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BACKGROUND: CDH1 and CTNNA1 remain as the main genes for hereditary gastric cancer. However, they only explain a small fraction of gastric cancer cases with suspected inherited basis. In this study, we aimed to identify new hereditary genes for early-onset gastric cancer patients (EOGC; < 50 years old). METHODS: After germline exome sequencing in 20 EOGC patients and replication of relevant findings by gene-panel sequencing in an independent cohort of 152 patients, CTNND1 stood out as an interesting candidate gene, since its protein product (p120ctn) directly interacts with E-cadherin. We proceeded with functional characterization by generating two knockout CTNND1 cellular models by gene editing and introducing the detected genetic variants using a lentiviral delivery system. We assessed ß-catenin and E-cadherin levels, cell detachment, as well as E-cadherin localization and cell-to-cell interaction by spheroid modeling. RESULTS: Three CTNND1 germline variants [c.28_29delinsCT, p.(Ala10Leu); c.1105C > T, p.(Pro369Ser); c.1537A > G, p.(Asn513Asp)] were identified in our EOGC cohorts. Cells encoding CTNND1 variants displayed altered E-cadherin levels and intercellular interactions. In addition, the p.(Pro369Ser) variant, located in a key region in the E-cadherin/p120ctn binding domain, showed E-cadherin mislocalization. CONCLUSIONS: Defects in CTNND1 could be involved in germline predisposition to gastric cancer by altering E-cadherin and, consequently, cell-to-cell interactions. In the present study, CTNND1 germline variants explained 2% (3/172) of the cases, although further studies in larger external cohorts are needed.
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Cadherinas , Cateninas , Catenina delta , Predisposición Genética a la Enfermedad , Mutación de Línea Germinal , Neoplasias Gástricas , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Humanos , Masculino , Cateninas/genética , Cateninas/metabolismo , Femenino , Persona de Mediana Edad , Adulto , Cadherinas/genética , Comunicación Celular , Edad de Inicio , Antígenos CDRESUMEN
Catecholamine-triggered ß-adrenoceptor (ß-AR) signaling is essential for the correct functioning of the heart. Although both ß1 - and ß2 -AR subtypes are expressed in cardiomyocytes, drugs selectively targeting ß1 -AR have proven this receptor as the main target for the therapeutic effects of beta blockers in the heart. Here, we report a new strategy for the light-control of ß1 -AR activation by means of photoswitchable drugs with a high level of ß1 -/ß2 -AR selectivity. All reported molecules allow for an efficient real-time optical control of receptor function in vitro. Moreover, using confocal microscopy we demonstrate that the binding of our best hit, pAzo-2, can be reversibly photocontrolled. Strikingly, pAzo-2 also enables a dynamic cardiac rhythm management on living zebrafish larvae using light, thus highlighting the therapeutic and research potential of the developed photoswitches. Overall, this work provides the first proof of precise control of the therapeutic target ß1 -AR in native environments using light.
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Receptores Adrenérgicos beta 2 , Pez Cebra , Antagonistas Adrenérgicos beta/farmacología , Animales , Ligandos , Miocitos Cardíacos/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Pez Cebra/metabolismoRESUMEN
The olfacto-genital syndrome (Kallmann syndrome) associates congenital hypogonadism due to gonadotropin-releasing hormone (GnRH) deficiency and anosmia. This is a genetically heterogeneous developmental disease with various modes of transmission, including oligogenic inheritance. Previous reports have involved defective cell signaling by semaphorin-3A in the disease pathogenesis. Here, we report that the embryonic phenotype of Plxna1-/- mutant mice lacking plexin-A1 (a major receptor of class 3 semaphorins), though not fully penetrant, resembles that of Kallmann syndrome fetuses. Pathohistological analysis indeed showed a strongly abnormal development of the peripheral olfactory system and defective embryonic migration of the neuroendocrine GnRH cells to the hypothalamic brain region in some of the mutant mice, which resulted in reduced fertility in adult males. We thus screened 250 patients for the presence of mutations in PLXNA1, and identified different nonsynonymous mutations (p.V349L, p.V437L, p.R528W, p.H684Y, p.G720E, p.R740H, p.R813H, p.R840Q, p.A854T, p.R897H, p.L1464V, p.K1618T, p.C1744F), all at heterozygous state, in 15 patients. Most of these mutations are predicted to affect plexin-A1 stability or signaling activity based on predictive algorithms and a structural model of the protein. Moreover, in vitro experiments allowed us to show the existence of deleterious effects of eight mutations (including a transcript splicing defect), none of which are expected to result in a complete loss of protein synthesis, targeting, or signaling activity, though. Our findings indicate that signaling insufficiency through plexin-A1 can contribute to the pathogenesis of Kallmann syndrome, and further substantiate the oligogenic pattern of inheritance in this developmental disorder.
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Síndrome de Kallmann/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Adulto , Animales , Movimiento Celular , Femenino , Hormona Liberadora de Gonadotropina/deficiencia , Heterocigoto , Humanos , Hipogonadismo/genética , Hipotálamo/metabolismo , Masculino , Ratones , Mutación , Proteínas del Tejido Nervioso/fisiología , Células Neuroendocrinas/metabolismo , Neuronas/metabolismo , Bulbo Olfatorio/fisiología , Receptores de Superficie Celular/fisiología , Reproducción , Semaforina-3A/genética , Semaforina-3A/metabolismo , Semaforinas/metabolismo , Transducción de SeñalRESUMEN
G protein-coupled receptors (GPCRs) are major players in cell communication. Although they form functional monomers, increasing evidence indicates that GPCR dimerization has a critical role in cooperative phenomena that are important for cell signal integration. However, the structural bases of these phenomena remain elusive. Here, using well-characterized receptor dimers, the metabotropic glutamate receptors (mGluRs), we show that structural changes at the dimer interface are linked to receptor activation. We demonstrate that the main dimer interface is formed by transmembrane α helix 4 (TM4) and TM5 in the inactive state and by TM6 in the active state. This major change in the dimer interface is required for receptor activity because locking the TM4-TM5 interface prevents activation by agonist, whereas locking the TM6 interface leads to a constitutively active receptor. These data provide important information on the activation mechanism of mGluRs and improve our understanding of the structural basis of the negative cooperativity observed in these GPCR dimers.
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Multimerización de Proteína , Receptores de Glutamato Metabotrópico/química , Alanina/genética , Animales , Células COS , Chlorocebus aethiops , Cisteína/genética , Células HEK293 , Humanos , Ligandos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Unión Proteica , Estructura Terciaria de Proteína , Subunidades de Proteína , Ratas , Receptor del Glutamato Metabotropico 5/agonistas , Receptor del Glutamato Metabotropico 5/química , Receptor del Glutamato Metabotropico 5/genética , Receptores de GABA-B/química , Receptores de GABA-B/genética , Receptores de GABA-B/metabolismo , Receptores de Glutamato Metabotrópico/agonistas , Receptores de Glutamato Metabotrópico/genética , Activación Transcripcional , TransfecciónRESUMEN
Controlling drug activity with light offers the possibility of enhancing pharmacological selectivity with spatial and temporal regulation, thus enabling highly localized therapeutic effects and precise dosing patterns. Here we report on the development and characterization of what is to our knowledge the first photoswitchable allosteric modulator of a G protein-coupled receptor. Alloswitch-1 is selective for the metabotropic glutamate receptor mGlu5 and enables the optical control of endogenous mGlu5 receptors.
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Compuestos Azo/química , Piridinas/farmacología , Receptor del Glutamato Metabotropico 5/metabolismo , Regulación Alostérica/efectos de la radiación , Sitio Alostérico , Animales , Animales Recién Nacidos , Astrocitos/citología , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Astrocitos/efectos de la radiación , Expresión Génica , Células HEK293 , Humanos , Larva/efectos de los fármacos , Larva/fisiología , Larva/efectos de la radiación , Luz , Procesos Fotoquímicos , Cultivo Primario de Células , Piridinas/síntesis química , Ratas , Receptor del Glutamato Metabotropico 5/agonistas , Receptor del Glutamato Metabotropico 5/antagonistas & inhibidores , Receptor del Glutamato Metabotropico 5/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Transfección , Xenopus/fisiologíaRESUMEN
Metabotropic glutamate receptors (mGluRs) play key roles in the modulation of many synapses. Chloride (Cl(-)) is known to directly bind and regulate the function of different actors of neuronal activity, and several studies have pointed to the possible modulation of mGluRs by Cl(-). Herein, we demonstrate that Cl(-) behaves as a positive allosteric modulator of mGluRs. For example, whereas glutamate potency was 3.08 ± 0.33 µM on metabotropic glutamate (mGlu) 4 receptors in high-Cl(-) buffer, signaling activity was almost abolished in low Cl(-) in cell-based assays. Cl(-) potency was 78.6 ± 3.5 mM. Cl(-) possesses a high positive cooperativity with glutamate (Hill slope ≈6 on mGlu4), meaning that small variations in [Cl(-)] lead to large variations in glutamate action. Using molecular modeling and mutagenesis, we have identified 2 well-conserved Cl(-) binding pockets in the extracellular domain of mGluRs. Moreover, modeling of activity-dependent Cl(-) variations at GABAergic synapses suggests that these variations may be compatible with a dynamic modulation of the most sensitive mGluRs present in these synapses. Taken together, these data reveal a necessary role of Cl(-) for the glutamate activation of many mGluRs. Exploiting Cl(-) binding pockets may yield to the development of innovative regulators of mGluR activity.
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Cloruros/metabolismo , Espacio Extracelular/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Regulación Alostérica , Sitio Alostérico , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Cloruros/farmacología , Células HEK293 , Humanos , Modelos Moleculares , Mutación , Unión Proteica , Estructura Terciaria de Proteína/efectos de los fármacos , Ratas , Receptores de Glutamato Metabotrópico/química , Receptores de Glutamato Metabotrópico/genética , Homología de Secuencia de AminoácidoRESUMEN
Type 4 metabotropic glutamate (mGlu4) receptors are emerging targets for the treatment of various disorders. Accordingly, numerous mGlu4-positive allosteric modulators (PAMs) have been identified, some of which also display agonist activity. To identify the structural bases for their allosteric action, we explored the relationship between the binding pockets of mGlu4 PAMs with different chemical scaffolds and their functional properties. By use of innovative mGlu4 biosensors and second-messenger assays, we show that all PAMs enhance agonist action on the receptor through different degrees of allosteric agonism and positive cooperativity. For example, whereas VU0155041 and VU0415374 display equivalent efficacies [log(τ(B)) = 1.15 ± 0.38 and 1.25 ± 0.44, respectively], they increase the ability of L-AP4 to stabilize the active conformation of the receptor by 4 and 39 times, respectively. Modeling and docking studies identify 2 overlapping binding pockets as follows: a first site homologous to the pocket of natural agonists of class A GPCRs linked to allosteric agonism and a second one pointing toward a site topographically homologous to the Na(+) binding pocket of class A GPCRs, occupied by PAMs exhibiting the strongest cooperativity. These results reveal that intrinsic efficacy and cooperativity of mGlu4 PAMs are correlated with their binding mode, and vice versa, integrating structural and functional knowledge from different GPCR classes.
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Receptores de Glutamato Metabotrópico/agonistas , Receptores de Glutamato Metabotrópico/metabolismo , Regulación Alostérica , Sitio Alostérico , Anilidas/farmacología , Técnicas Biosensibles , Ácidos Ciclohexanocarboxílicos/farmacología , Transferencia Resonante de Energía de Fluorescencia , Células HEK293 , Humanos , Ligandos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Conformación Proteica , Receptores de Glutamato Metabotrópico/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sistemas de Mensajero SecundarioRESUMEN
Metabotropic glutamate receptors (mGlu) regulate multiple functions in the nervous systems and are involved in several neurological disorders. However, selectively targeting individual mGlu subtypes with spatiotemporal precision is still an unmet need. Photopharmacology can address this concern through the utilization of photoswitchable compounds such as optogluram, which is a positive allosteric modulator (PAM) of mGlu4 that enables the precise control of physiological responses using light but does not have an optimal selectivity profile. Optogluram analogs were developed to obtain photoswitchable PAMs of mGlu4 receptor with an improved selectivity. Among them, optogluram-2 emerged as a photoswitchable ligand for mGlu4 receptor with activity as both PAM and allosteric agonists. It presents a higher selectivity and offers improved photoswitching of mGlu4 activity. These improved properties make optogluram-2 an excellent candidate to study the role of mGlu4 with a high spatiotemporal precision in systems where mGlu4 can be co-expressed with other mGlu receptors.
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Knowing the site of drug action is important to optimize effectiveness and address any side effects. We used light-sensitive drugs to identify the brain region-specific role of mGlu5 metabotropic glutamate receptors in the control of pain. Optical activation of systemic JF-NP-26, a caged, normally inactive, negative allosteric modulator (NAM) of mGlu5 receptors, in cingulate, prelimbic and infralimbic cortices and thalamus inhibited neuropathic pain hypersensitivity. Systemic treatment of alloswitch-1, an intrinsically active mGlu5 receptor NAM, caused analgesia, and the effect was reversed by light-induced drug inactivation in in the prelimbic and infralimbic cortices, and thalamus. This demonstrates that mGlu5 receptor blockade in the medial prefrontal cortex and thalamus is both sufficient and necessary for the analgesic activity of mGlu5 receptor antagonists. Surprisingly, when light was delivered in the basolateral amygdala, local activation of systemic JF-NP-26 reduced pain thresholds, whereas inactivation of alloswitch-1 enhanced analgesia. Electrophysiological analysis showed that alloswitch-1 increased excitatory synaptic responses in prelimbic pyramidal neurons evoked by stimulation of BLA input, and decreased feedforward inhibition of amygdala output neurons by BLA. Both effects were reversed by optical silencing and reinstated by optical reactivation of alloswitch-1. These findings demonstrate for the first time that the action of mGlu5 receptors in the pain neuraxis is not homogenous, and suggest that blockade of mGlu5 receptors in the BLA may limit the overall analgesic activity of mGlu5 receptor antagonists. This could explain the suboptimal effect of mGlu5 NAMs on pain in human studies and validate photopharmacology as an important tool to determine ideal target sites for systemic drugs.
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Allosteric modulators bear great potential to fine-tune neurotransmitter action. Promising targets are metabotropic glutamate (mGlu) receptors, which are associated with numerous brain diseases. Orthosteric and allosteric ligands act in synergy to control the activity of these multidomain dimeric GPCRs. Here, we analyzed the effect of such molecules on the concerted conformational changes of full-length mGlu2 at the single-molecule level. We first established FRET sensors through genetic code expansion combined with click chemistry to monitor conformational changes on live cells. We then used single-molecule FRET and show that orthosteric agonist binding leads to the stabilization of most of the glutamate binding domains in their closed state, while the reorientation of the dimer into the active state remains partial. Allosteric modulators, interacting with the transmembrane domain, are required to stabilize the fully reoriented active dimer. These results illustrate how concerted conformational changes within multidomain proteins control their activity, and how these are modulated by allosteric ligands.
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Receptores de Glutamato Metabotrópico , Regulación Alostérica , Ligandos , Receptores de Glutamato Metabotrópico/genética , Receptores de Glutamato Metabotrópico/agonistas , Receptores de Glutamato Metabotrópico/metabolismo , GlutamatosRESUMEN
BACKGROUND: The original SARS-CoV-2 vaccination regimen (2 doses) induces insufficient short-term response in kidney transplant (KT) recipients. This study assessed the response to a third dose and the long-term immunogenicity after 2 doses in KT. METHODS: We analyzed the dynamics of the humoral and cellular response by monitoring SARS-CoV-2 IgG antibodies against the Spike-protein (IgG-Spike) and QuantiFERON SARS-CoV-2 IFN-γ release assay 6 mo after the second dose (T2) and 28 d after the third dose of mRNA vaccines (T3) to KT and controls (dialysis patients and healthy individuals). RESULTS: At T2, the percentage of IgG-Spike+ KT and dialysis patients decreased (KT 65.8%-52.6%, hemodialysis 92.6-81.5%, and peritoneal dialysis 100%-90%), whereas 100% of healthy controls remained positive. About the cellular response, the percentage of responders decreased in all groups, especially in KT (22.4%-9.2%, P = 0.081). At T3, 92% of KT, 94%-98% of dialysis patients, and 100% of healthy controls were IgG-Spike+. In terms of antibody titers, patients and controls showed a reduction between T2 and T3 and about 80% of dialysis patients and 100% of controls achieved high titers after the third dose (>1479.5 Binding Antibody Units/mL), whereas this percentage was only 50% in KT. With respect to the cellular response, only KT displayed a significant rise after the third dose. CONCLUSIONS: The third dose of mRNA vaccine improves both humoral and cellular responses, but less effectively in KT compared with dialysis patients and healthy controls.
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COVID-19 , Trasplante de Riñón , Humanos , Vacunas contra la COVID-19 , SARS-CoV-2 , Trasplante de Riñón/efectos adversos , COVID-19/diagnóstico , COVID-19/prevención & control , Diálisis Renal , Vacunas de ARNm , Anticuerpos Antivirales , Inmunoglobulina G , Receptores de Trasplantes , VacunaciónRESUMEN
Metabotropic glutamate receptors (mGlu) are a family of class C G protein-coupled receptors (GPCRs) with important biological functions and widespread expression. The mechanisms of mGlu activation and the development of allosteric modulators for these dimeric proteins have attracted singular attention including the use of light regulated ligands. Photopharmacology involves the integration of a photoactive moiety into the ligand structure that following specific illumination undergoes a structural rearrangement and changes its biological activity. The use of light-regulated allosteric ligands offers the opportunity to manipulate mGlu signalling with spatiotemporal precision, unattainable with classical pharmacological approaches. In this review, we will discuss some of the innovations that have been made in the allosteric photopharmacology of mGlu receptors to date. We discuss the prospects of these molecular tools in the control of mGluRs and the new perspectives in understanding mGlu mechanisms, pharmacology and (patho)physiology that can ultimately result in innovative drug discovery concepts.
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Regulación Alostérica , Receptores de Glutamato Metabotrópico , Descubrimiento de Drogas , Humanos , Ligandos , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Glutamato Metabotrópico/metabolismoRESUMEN
Adrenoceptors are G protein-coupled receptors involved in a large variety of physiological processes, also under pathological conditions. This is due in large part to their ubiquitous expression in the body exerting numerous essential functions. Therefore, the possibility to control their activity with high spatial and temporal precision would constitute a valuable research tool. In this study, we present a caged version of the approved non-selective ß-adrenoceptor antagonist carvedilol, synthesized by alkylation of its secondary amine with a coumarin derivative. Introducing this photo-removable group abolished carvedilol physiological effects in cell cultures, mouse isolated perfused hearts and living zebrafish larvae. Only after visible light application, carvedilol was released and the different physiological systems were pharmacologically modulated in a similar manner as the control drug. This research provides a new photopharmacological tool for a wide range of research applications that may help in the development of future precise therapies.
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The insecticide carbaryl is commonly found in indirectly exposed freshwater ecosystems at low concentrations considered safe for fish communities. In this study, we showed that after only 24 h of exposure to environmental concentrations of carbaryl (0.066-660 ng/L), zebrafish larvae exhibit impairments in essential behaviours. Interestingly, the observed behavioural effects induced by carbaryl were acetylcholinesterase-independent. To elucidate the molecular initiating event that resulted in the observed behavioural effects, in silico predictions were followed by in vitro validation. We identified two target proteins that potentially interacted with carbaryl, the α2B adrenoceptor (ADRA2B) and the serotonin 2B receptor (HTR2B). Using a pharmacological approach, we then tested the hypothesis that carbaryl had antagonistic interactions with both receptors. Similar to yohimbine and SB204741, which are prototypic antagonists of ADRA2B and HTR2B, respectively, carbaryl increased the heart rate of zebrafish larvae. When we compared the behavioural effects of a 24-h exposure to these pharmacological antagonists with those of carbaryl, a high degree of similarity was found. These results strongly suggest that antagonism of both ADRA2B and HTR2B is the molecular initiating event that leads to adverse outcomes in zebrafish larvae that have undergone 24 h of exposure to environmentally relevant levels of carbaryl.
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Carbaril , Pez Cebra , Acetilcolinesterasa , Animales , Carbaril/toxicidad , Ecosistema , LarvaRESUMEN
G protein-coupled receptors (GPCRs) are among the most promising drug targets. They often form homo- and heterodimers with allosteric cross-talk between receptor entities, which contributes to fine-tuning of transmembrane signaling. Specifically controlling the activity of GPCR dimers with ligands is a good approach to clarify their physiological roles and validate them as drug targets. Here, we examined the mode of action of positive allosteric modulators (PAMs) that bind at the interface of the transmembrane domains of the heterodimeric GABAB receptor. Our site-directed mutagenesis results show that mutations of this interface impact the function of the three PAMs tested. The data support the inference that they act at the active interface between both transmembrane domains, the binding site involving residues of the TM6s of the GABAB1 and the GABAB2 subunit. Importantly, the agonist activity of these PAMs involves a key region in the central core of the GABAB2 transmembrane domain, which also controls the constitutive activity of the GABAB receptor. This region corresponds to the sodium ion binding site in class A GPCRs that controls the basal state of the receptors. Overall, these data reveal the possibility of developing allosteric compounds able to specifically modulate the activity of GPCR homo- and heterodimers by acting at their transmembrane interface.
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Receptores de GABA-B/genética , Regulación Alostérica , Células HEK293 , Humanos , Ligandos , Dominios Proteicos , Receptores de GABA-B/metabolismoRESUMEN
ß2-Adrenoceptors (ß2-AR) are prototypical G-protein-coupled receptors and important pharmacological targets with relevant roles in physiological processes and diseases. Herein, we introduce Photoazolol-1-3, a series of photoswitchable azobenzene ß2-AR antagonists that can be reversibly controlled with light. These new photochromic ligands are designed following the azologization strategy, with a p-acetamido azobenzene substituting the hydrophobic moiety present in many ß2-AR antagonists. Using a fluorescence resonance energy transfer (FRET) biosensor-based assay, a variety of photopharmacological properties are identified. Two of the light-regulated molecules show potent ß2-AR antagonism and enable a reversible and dynamic control of cellular receptor activity with light. Their photopharmacological properties are opposite, with Photoazolol-1 being more active in the dark and Photoazolol-2 demonstrating higher antagonism upon illumination. In addition, we provide a molecular rationale for the interaction of the different photoisomers with the receptor. Overall, we present innovative tools and a proof of concept for the precise control of ß2-AR by means of light.
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Antagonistas de Receptores Adrenérgicos beta 2/farmacología , Compuestos Azo/farmacología , Receptores Adrenérgicos beta 2/metabolismo , Antagonistas de Receptores Adrenérgicos beta 2/química , Compuestos Azo/química , Descubrimiento de Drogas , Transferencia Resonante de Energía de Fluorescencia , Células HEK293 , Humanos , Ligandos , Luz , Modelos MolecularesRESUMEN
G protein-coupled receptors (GPCR), including the metabotrobic glutamate 5 receptor (mGlu5), are important therapeutic targets and the development of allosteric ligands for targeting GPCRs has become a desirable approach toward modulating receptor activity. Traditional pharmacological approaches toward modulating GPCR activity are still limited since precise spatiotemporal control of a ligand is lost as soon as it is administered. Photopharmacology proposes the use of photoswitchable ligands to overcome this limitation, since their activity can be reversibly controlled by light with high precision. As this is still a growing field, our understanding of the molecular mechanisms underlying the light-induced changes of different photoswitchable ligand pharmacology is suboptimal. For this reason, we have studied the mechanisms of action of alloswitch-1 and MCS0331; two freely diffusible, mGlu5 phenylazopyridine photoswitchable negative allosteric modulators. We combined photochemical, cell-based, and in vivo photopharmacological approaches to investigate the effects of trans-cis azobenzene photoisomerization on the functional activity and binding ability of these ligands to the mGlu5 allosteric pocket. From these results, we conclude that photoisomerization can take place inside and outside the ligand binding pocket, and this leads to a reversible loss in affinity, in part, due to changes in dissociation rates from the receptor. Ligand activity for both photoswitchable ligands deviates from high-affinity mGlu5 negative allosteric modulation (in the trans configuration) to reduced affinity for the mGlu5 in their cis configuration. Importantly, this mechanism translates to dynamic and reversible control over pain following local injection and illumination of negative allosteric modulators into a brain region implicated in pain control.
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Glycine receptors (GlyRs) are indispensable for maintaining excitatory/inhibitory balance in neuronal circuits that control reflexes and rhythmic motor behaviors. Here we have developed Glyght, a GlyR ligand controlled with light. It is selective over other Cys-loop receptors, is active in vivo, and displays an allosteric mechanism of action. The photomanipulation of glycinergic neurotransmission opens new avenues to understanding inhibitory circuits in intact animals and to developing drug-based phototherapies.
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Compuestos Azo/farmacología , Receptores de Glicina/antagonistas & inhibidores , Animales , Compuestos Azo/síntesis química , Compuestos Azo/química , Células Cultivadas , Cricetulus , Femenino , Ligandos , Masculino , Ratones , Ratones Endogámicos ICR , Simulación del Acoplamiento Molecular , Estructura Molecular , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Procesos Fotoquímicos , Receptores de Glicina/metabolismo , Transmisión Sináptica/efectos de los fármacosRESUMEN
G protein-coupled receptors (GPCRs) can integrate extracellular signals via allosteric interactions within dimers and higher-order oligomers. However, the structural bases of these interactions remain unclear. Here, we use the GABAB receptor heterodimer as a model as it forms large complexes in the brain. It is subjected to genetic mutations mainly affecting transmembrane 6 (TM6) and involved in human diseases. By cross-linking, we identify the transmembrane interfaces involved in GABAB1-GABAB2, as well as GABAB1-GABAB1 interactions. Our data are consistent with an oligomer made of a row of GABAB1. We bring evidence that agonist activation induces a concerted rearrangement of the various interfaces. While the GB1-GB2 interface is proposed to involve TM5 in the inactive state, cross-linking of TM6s lead to constitutive activity. These data bring insight for our understanding of the allosteric interaction between GPCRs within oligomers.
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Modelos Moleculares , Dominios Proteicos/fisiología , Multimerización de Proteína/fisiología , Receptores de GABA-B/metabolismo , Regulación Alostérica/efectos de los fármacos , Regulación Alostérica/fisiología , Sitio Alostérico/efectos de los fármacos , Sitio Alostérico/fisiología , Reactivos de Enlaces Cruzados/química , Agonistas de Receptores GABA-B/farmacología , Células HEK293 , Humanos , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Dominios Proteicos/efectos de los fármacos , Multimerización de Proteína/efectos de los fármacos , Ácido gamma-Aminobutírico/metabolismoRESUMEN
A mathematical model for the binding and function of metabotropic glutamate receptors was developed, with the aim to gain new insights into the functioning of these complex receptors. These receptors are homodimers, and each subunit is composed of a ligand binding [Venus flytrap (VFT)] domain and a heptahelical domain (HD) responsible for G-protein activation. Our mechanistic model integrates all structural information available so far: the various states of the VFT dimer (open-open, closed-open, and closed-closed), as well as the fact that a single HD is active at a time. To provide the model with parameters with biological meaning, two published experimental studies were reanalyzed. The first one reports a negative cooperativity in agonist binding (J Biol Chem 279:35526-35534, 2004), whereas the other indicates a positive cooperativity in agonist-mediated response (Nat Struct Mol Biol 11:706-713, 2004). The former study allowed us to explain the mechanistic features associated with VFT recognition by agonists and antagonists integrating a negative allosteric interaction for agonist binding. The second study helped us to quantitatively describe the functional dynamics of transduction of the VFT occupation into functional response, confirming a putative positive cooperativity at the level of receptor coupling efficacy. This model will help both to better understand the functioning of these receptors and to characterize the mechanism of action of various types of allosteric modulators. Moreover, this model may be of general utility for oligomeric systems in which the ligand binding and effector domains correspond to distinct structural domains.