The mutational landscape of human olfactory G protein-coupled receptors

Olfactory receptors (ORs) constitute a large family of sensory proteins that enable us to recognize a wide range of chemical volatiles in the environment. By contrast to the extensive information about human olfactory thresholds for thousands of odorants, studies of the genetic influence on olfaction are limited to a few examples. To annotate on a broad scale the impact of mutations at the structural level, here we analyzed a compendium of 119,069 natural variants in human ORs collected from the public domain. OR mutations were categorized depending on their genomic and protein contexts, as well as their frequency of occurrence in several human populations. Functional interpretation of the natural changes was estimated from the increasing knowledge of the structure and function of the G protein-coupled receptor (GPCR) family, to which ORs belong. Our analysis reveals an extraordinary diversity of natural variations in the olfactory gene repertoire between individuals and populations, with a significant number of changes occurring at the structurally conserved regions. A particular attention is paid to mutations in positions linked to the conserved GPCR activation mechanism that could imply phenotypic variation in the olfactory perception. An interactive web application (hORMdb, Human Olfactory Receptor Mutation Database) was developed for the management and visualization of this mutational dataset. We performed topological annotations and population analysis of natural variants of human olfactory receptors and provide an interactive application to explore human OR mutation data. We envisage that the utility of this information will increase as the amount of available pharmacological data for these receptors grow. This effort, together with ongoing research in the study of genetic changes in other sensory receptors could shape an emerging sensegenomics field of knowledge, which should be considered by food and cosmetic consumer product manufacturers for the benefit of the general population. https://doi.org/10.13039/5011000110335https://doi.org/10.13039/5011000110333https://doi.org/10.13039/5011000110336https://doi.org/10.13039/501100011033 https://doi.org/10.13039/501100011033_https://doi.org/10.13039/501100011033_https://doi.org/10.13039/501100011033 https://doi.org/10.13039/501100011033$https://doi.org/10.13039/501100011033ahttps://doi.org/10.13039/501100011033 https://doi.org/10.13039/501100011033Ahttps://doi.org/10.13039/501100011033ghttps://doi.org/10.13039/501100011033ehttps://doi.org/10.13039/501100011033nhttps://doi.org/10.13039/501100011033chttps://doi.org/10.13039/501100011033ihttps://doi.org/10.13039/501100011033ahttps://doi.org/10.13039/501100011033 https://doi.org/10.13039/501100011033Ehttps://doi.org/10.13039/501100011033shttps://doi.org/10.13039/501100011033thttps://doi.org/10.13039/501100011033ahttps://doi.org/10.13039/501100011033thttps://doi.org/10.13039/501100011033ahttps://doi.org/10.13039/501100011033lhttps://doi.org/10.13039/501100011033 https://doi.org/10.13039/501100011033dhttps://doi.org/10.13039/501100011033ehttps://doi.org/10.13039/501100011033 https://doi.org/10.13039/501100011033Ihttps://doi.org/10.13039/501100011033nhttps://doi.org/10.13039/501100011033vhttps://doi.org/10.13039/501100011033ehttps://doi.org/10.13039/501100011033shttps://doi.org/10.13039/501100011033thttps://doi.org/10.13039/501100011033ihttps://doi.org/10.13039/501100011033ghttps://doi.org/10.13039/501100011033ahttps://doi.org/10.13039/501100011033chttps://doi.org/10.13039/501100011033ihttps://doi.org/10.13039/501100011033óhttps://doi.org/10.13039/501100011033nhttps://doi.org/10.13039/501100011033 https://doi.org/10.13039/501100011033$https://doi.org/10.13039/501100011033dhttps://doi.org/10.13039/501100011033 https://doi.org/10.13039/501100011033 https://doi.org/10.13039/501100011033$https://doi.org/10.13039/501100011033fhttps://doi.org/10.13039/501100011033 https://doi.org/10.13039/501100011033Phttps://doi.org/10.13039/501100011033Ihttps://doi.org/10.13039/501100011033Dhttps://doi.org/10.13039/5011000110332https://doi.org/10.13039/5011000110330https://doi.org/10.13039/5011000110331https://doi.org/10.13039/5011000110339https://doi.org/10.13039/501100011033-https://doi.org/10.13039/5011000110331https://doi.org/10.13039/5011000110330https://doi.org/10.13039/5011000110339https://doi.org/10.13039/5011000110332https://doi.org/10.13039/5011000110334https://doi.org/10.13039/5011000110330https://doi.org/10.13039/501100011033Rhttps://doi.org/10.13039/501100011033Bhttps://doi.org/10.13039/501100011033-https://doi.org/10.13039/501100011033Ihttps://doi.org/10.13039/5011000110330https://doi.org/10.13039/5011000110330https://doi.org/10.13039/50110001103

Experimental and computational analysis of biased agonism on full-length and a C-terminally truncated adenosine A receptor

Funding: This work was partially supported by grants from the Spanish Ministry of Economy and Competitiveness (BFU2015-64405-R, SAF2017-84117-R, RTI2018-094204-B-I00 and PID2019- 109240RB-I00; they may include FEDER funds), the Alzheimer's Association (AARFD-17-503612) and by a grant from Fundacio "la Marato" de TV3 (201413-30).Biased agonism, the ability of agonists to differentially activate downstream signaling pathways by stabilizing specific receptor conformations, is a key issue for G protein-coupled receptor (GPCR) signaling. The C-terminal domain might influence this functional selectivity of GPCRs as it engages G proteins, GPCR kinases, β-arrestins, and several other proteins. Thus, the aim of this paper is to compare the agonist-dependent selectivity for intracellular pathways in a heterologous system expressing the full-length (AR) and a C-tail truncated (A Δ40 R lacking the last 40 amino acids) adenosine A receptor, a GPCR that is already targeted in Parkinson's disease using a first-in-class drug. Experimental data such as ligand binding, cAMP production, β-arrestin recruitment, ERK1/2 phosphorylation and dynamic mass redistribution assays, which correspond to different aspects of signal transduction, were measured upon the action of structurally diverse compounds (the agonists adenosine, NECA, CGS-21680, PSB-0777 and LUF-5834 and the SCH-58261 antagonist) in cells expressing AR and A Δ40 R. The results show that taking cAMP levels and the endogenous adenosine agonist as references, the main difference in bias was obtained with PSB-0777 and LUF-5834. The C-terminus is dispensable for both G-protein and β-arrestin recruitment and also for MAPK activation. Unrestrained molecular dynamics simulations, at the μs timescale, were used to understand the structural arrangements of the binding cavity, triggered by these chemically different agonists, facilitating G protein binding with different efficacy

A Single Point Mutation Blocks the Entrance of Ligands to the Cannabinoid CB Receptor via the Lipid Bilayer

Molecular dynamic (MD) simulations have become a common tool to study the pathway of ligand entry to the orthosteric binding site of G protein-coupled receptors. Here, we have combined MD simulations and site-directed mutagenesis to study the binding process of the potent JWH-133 agonist to the cannabinoid CB receptor (CBR). In CBR, the N-terminus and extracellular loop 2 fold over the ligand binding pocket, blocking access to the binding cavity from the extracellular environment. We, thus, hypothesized that the binding pathway is a multistage process consisting of the hydrophobic ligand diffusing in the lipid bilayer to contact a lipid-facing vestibule, from which the ligand enters an allosteric site inside the transmembrane bundle through a tunnel formed between TMs 1 and 7 and finally moving from the allosteric to the orthosteric binding cavity. This pathway was experimentally validated by the Ala282 7.36 Phe mutation that blocks the entrance of the ligand, as JWH-133 was not able to decrease the forskolin-induced cAMP levels in cells expressing the mutant receptor. This proposed ligand entry pathway defines transient binding sites that are potential cavities for the design of synthetic modulators

Preferential Gs protein coupling of the galanin Gal1 receptor in the μ-opioid-Gal1 receptor heterotetramer

Recent studies have proposed that heteromers of μ-opioid receptors (MORs) and galanin Gal1 receptors (Gal1Rs) localized in the mesencephalon mediate the dopaminergic effects of opioids. The present study reports converging evidence, using a peptide-interfering approach combined with biophysical and biochemical techniques, including total internal reflection fluorescence microscopy, for a predominant homodimeric structure of MOR and Gal1R when expressed individually, and for their preference to form functional heterotetramers when co-expressed. Results show that a heteromerization-dependent change in the Gal1R homodimeric interface leads to a switch in G-protein coupling from inhibitory Gi to stimulatory Gs proteins. The MOR-Gal1R heterotetramer, which is thus bound to Gs via the Gal1R homodimer and Gi via the MOR homodimer, provides the framework for a canonical Gs-Gi antagonist interaction at the adenylyl cyclase level. These novel results shed light on the intense debate about the oligomeric quaternary structure of G protein-coupled receptors, their predilection for heteromer formation, and the resulting functional significance

Heterobivalent Ligand for the Adenosine A2A-Dopamine D2 Receptor Heteromer

A G protein-coupled receptor heteromer that fulfills the established criteria for its existence in vivo is the complex between adenosine A2A (A2AR) and dopamine D2 (D2R) receptors. Here, we have designed and synthesized heterobivalent ligands for the A2AR-D2R heteromer with various spacer lengths. The indispensable simultaneous binding of these ligands to the two different orthosteric sites of the heteromer has been evaluated by radioligand competition-binding assays in the absence and presence of specific peptides that disrupt the formation of the heteromer, label-free dynamic mass redistribution assays in living cells, and molecular dynamic simulations. This combination of techniques has permitted us to identify compound 26 [KDB1 (A2AR) = 2.1 nM, KDB1 (D2R) = 0.13 nM], with a spacer length of 43-atoms, as a true bivalent ligand that simultaneously binds to the two different orthosteric sites. Moreover, bioluminescence resonance energy transfer experiments indicate that 26 favors the stabilization of the A2AR-D2R heteromer.This work is supported by the Spanish Ministerio de Ciencia e Innovación (RTI2018-093831-B-I00 to MR, SAF2017-87629-R to VC and PID2019-109240RB-I00 to LP; they might include FEDER founds), CIBER BBN (CB06-01-0074); Generalitat de Catalunya (2017SGR1439 and 2017SGR1497); and intramural funds of the National Institute on Drug Abuse. MG-A acknowledges the Universitat Autònoma de Barcelona for his pre-doctoral grant.Peer reviewe

Unique effect of clozapine on adenosine A2A-dopamine D2 receptor heteromerization

The striatal dopamine D2 receptor (D2R) is generally accepted to be involved in positive symptoms of schizophrenia and is a main target for clinically used antipsychotics. D2R are highly expressed in the striatum, where they form heteromers with the adenosine A2A receptor (A2AR). Changes in the density of A2AR-D2R heteromers have been reported in postmortem tissue from patients with schizophrenia, but the degree to which A2R are involved in schizophrenia and the effect of antipsychotic drugs is unknown. Here, we examine the effect of exposure to three prototypical antipsychotic drugs on A2AR-D2R heteromerization in mammalian cells using a NanoBiT assay. After 16 h of exposure, a significant increase in the density of A2AR-D2R heteromers was found with haloperidol and aripiprazole, but not with clozapine. On the other hand, clozapine, but not haloperidol or aripiprazole, was associated with a significant decrease in A2AR-D2R heteromerization after 2 h of treatment. Computational binding models of these compounds revealed distinctive molecular signatures that explain their different influence on heteromerization. The bulky tricyclic moiety of clozapine displaces TM 5 of D2R, inducing a clash with A2AR, while the extended binding mode of haloperidol and aripiprazole stabilizes a specific conformation of the second extracellular loop of D2R that enhances the interaction with A2AR. It is proposed that an increase in A2AR-D2R heteromerization is involved in the extrapyramidal side effects (EPS) of antipsychotics and that the specific clozapine-mediated destabilization of A2AR-D2R heteromerization can explain its low EPS liability

A Single Point Mutation Blocks the Entrance of Ligands to the Cannabinoid CB2 Receptor via the Lipid Bilayer

Molecular dynamic (MD) simulations have become a common tool to study the pathway of ligand entry to the orthosteric binding site of G protein-coupled receptors. Here, we have combined MD simulations and site-directed mutagenesis to study the binding process of the potent JWH-133 agonist to the cannabinoid CB2 receptor (CB2R). In CB2R, the N-terminus and extracellular loop 2 fold over the ligand binding pocket, blocking access to the binding cavity from the extracellular environment. We, thus, hypothesized that the binding pathway is a multistage process consisting of the hydrophobic ligand diffusing in the lipid bilayer to contact a lipid-facing vestibule, from which the ligand enters an allosteric site inside the transmembrane bundle through a tunnel formed between TMs 1 and 7 and finally moving from the allosteric to the orthosteric binding cavity. This pathway was experimentally validated by the Ala2827.36Phe mutation that blocks the entrance of the ligand, as JWH-133 was not able to decrease the forskolin-induced cAMP levels in cells expressing the mutant receptor. This proposed ligand entry pathway defines transient binding sites that are potential cavities for the design of synthetic modulators

Design of negative and positive allosteric modulators of the cannabinoid CB2 Receptor derived from the natural product cannabidiol

Cannabidiol (CBD), the second most abundant of the active compounds found in the Cannabis sativa plant, is of increasing interest because it is approved for human use and is neither euphorizing nor addictive. Here, we design and synthesize novel compounds taking into account that CBD is both a partial agonist, when it binds to the orthosteric site, and a negative allosteric modulator, when it binds to the allosteric site of the cannabinoid CB2 receptor. Molecular dynamic simulations and site-directed mutagenesis studies have identified the allosteric site near the receptor entrance. This knowledge has permitted to perform structure-guided design of negative and positive allosteric modulators of the CB2 receptor with potential therapeutic utility

Unique pharmacodynamic properties and low abuse liability of the μ-opioid receptor ligand (S)-methadone

(R,S)-methadone ((R,S)-MTD) is a racemic μ-opioid receptor (MOR) agonist comprised of (R)-MTD and (S)-MTD enantiomers used for the treatment of opioid use disorder (OUD) and pain. (R)-MTD is used as an OUD treatment, has high MOR potency, and is believed to mediate (R,S)-MTD's therapeutic efficacy. (S)-MTD is in clinical development as an antidepressant and is considered an N-methyl-D-aspartate receptor (NMDAR) antagonist. In opposition to this purported mechanism of action, we found that (S)-MTD does not occupy NMDARs in vivo in rats. Instead, (S)-MTD produced MOR occupancy and induced analgesia with similar efficacy as (R)-MTD. Unlike (R)-MTD, (S)-MTD was not self-administered and failed to increase locomotion or extracellular dopamine levels indicating low abuse liability. Moreover, (S)-MTD antagonized the effects of (R)-MTD in vivo and exhibited unique pharmacodynamic properties, distinct from those of (R)-MTD. Specifically, (S)-MTD acted as a MOR partial agonist with a specific loss of efficacy at the MOR-galanin 1 receptor (Gal1R) heteromer, a key mediator of the dopaminergic effects of opioids. In sum, we report novel and unique pharmacodynamic properties of (S)-MTD that are relevant to its potential mechanism of action and therapeutic use, as well as those of (R,S)-MTD

Pharmacological targeting of G protein-coupled receptor heteromers.

A main rationale for the role of G protein-coupled receptor (GPCR) heteromers as targets for drug development is the putative ability of selective ligands for specific GPCRs to change their pharmacological properties upon GPCR heteromerization. The present study provides a proof of concept for this rationale by demonstrating that heteromerization of dopamine D1 and D3 receptors (D1R and D3R) influences the pharmacological properties of three structurally similar selective dopamine D3R ligands, the phenylpiperazine derivatives PG01042, PG01037 and VK4-116. By using D1R-D3R heteromer-disrupting peptides, it could be demonstrated that the three D3R ligands display different D1R-D3R heteromer-dependent pharmacological properties: PG01042, acting as G protein-biased agonist, counteracted D1R-mediated signaling in the D1R-D3R heteromer; PG01037, acting as a D3R antagonist cross-antagonized D1R-mediated signaling in the D1R-D3R heteromer; and VK4-116 specifically acted as a ß-arrestin-biased agonist in the D1R-D3R heteromer. Molecular dynamics simulations predicted potential molecular mechanisms mediating these qualitatively different pharmacological properties of the selective D3R ligands that are dependent on D1R-D3R heteromerization. The results of in vitro experiments were paralleled by qualitatively different pharmacological properties of the D3R ligands in vivo. The results supported the involvement of D1R-D3R heteromers in the locomotor activation by D1R agonists in reserpinized mice and L-DOPA-induced dyskinesia in rats, highlighting the D1R-D3R heteromer as a main pharmacological target for L-DOPA-induced dyskinesia in Parkinsons disease. More generally, the present study implies that when suspecting its pathogenetic role, a GPCR heteromer, and not its individual GPCR units, should be considered as main target for drug development