanisms whereby mutations in GPCRs involved in endocrine function in humans lead to misfolding, decreased plasma membrane expression of the receptor protein, and loss-of-function diseases, and also describe several experimental approaches employed to rescue trafficking and function of the misfolded receptors. Special attention is given to misfolded GPCRs that regulate reproductive function, given the key role played by these particular membrane receptors in sexual development and fertility, and recent reports on promising therapeutic interventions targeting trafficking of these defective proteins to rescue completely or partially their normal function.

Rescue of Cell Surface Expression and Signaling of Mutant Follicle-Stimulating Hormone Receptors.

Mutations in G protein-coupled receptors (GPCRs) underlie numerous diseases. Many cause receptor misfolding and failure to reach the cell surface. Pharmacological chaperones are cell-permeant small molecules that engage nascent mutant GPCRs in the endoplasmic reticulum, stabilizing folding and "rescuing" cell surface expression. We previously demonstrated rescue of cell surface expression of luteinizing hormone receptor mutants by an allosteric agonist. Here we demonstrate that a similar approach can be employed to rescue mutant follicle-stimulating hormone receptors (FSHRs) with poor cell surface expression using a small-molecule FSHR agonist, CAN1404. Seventeen FSHR mutations described in patients with reproductive dysfunction were expressed in HEK 293T cells, and cell surface expression was determined by enzyme-linked immunosorbent assay of epitope-tagged FSHRs before/after treatment with CAN1404. Cell surface expression was severely reduced to ≤18% of wild-type (WT) for 11, modestly reduced to 66% to 84% of WT for 4, and not reduced for 2. Of the 11 with severely reduced cell surface expression, restoration to ≥57% of WT levels was achieved for 6 by treatment with 1 µM CAN1404 for 24 h, and a corresponding increase in FSH-induced signaling was observed for 4 of these, indicating restored functionality. Therefore, CAN1404 acts as a pharmacological chaperone and can rescue cell surface expression and function of certain mutant FSHRs with severely reduced cell surface expression. These findings aid in advancing the understanding of the effects of genetic mutations on GPCR function and provide a proof of therapeutic principle for FSHR pharmacological chaperones.

Allosteric communication regulates ligand-specific GPCR activity.

G protein-coupled receptors (GPCRs) are membrane-bound proteins that are ubiquitously expressed in many cell types and take part in mediating multiple signaling pathways. GPCRs are dynamic proteins and exist in an equilibrium between an ensemble of conformational states such as inactive and fully active states. This dynamic nature of GPCRs is one of the factors that confers their basal activity even in the absence of any ligand-mediated activation. Ligands selectively bind and stabilize a subset of the conformations from the ensemble leading to a shift in the equilibrium toward the inactive or the active state depending on the nature of the ligand. This ligand-selective effect is achieved through allosteric communication between the ligand binding site and G protein or β-arrestin coupling site. Similarly, the G protein coupling to the receptor exerts the allosteric effect on the ligand binding region leading to increased binding affinity for agonists and decreased affinity for antagonists or inverse agonists. In this review, we enumerate the current state of our understanding of the mechanism of allosteric communication in GPCRs with a specific focus on the critical role of computational methods in delineating the residues involved in allosteric communication. Analyzing allosteric communication mechanism using molecular dynamics simulations has revealed (a) a structurally conserved mechanism of allosteric communication that regulates the G protein coupling, (b) a rational structure-based approach to designing selective ligands, and (c) an approach to designing allosteric GPCR mutants that are either ligand and G protein or β-arrestin selective.

Thyrotropin Receptor p.N432D Retained Variant Is Degraded Through an Alternative Lysosomal/Autophagosomal Pathway and Can Be Functionally Rescued by Chemical Chaperones.

Background: Loss-of-function mutations of thyrotropin receptor (TSHR) are one of the main causes of congenital hypothyroidism. As for many disease-associated G-protein coupled receptors (GPCRs), these mutations often affect the correct trafficking and maturation of the receptor, thus impairing the expression on the cell surface. Several retained GPCR mutants are able to effectively bind their ligands and to transduce signals when they are forced to the cell surface by degradation inhibition or by treatment with chaperones. Despite the large number of well-characterized retained TSHR mutants, no attempts have been made for rescue. Further, little is known about TSHR degradation pathways. We hypothesize that, similar to other GPCRs, TSHR retained mutants may be at least partially functional if their maturation and membrane expression is facilitated by chaperones or degradation inhibitors. Methods: We performed in silico predictions of the functionality of known TSHR variants and compared the results with available in vitro data. Western blot, confocal microscopy, enzyme-linked immunosorbent assays, and dual luciferase assays were used to investigate the effects of degradation pathways inhibition and of chemical chaperone treatments on TSHR variants' maturation and functionality. Results: We found a high discordance rate between in silico predictions and in vitro data for retained TSHR variants, a fact indicative of a conserved potential to initiate signal transduction if these mutants were expressed on the cell surface. We show experimentally that some maturation defective TSHR mutants are able to effectively transduce Gs/cAMP signaling if their maturation and expression are enhanced by using chemical chaperones. Further, through the characterization of the intracellular retained p.N432D variant, we provide new insights on the TSHR degradation mechanism, as our results suggest that aggregation-prone mutant can be directed toward the autophagosomal pathway instead of the canonical proteasome system. Conclusions: Our study reveals alternative pathways for TSHR degradation. Retained TSHR variants can be functional when expressed on the cell surface membrane, thus opening the possibility of further studies on the pharmacological modulation of TSHR expression and functionality in patients in whom TSHR signaling is disrupted.

Update on GNA Alterations in Cancer: Implications for Uveal Melanoma Treatment.

Tumorigenesis is correlated with abnormal expression and activity of G protein-coupled receptors (GPCRs) and associated G proteins. Oncogenic mutations in both GPCRs and G proteins (GNAS, GNAQ or GNA11) encoding genes have been identified in a significant number of tumors. Interestingly, uveal melanoma driver mutations in GNAQ/GNA11 were identified for a decade, but their discovery did not lead to mutation-specific drug development, unlike it the case for BRAF mutations in cutaneous melanoma which saw enormous success. Moreover, new immunotherapies strategies such as immune checkpoint inhibitors have given underwhelming results. In this review, we summarize the current knowledge on cancer-associated alterations of GPCRs and G proteins and we focus on the case of uveal melanoma. Finally, we discuss the possibilities that this signaling might represent in regard to novel drug development for cancer prevention and treatment.

Molecular chaperones and G protein-coupled receptor maturation and pharmacology

G protein-coupled receptors (GPCRs) are highly conserved versatile signaling molecules located at the plasma membrane that respond to diverse extracellular signals. They regulate almost all physiological processes in the vertebrates. About 35% of current drugs target these receptors. Mutations in these genes have been identified as causes of numerous diseases. The seven transmembrane domain structure of GPCRs implies that the folding of these transmembrane proteins is extremely complicated and difficult. Indeed, many wild type GPCRs are not folded optimally. The most common defect in genetic diseases caused by GPCR mutations is misfolding and failure to reach the plasma membrane where it functions. General molecular chaperones aid the folding of all proteins, including GPCRs, by preventing aggregation, promoting folding and disaggregating small aggregates. Some GPCRs need additional receptor-specific chaperones to assist their folding. Many of these receptor-specific chaperones interact with additional receptors and alter receptor pharmacology, expanding the understanding of these chaperone proteins.

Machine Learning for Prioritization of Thermostabilizing Mutations for G-Protein Coupled Receptors

Although the three-dimensional structures of G-protein coupled receptors (GPCRs), the largest superfamily of drug targets, have enabled structure-based drug design, there are no structures available for 87% of GPCRs. This is due to the stiff challenge in purifying the inherently flexible GPCRs. Identifying thermostabilized mutant GPCRs via systematic alanine scanning mutations has been a successful strategy in stabilizing GPCRs, but it remains a daunting task for each GPCR. We developed a computational method that combines sequence-, structure-, and dynamics-based molecular properties of GPCRs that recapitulate GPCR stability, with four different machine learning methods to predict thermostable mutations ahead of experiments. This method has been trained on thermostability data for 1231 mutants, the largest publicly available data set. A blind prediction for thermostable mutations of the complement factor C5a receptor 1 retrieved 36% of the thermostable mutants in the top 50 prioritized mutants compared to 3% in the first 50 attempts using systematic alanine scanning.

GPCRs in Autocrine and Paracrine Regulations.

G protein-coupled receptors (GPCRs) constitute the largest superfamily of integral membrane protein receptors. As signal detectors, the several 100 known GPCRs are responsible for sensing the plethora of endogenous ligands that are critical for the functioning of our endocrine system. Although GPCRs are typically considered as detectors for first messengers in classical signal transduction pathways, they seldom operate in isolation in complex biological systems. Intercellular communication between identical or different cell types is often mediated by autocrine or paracrine signals that are generated upon activation of specific GPCRs. In the context of energy homeostasis, the distinct complement of GPCRs in each cell type bridges the autocrine and paracrine communication within an organ, and the various downstream signaling mechanisms regulated by GPCRs can be integrated in a cell to produce an ultimate output. GPCRs thus act as gatekeepers that coordinate and fine-tune a response. By examining the role of GPCRs in activating and receiving autocrine and paracrine signals, one may have a better understanding of endocrine diseases that are associated with GPCR mutations, thereby providing new insights for treatment regimes.

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.

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.

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.

Therapeutic strategies for diseases caused by loss-of-function mutations in G protein-coupled receptors.

As one of the largest families of cell membrane proteins, G protein-coupled receptors (GPCRs) are involved in regulating almost all physiological processes by transducing extracellular signals into the cytoplasm. Since the first discovery of naturally occurring mutations in Rhodopsin gene in 1990, hundreds of loss-of-function mutations in multiple GPCRs have been identified to be pathogenic for more than 30 diverse human diseases, making these defective receptors important drug targets for personalized medicine. In this review, we aim to elucidate the etiologies of five common inherited diseases caused by six of the most extensively studied GPCRs. The molecular basis and classification of inactivating mutations in GPCRs are also reviewed. The available therapeutic approaches directed against different classes of mutants, especially pharmacological chaperones targeting intracellularly retained mutants, reported during the past two decades, are systematically summarized.