The ability of vertebrates, including humans, to see light relies on a visual transduction cascade in which rhodopsin’s ability to stimulate the nervous system plays a central role. Rhodopsin is a 348 amino acid protein composed of seven transmembrane alpha helices located within the rod photoreceptor cells of the retina. Rhodopsin is a member of the much broader protein superfamily, the G‐protein coupled receptors (GPCRs). GPCRs are cell surface signaling receptors that convert extracellular signals into intracellular signaling pathways through the activation of G proteins. There are about 800 different human GPCRs and members of this superfamily are responsible for our sense of smell, taste, and vision. The “wild type” rhodopsin uses its retinal pigment, a derivative of vitamin A, in conjunction with its G protein partner, transducin, to detect light, particularly in night vision. Retinal serves as the “cofactor” of rhodopsin that directly absorbs a photon, causing its isomerization and a change in the structure of rhodopsin that is sensed by transducin, leading to an intracellular signaling cascade that results in a signal being sent through the optic nerve to the brain that light has been detected. Any one of over 150 rhodopsin mutations already documented in humans are known to result in loss of rhodopsin’s light‐sensing function, leading to Retinitis Pigmentosa (RP). RP is a rare, slow, progressive, genetic disease which damages the retina leading to loss of vision, particularly among young adults. The most common cause of RP is a mutation‐induced misfolding of rhodopsin. The many forms of proposed treatment of RP are largely focused on improving folding of rhodopsin and inducing proteostasis, the process in the cell that promotes correct folding and quantities of proteins. Pharmacological chaperones, such as retinoids or non‐isomerrizable retinoid analogues directly target protein structure and have produced lab results that show enhanced folding and protection of other cells from the effects of mutated rhodopsin. However, replicating this in living organisms has not been possible yet. Nutritional supplements, histone deacetylase inhibitors, such as Valproate, and molecular chaperone inducers have also been tested, but only produce laboratory results that positively affect only a few aspects of mutated rhodopsin and have not yet yielded promising results in humans. Seemingly, the most promising forms of treatment for RP and rhodopsin mutations are gene therapy and CRISPR Cas‐9 genome editing to correct the genetic defects that underlie RP. However, whether any of these approaches will eventually form the basis for effective treatment or cure of RP is not yet known. The Walton High School MSOE Center for Biomolecular Modeling SMART Team has designed a 3‐D model of rhodopsin to further investigate and examine the structure‐function relationship of the protein.Support or Funding InformationMSOE Center for Biomolecular Modeling
1488. In Vitro and In Vivo Genome Editing of the RHO Gene to Downregulate Dominant Mutations
488. In Vitro and In Vivo Genome Editing of the RHO Gene to Downregulate Dominant Mutations
41Structure and function of G protein-coupled receptors using NMR spectroscopy
Structure and function of G protein-coupled receptors using NMR spectroscopy
150Inactivating mutations of G protein-coupled receptors and diseases: Structure-function insights and therapeutic implications
Inactivating mutations of G protein-coupled receptors and diseases: Structure-function insights and therapeutic implications
Read more247A chemogenomic analysis of the transmembrane binding cavity of human G‐protein‐coupled receptors
The amino acid sequences of 369 human nonolfactory G-protein-coupled receptors (GPCRs) have been aligned at the seven transmembrane domain (TM) and used to extract the nature of 30 critical residues supposed--from the X-ray structure of bovine rhodopsin bound to retinal--to line the TM binding cavity of ground-state receptors. Interestingly, the clustering of human GPCRs from these 30 residues mirrors the recently described phylogenetic tree of full-sequence human GPCRs (Fredriksson et al., Mol Pharmacol 2003;63:1256-1272) with few exceptions. A TM cavity could be found for all investigated GPCRs with physicochemical properties matching that of their cognate ligands. The current approach allows a very fast comparison of most human GPCRs from the focused perspective of the predicted TM cavity and permits to easily detect key residues that drive ligand selectivity or promiscuity.
Read more41Different Amino Acid Substitutions at the Same Position in Rhodopsin Lead to Distinct Phenotypes
Identification of a novel rhodopsin mutation in a family with retinitis pigmentosa and comparison of the clinical phenotype to a known mutation at the same amino acid position. Screening for mutations in rhodopsin was performed in 78 patients with retinitis pigmentosa. All exons and flanking intronic regions were amplified by PCR, sequenced, and compared to the reference sequence derived from the National Center for Biotechnology Information (NCBI, Bethesda, MD) database. Patients were characterized clinically according to the results of best corrected visual acuity testing (BCVA), slit lamp examination (SLE), funduscopy, Goldmann perimetry (GP), dark adaptometry (DA), and electroretinography (ERG). Structural analyses of the rhodopsin protein were performed with the Swiss-Pdb Viewer program available on-line (http://www.expasy.org.spdvbv/ provided in the public domain by Swiss Institute of Bioinformatics, Geneva, Switzerland). A novel rhodopsin mutation (Gly90Val) was identified in a Swiss family of three generations. The pedigree indicated autosomal dominant inheritance. No additional mutation was found in this family in other autosomal dominant genes. The BCVA of affected family members ranged from 20/25 to 20/20. Fundus examination showed fine pigment mottling in patients of the third generation and well-defined bone spicules in patients of the second generation. GP showed concentric constriction. DA demonstrated monophasic cone adaptation only. ERG revealed severely reduced rod and cone signals. The clinical picture is compatible with retinitis pigmentosa. A previously reported amino acid substitution at the same position in rhodopsin leads to a phenotype resembling night blindness in mutation carriers, whereas patients reported in the current study showed the classic retinitis pigmentosa phenotype. The effect of different amino acid substitutions on the three-dimensional structure of rhodopsin was analyzed by homology modeling. Distinct distortions of position 90 (shifts in amino acids 112 and 113) and additional hydrogen bonds were found. Different amino acid substitutions at position 90 of rhodopsin can lead to night blindness or retinitis pigmentosa. The data suggest that the property of the substituted amino acid distinguishes between the phenotypes.
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4Pupil size following dark adaptation in patients with retinitis pigmentosa.
According to the equivalent light hypothesis, molecular defects in the photoreceptor lead to a continuous activation of the photoreceptor cascade in a manner equivalent to real light. The consequences in diseases such as retinitis pigmentosa (RP) are as disruptive to the cells as real light. Two forms of the equivalent light hypothesis can be distinguished: strong - mutations in rhodopsin or other cascade proteins in some forms of RP continuously excite the visual phototransduction cascade; weak - disruption of outer segments in all patients with RP eliminates circulating dark current and blocks neurotransmitter release in a manner similar to real light. Both forms of the equivalent light hypothesis predict that pupils of patients with RP will be constricted like those of normal subjects in the light. The purpose of this study was to test the equivalent light hypothesis by determining whether steady-state pupil diameter following full dark adaptation is abnormally small in any of a sample of patients with RP. Thirty-five patients with RP and 15 normal subjects were tested. Direct steady-state pupillometric measures were obtained from one eye in a full-field dome after 45 min of dark adaptation by videotaping the pupil with an infrared camera. Mean pupil diameter in the dark was comparable (t = -0.15, P = 0.88) between patients with RP (6.85 +/- 0.58 mm) and normal subjects (6.82 +/- 0.76 mm). The results of the present study are clearly counter to the prediction of the second (weaker) form of the equivalent light hypothesis.
Read more430Comprehensive repertoire and phylogenetic analysis of the G protein-coupled receptors in human and mouse
Comprehensive repertoire and phylogenetic analysis of the G protein-coupled receptors in human and mouse
170Novel mutations in the long isoform of the USH2A gene in patients with Usher syndrome type II or non-syndromic retinitis pigmentosa
BackgroundUsher syndrome type II (USH2) is an autosomal recessive disorder characterised by retinitis pigmentosa (RP) and mild to moderate sensorineural hearing loss. Mutations in the USH2A gene are the most...
Read more40Genome wide survey of G protein-coupled receptors in Tetraodon nigroviridis
BackgroundThe G-protein-coupled receptors (GPCRs) constitute one of the largest and most ancient superfamilies of membrane proteins. They play a central role in physiological processes affecting almost all aspects of the life cycle of an organism. Availability of the complete sets of putative members of a family from diverse species provides the basis for cross genome comparative studies.ResultsWe have defined the repertoire of GPCR superfamily of Tetraodon complement with the availability of complete sequence of the freshwater puffer fish Tetraodon nigroviridis. Almost all 466 Tetraodon GPCRs (Tnig-GPCRs) identified had a clear human homologue. 189 putative human and Tetraodon GPCR orthologous pairs could be identified. Tetraodon GPCRs are classified into five GRAFS families, by phylogenetic analysis, concurrent with human GPCR classification.ConclusionDirect comparison of GPCRs in Tetraodon and human genomes displays a high level of orthology and supports large-scale gene duplications in Tetraodon. Examples of lineage specific gene expansions were also observed in opsin and odorant receptors. The human and Tetraodon GPCR sequences are analogous in terms of GPCR subfamilies but display disproportionate numbers of receptors at the subfamily level. The teleost genome with its expanded set of GPCRs provides additional and interesting comparators to study both evolution and function of these receptors.
Read moreHigh 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.
Read more58The G protein-coupled receptor subset of the dog genome is more similar to that in humans than rodents
BackgroundThe dog is an important model organism and it is considered to be closer to humans than rodents regarding metabolism and responses to drugs. The close relationship between humans and dogs over many centuries has lead to the diversity of the canine species, important genetic discoveries and an appreciation of the effects of old age in another species. The superfamily of G protein-coupled receptors (GPCRs) is one of the largest gene families in most mammals and the most exploited in terms of drug discovery. An accurate comparison of the GPCR repertoires in dog and human is valuable for the prediction of functional similarities and differences between the species.ResultsWe searched the dog genome for non-olfactory GPCRs and obtained 353 full-length GPCR gene sequences, 18 incomplete sequences and 13 pseudogenes. We established relationships between human, dog, rat and mouse GPCRs resolving orthologous pairs and species-specific duplicates. We found that 12 dog GPCR genes are missing in humans while 24 human GPCR genes are not part of the dog GPCR repertoire. There is a higher number of orthologous pairs between dog and human that are conserved as compared with either mouse or rat. In almost all cases the differences observed between the dog and human genomes coincide with other variations in the rodent species. Several GPCR gene expansions characteristic for rodents are not found in dog.ConclusionThe repertoire of dog non-olfactory GPCRs is more similar to the repertoire in humans as compared with the one in rodents. The comparison of the dog, human and rodent repertoires revealed several examples of species-specific gene duplications and deletions. This information is useful in the selection of model organisms for pharmacological experiments.
Read more4Identification of RP1 as the genetic cause of retinitis pigmentosa in a multi-generational pedigree using Extremely Low-Coverage Whole Genome Sequencing (XLC-WGS)
Identification of RP1 as the genetic cause of retinitis pigmentosa in a multi-generational pedigree using Extremely Low-Coverage Whole Genome Sequencing (XLC-WGS)
Read more64Nonsyndromic retinitis pigmentosa is highly prevalent in the Jerusalem region with a high frequency of founder mutations
PurposeNonsyndromic retinitis pigmentosa (RP) is the most common inherited retinal degeneration, and prevalence of the disease has been reported in populations of American and European origin with a relatively low consanguinity rate. Our aim was to determine the prevalence of nonsyndromic RP in the Jerusalem region, which has a population of about 1 million individuals with a high rate of consanguinity.MethodsThe patients’ clinical data included eye exam findings (visual acuity, anterior segment, and funduscopy) as well as electroretinographic (ERG) testing results under scotopic and photopic conditions. Mutation analysis on a subgroup of patients was performed mainly with candidate gene analysis and homozygosity mapping.ResultsWe evaluated the medical records of patients with degenerative retinal diseases residing in the Jerusalem region who were examined over the past 20 years in a large tertiary medical center. A total of 453 individuals affected with nonsyndromic RP were diagnosed at our center, according to funduscopic findings and ERG testing. Based on the estimated population size of 945,000 individuals who reside in the vicinity of Jerusalem, the prevalence of nonsyndromic RP in this region is 1:2,086. The prevalence of RP was higher among Arab Muslims (1:1,798) compared to Jews (1:2,230), mainly due to consanguineous marriages that are more common in the Arab Muslim population. To identify the genetic causes of RP in our cohort, we recruited 383 patients from 183 different families for genetic analysis: 70 with autosomal recessive (AR) inheritance, 15 with autosomal dominant, 86 isolate cases, and 12 with an X-linked inheritance pattern. In 64 (35%) of the families, we identified the genetic cause of the disease, and we revised the inheritance pattern of 20 isolate cases to the AR pattern; 49% of the families in our cohort had AR inheritance. Interestingly, in 42 (66%) of the genetically identified families, the cause of disease was a founder mutation.ConclusionsPrevious studies showed an approximate prevalence of 1:5,260 on average for nonsyndromic RP in American and European populations. We show that the prevalence in the vicinity of Jerusalem is two-and-a-half times higher due to a high rate of consanguinity and highly prevalent founder mutations within the historically semi-isolated subpopulations we serve.
Read more15Molecular analysis of the rhodopsin gene in southern France: identification of the first duplication responsible for retinitis pigmentosa, c.998^999ins4
Purpose: Mutations in the gene encoding rhodopsin, the visual pigment in rod photoreceptors, were shown to be the most common cause of autosomal retinitis pigmentosa (RP). In order to determine the prevalence of rhodopsin alterations in southern French populations, we examined 52 unrelated patients/families with autosomal dominant RP (adRP=29), RP simplex (6), or unclassified RP (17). Methods: The full coding and flanking sequences of the rhodopsin (RHO) gene were scanned using an improved DGGE (denaturing gradient gel electrophoresis) assay, followed by sequencing of abnormal fragments. Results: This study revealed three RHO mutations in patients with adRP (G106R, R135W, and c.998^999ins4) and a number of frequent or rare polymorphisms. No disease-causing sequence variation was found in simplex and unclassified RP pedigrees. Mutation c.998^999ins4 has not been previously reported, and appears as the first duplication identified so far in the RHO gene. This frameshift mutation, which is associated with a severe RP, alters the carboxy terminus and predicts a 353-amino acid mutant rhodopsin instead of 348. Discussion: Our study demonstrates that rhodopsin mutations are responsible for only 10.3% of adRP in French populations living in the Mediterranean area in contrast to the 25-35% reported in other populations.
Read more208Evolutionary Trace of G Protein-coupled Receptors Reveals Clusters of Residues That Determine Global and Class-specific Functions
G protein-coupled receptor (GPCR) activation mediated by ligand-induced structural reorganization of its helices is poorly understood. To determine the universal elements of this conformational switch, we used evolutionary tracing (ET) to identify residue positions commonly important in diverse GPCRs. When mapped onto the rhodopsin structure, these trace residues cluster into a network of contacts from the retinal binding site to the G protein-coupling loops. Their roles in a generic transduction mechanism were verified by 211 of 239 published mutations that caused functional defects. When grouped according to the nature of the defects, these residues sub-divided into three striking sub-clusters: a trigger region, where mutations mostly affect ligand binding, a coupling region near the cytoplasmic interface to the G protein, where mutations affect G protein activation, and a linking core in between where mutations cause constitutive activity and other defects. Differential ET analysis of the opsin family revealed an additional set of opsin-specific residues, several of which form part of the retinal binding pocket, and are known to cause functional defects upon mutation. To test the predictive power of ET, we introduced novel mutations in bovine rhodopsin at a globally important position, Leu-79, and at an opsin-specific position, Trp-175. Both were functionally critical, causing constitutive G protein activation of the mutants and rapid loss of regeneration after photobleaching. These results define in GPCRs a canonical signal transduction mechanism where ligand binding induces conformational changes propagated through adjacent trigger, linking core, and coupling regions.
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