Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 23
Filtrar
1.
Hum Mutat ; 42(2): 164-176, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33252155

RESUMEN

Biallelic mutations in G-Protein coupled receptor kinase 1 (GRK1) cause Oguchi disease, a rare subtype of congenital stationary night blindness (CSNB). The purpose of this study was to identify disease causing GRK1 variants and use in-depth bioinformatic analyses to evaluate how their impact on protein structure could lead to pathogenicity. Patients' genomic DNA was sequenced by whole genome, whole exome or focused exome sequencing. Disease associated variants, published and novel, were compared to nondisease associated missense variants. The impact of GRK1 missense variants at the protein level were then predicted using a series of computational tools. We identified twelve previously unpublished cases with biallelic disease associated GRK1 variants, including eight novel variants, and reviewed all GRK1 disease associated variants. Further structure-based scoring revealed a hotspot for missense variants in the kinase domain. In addition, to aid future clinical interpretation, we identified the bioinformatics tools best able to differentiate disease associated from nondisease associated variants. We identified GRK1 variants in Oguchi disease patients and investigated how disease-causing variants may impede protein function in-silico.


Asunto(s)
Enfermedades Hereditarias del Ojo , Quinasa 1 del Receptor Acoplado a Proteína-G , Ceguera Nocturna , Enfermedades Hereditarias del Ojo/genética , Quinasa 1 del Receptor Acoplado a Proteína-G/genética , Humanos , Ceguera Nocturna/genética
2.
Pharmacol Rev ; 68(4): 954-1013, 2016 10.
Artículo en Inglés | MEDLINE | ID: mdl-27630114

RESUMEN

The glucagon-like peptide (GLP)-1 receptor (GLP-1R) is a class B G protein-coupled receptor (GPCR) that mediates the action of GLP-1, a peptide hormone secreted from three major tissues in humans, enteroendocrine L cells in the distal intestine, α cells in the pancreas, and the central nervous system, which exerts important actions useful in the management of type 2 diabetes mellitus and obesity, including glucose homeostasis and regulation of gastric motility and food intake. Peptidic analogs of GLP-1 have been successfully developed with enhanced bioavailability and pharmacological activity. Physiologic and biochemical studies with truncated, chimeric, and mutated peptides and GLP-1R variants, together with ligand-bound crystal structures of the extracellular domain and the first three-dimensional structures of the 7-helical transmembrane domain of class B GPCRs, have provided the basis for a two-domain-binding mechanism of GLP-1 with its cognate receptor. Although efforts in discovering therapeutically viable nonpeptidic GLP-1R agonists have been hampered, small-molecule modulators offer complementary chemical tools to peptide analogs to investigate ligand-directed biased cellular signaling of GLP-1R. The integrated pharmacological and structural information of different GLP-1 analogs and homologous receptors give new insights into the molecular determinants of GLP-1R ligand selectivity and functional activity, thereby providing novel opportunities in the design and development of more efficacious agents to treat metabolic disorders.


Asunto(s)
Péptido 1 Similar al Glucagón , Receptores Acoplados a Proteínas G , Animales , Péptido 1 Similar al Glucagón/química , Péptido 1 Similar al Glucagón/genética , Péptido 1 Similar al Glucagón/metabolismo , Humanos , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo
3.
Biochem Biophys Res Commun ; 498(2): 359-365, 2018 03 29.
Artículo en Inglés | MEDLINE | ID: mdl-29397068

RESUMEN

The receptor for glucagon-like peptide 1 (GLP-1R) is a validated drug target for the treatment of type 2 diabetes and obesity. Recently the first three structures of GLP-1R were published - an X-ray structure of the apo transmembrane domain in the inactive conformation; an X-ray structure of the full-length receptor bound to a truncated peptide agonist; and a cryo-EM structure of the full-length receptor bound with GLP-1 and coupled to the G protein Gs. Since the inactive structure was incomplete, and the two active-state structures shared significant differences, we utilised all available knowledge to build hybrid models of the full length active and inactive state receptors. The two models were simulated using molecular dynamics and the output trajectories analysed and compared to reveal insights into the mechanism for agonist-mediated receptor activation. His-7, Glu-9 and Asp-15 of GLP-1 act together to destabilise transmembrane helix 6 and extracellular loop 3 in order to generate an active conformation of GLP-1R.


Asunto(s)
Receptor del Péptido 1 Similar al Glucagón/agonistas , Receptor del Péptido 1 Similar al Glucagón/química , Simulación de Dinámica Molecular , Péptido 1 Similar al Glucagón/química , Péptido 1 Similar al Glucagón/metabolismo , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Humanos , Modelos Moleculares , Conformación Proteica , Dominios Proteicos
4.
Biochem Biophys Res Commun ; 391(1): 437-42, 2010 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-19914210

RESUMEN

The receptor for calcitonin gene-related peptide (CGRP) has been the target for the development of novel small molecule antagonists for the treatment of migraine. Two such antagonists, BIBN4096BS and MK-0974, have shown great promise in clinical trials and hence a deeper understanding of the mechanism of their interaction with the receptor is now required. The structure of the CGRP receptor is unusual since it is comprised of a hetero-oligomeric complex between the calcitonin receptor-like receptor (CRL) and an accessory protein (RAMP1). Both the CLR and RAMP1 components have extracellular domains which interact with each other and together form part of the peptide-binding site. It seems likely that the antagonist binding site will also be located on the extracellular domains and indeed Trp-74 of RAMP1 has been shown to form part of the binding site for BIBN4096BS. However, despite a chimeric study demonstrating the role of the N-terminal domain of CLR in antagonist binding, no specific residues have been identified. Here we carry out a mutagenic screen of the extreme N-terminal domain of CLR (residues 23-63) and identify a mutant, Met-42-Ala, which displays 48-fold lower affinity for BIBN4096BS and almost 900-fold lower affinity for MK-0974. In addition, we confirm that the Trp-74-Lys mutation at human RAMP1 reduces BIBN4096BS affinity by over 300-fold and show for the first time a similar effect for MK-0974 affinity. The data suggest that the non-peptide antagonists occupy a binding site close to the interface of the N-terminal domains of CLR and RAMP1.


Asunto(s)
Azepinas/metabolismo , Antagonistas del Receptor Peptídico Relacionado con el Gen de la Calcitonina , Imidazoles/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Piperazinas/metabolismo , Quinazolinas/metabolismo , Receptores de Calcitonina/metabolismo , Azepinas/química , Azepinas/farmacología , Proteína Similar al Receptor de Calcitonina , Humanos , Imidazoles/química , Imidazoles/farmacología , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana/genética , Metionina/genética , Metionina/metabolismo , Piperazinas/química , Piperazinas/farmacología , Estructura Terciaria de Proteína , Quinazolinas/química , Quinazolinas/farmacología , Proteína 1 Modificadora de la Actividad de Receptores , Proteínas Modificadoras de la Actividad de Receptores , Receptores de Calcitonina/genética , Triptófano/genética , Triptófano/metabolismo
5.
Mol Pharmacol ; 74(3): 605-13, 2008 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-18539702

RESUMEN

Parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP) bind and activate the PTH/PTHrP receptor (PTH-1R). However, while the related receptor PTH-2R responds potently to PTH, it is not activated by PTHrP. Two hormone sites are known to be responsible for these different potencies. First, the absence of efficacy for PTHrP at PTH-2R is due to the presence of His-5 in PTHrP (Ile-5 in PTH), which interacts with the receptor's juxtamembrane domain. Second, PTHrP has lower affinity than PTH for PTH-2R because of the presence of Phe-23 (Trp-23 in PTH), which interacts with the receptor's N-terminal extracellular domain. We used these different receptor subtype properties to demonstrate that residue 41 in PTH-1R, when either the native Leu or substituted by Ile or Met, can accommodate either Phe or Trp at position 23 of the ligand. However, when Leu-41 is substituted by a smaller side chain, either Ala or Val (its equivalent residue in PTH-2R), the receptor becomes highly selective for those peptide ligands with Trp-23. Hence, despite the conservative nature of the substitutions found in the native ligands (Phe for Trp) and receptors (Leu for Val), they nevertheless enable a significant degree of selectivity to be achieved. Analysis of this functionally important ligand-receptor contact, within the context of the recent X-ray structure of the peptide-bound PTH-1R N domain, reveals the nature of the selectivity filter and how it is by-passed in PTH-1R.


Asunto(s)
Aminoácidos/metabolismo , Receptores de Hormona Paratiroidea/química , Receptores de Hormona Paratiroidea/metabolismo , Secuencia de Aminoácidos , Unión Competitiva , Línea Celular , Membrana Celular/metabolismo , Cristalografía por Rayos X , Humanos , Ligandos , Datos de Secuencia Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Péptidos/química , Péptidos/metabolismo , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Relación Estructura-Actividad
6.
Biochem Pharmacol ; 127: 71-81, 2017 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-28012961

RESUMEN

TIP39 ("tuberoinfundibular peptide of 39 residues") acts via the parathyroid hormone 2 receptor, PTH2, a Family B G protein-coupled receptor (GPCR). Despite the importance of GPCRs in human physiology and pharmacotherapy, little is known about the molecular details of the TIP39-PTH2 interaction. To address this, we utilised the different pharmacological profiles of TIP39 and PTH(1-34) at PTH2 and its related receptor PTH1: TIP39 being an agonist at the former but an antagonist at the latter, while PTH(1-34) activates both. A total of 23 site-directed mutations of PTH2, in which residues were substituted to the equivalent in PTH1, were made and pharmacologically screened for agonist activity. Follow-up mutations were analysed by radioligand binding and cAMP assays. A model of the TIP39-PTH2 complex was built and analysed using molecular dynamics. Only Tyr318-Ile displayed reduced TIP39 potency, despite having increased PTH(1-34) potency, and further mutagenesis and analysis at this site demonstrated that this was due to reduced TIP39 affinity at Tyr318-Ile (pIC50=6.01±0.03) compared with wild type (pIC50=7.81±0.03). The hydroxyl group of the Tyr-318's side chain was shown to be important for TIP39 binding, with the Tyr318-Phe mutant displaying 13-fold lower affinity and 35-fold lower potency compared with wild type. TIP39 truncated by up to 5 residues at the N-terminus was still sensitive to the mutations at Tyr-318, suggesting that it interacts with a region within TIP39(6-39). Molecular modelling and molecular dynamics simulations suggest that the selectivity is based on an interaction between the Tyr-318 hydroxyl group with the carboxylate side chain of Asp-7 of the peptide.


Asunto(s)
Neuropéptidos/farmacología , Receptor de Hormona Paratiroídea Tipo 2/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Mutación , Neuropéptidos/química , Neuropéptidos/genética , Estructura Secundaria de Proteína , Ensayo de Unión Radioligante , Receptor de Hormona Paratiroídea Tipo 1/química , Receptor de Hormona Paratiroídea Tipo 1/metabolismo , Receptor de Hormona Paratiroídea Tipo 2/agonistas , Receptor de Hormona Paratiroídea Tipo 2/química , Tirosina/química , Tirosina/genética
7.
Biochem Pharmacol ; 71(4): 464-71, 2006 Feb 14.
Artículo en Inglés | MEDLINE | ID: mdl-16343447

RESUMEN

It is currently unclear whether activation of the AT1 receptor by agonists involves conformational selection or induction. We evaluated the pharmacological properties of wild type and N111G CAM human AT1 receptors stably expressed in HEK293 cells. Although [Sar1]-Ang II and Ang IV were full agonists at both receptors, the potency of Ang IV was 280-fold lower at the wild type receptor. [Sar1, Ile8]-Ang II was only a full agonist at the N111G CAM AT1 receptor. [Sar1]-Ang II and [Sar1, Ile8]-Ang II displayed similar high affinity binding to both receptors. In contrast, Ang IV displayed low affinity binding to the wild type and high affinity binding to the N111G CAM AT1 receptor. Based on these observations we provide strong evidence that conformational induction is the key process for activation of the AT1 receptor. Only by the creation of CAMs can conformational selection be envisaged to take place.


Asunto(s)
Conformación Proteica , Receptor de Angiotensina Tipo 1/metabolismo , Angiotensina II/análogos & derivados , Angiotensina II/farmacología , Unión Competitiva , Línea Celular , Humanos , Fosfatos de Inositol/metabolismo , Modelos Biológicos , Receptor de Angiotensina Tipo 1/agonistas , Receptor de Angiotensina Tipo 1/genética , Factores de Tiempo
8.
FEBS Lett ; 579(1): 285-91, 2005 Jan 03.
Artículo en Inglés | MEDLINE | ID: mdl-15620728

RESUMEN

We have investigated the effect of the sulfhydryl-reactive reagent, methyl thiosulfonate ethylammonium (MTSEA), on ligand binding to the human melanocortin-4 (MC4) receptor stably expressed in HEK-293 cells. MTSEA inhibited binding of the agonist, 125I-NDPalpha-MSH, and the antagonist, 125I-SHU9119, in a concentration-dependent manner. Pre-incubation of cells with either the agonist or antagonist protected from subsequent MTSEA inhibition of radioligand binding. Mutation of Cys130 in transmembrane helix 3 to alanine, whilst not affecting ligand binding, led to a complete loss of the inhibitory effect of MTSEA. Since other types of sulfhydryl-reactive reagents had no effect on ligand binding, we conclude that covalent modification of Cys130 by MTSEA disrupts ligand binding by neutralising a close-by negative charge, most likely on Asp126.


Asunto(s)
Cisteína/efectos de los fármacos , Metanosulfonato de Etilo/análogos & derivados , Metanosulfonato de Etilo/farmacología , Receptor de Melanocortina Tipo 4/química , Receptor de Melanocortina Tipo 4/efectos de los fármacos , Secuencias de Aminoácidos/genética , Secuencia de Aminoácidos , Sitios de Unión/genética , Cisteína/química , Cisteína/genética , Humanos , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación/genética , Estructura Secundaria de Proteína/genética , Receptor de Melanocortina Tipo 4/metabolismo
9.
Biosci Rep ; 36(1): e00285, 2015 Nov 23.
Artículo en Inglés | MEDLINE | ID: mdl-26598711

RESUMEN

Glucagon-like peptide-1 (7-36)amide (GLP-1) plays a central role in regulating blood sugar levels and its receptor, GLP-1R, is a target for anti-diabetic agents such as the peptide agonist drugs exenatide and liraglutide. In order to understand the molecular nature of the peptide-receptor interaction, we used site-directed mutagenesis and pharmacological profiling to highlight nine sites as being important for peptide agonist binding and/or activation. Using a knowledge-based approach, we constructed a 3D model of agonist-bound GLP-1R, basing the conformation of the N-terminal region on that of the receptor-bound NMR structure of the related peptide pituitary adenylate cyclase-activating protein (PACAP21). The relative position of the extracellular to the transmembrane (TM) domain, as well as the molecular details of the agonist-binding site itself, were found to be different from the model that was published alongside the crystal structure of the TM domain of the glucagon receptor, but were nevertheless more compatible with published mutagenesis data. Furthermore, the NMR-determined structure of a high-potency cyclic conformationally-constrained 11-residue analogue of GLP-1 was also docked into the receptor-binding site. Despite having a different main chain conformation to that seen in the PACAP21 structure, four conserved residues (equivalent to His-7, Glu-9, Ser-14 and Asp-15 in GLP-1) could be structurally aligned and made similar interactions with the receptor as their equivalents in the GLP-1-docked model, suggesting the basis of a pharmacophore for GLP-1R peptide agonists. In this way, the model not only explains current mutagenesis and molecular pharmacological data but also provides a basis for further experimental design.


Asunto(s)
Receptor del Péptido 1 Similar al Glucagón/química , Simulación del Acoplamiento Molecular , Sustitución de Aminoácidos , Receptor del Péptido 1 Similar al Glucagón/agonistas , Receptor del Péptido 1 Similar al Glucagón/genética , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Células HEK293 , Humanos , Mutagénesis Sitio-Dirigida , Resonancia Magnética Nuclear Biomolecular , Estructura Terciaria de Proteína , Relación Estructura-Actividad
10.
FEBS Lett ; 530(1-3): 244-8, 2002 Oct 23.
Artículo en Inglés | MEDLINE | ID: mdl-12387900

RESUMEN

The mutation of Asp198 to Asn in the receptor for glucagon-like peptide-1(7-36)amide (GLP-1) had no effect upon GLP-1 affinity whereas substitution with Ala greatly reduced affinity, demonstrating the importance of polarity rather than negative charge at Asp198. However, the Asp198-Ala mutation had less effect upon the affinity of Exendin-4, a peptide agonist that has been shown previously not to require its N-terminus for high affinity. Moreover, the affinity of a truncated GLP-1 analogue lacking the first eight residues was not affected by the Asp198-Ala mutation, demonstrating that Asp198 is required for maintaining the binding site of the N-terminal region of GLP-1.


Asunto(s)
Ácido Aspártico/metabolismo , Glucagón/metabolismo , Fragmentos de Péptidos/metabolismo , Precursores de Proteínas/metabolismo , Receptores de Glucagón/metabolismo , Línea Celular , Glucagón/química , Péptido 1 Similar al Glucagón , Receptor del Péptido 1 Similar al Glucagón , Humanos , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/química , Precursores de Proteínas/química , Ensayo de Unión Radioligante , Receptores de Glucagón/genética
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA