RESUMEN
Two aspartates in the third extracellular loop of the rat B2 bradykinin (BK) receptor have been implicated as important residues for agonist binding. Asp268 and Asp286 were mutated to alanine residues and changes in agonist and antagonist binding affinity were examined. The IC50 value for BK as a competitor of [3H] NPC 17731 binding to the rat wild type receptor was 1.1 nM, while the Ala268 and Ala286 receptor mutants exhibited IC50 values of 19 nM and 28 nM, respectively. The Ala268Ala268 receptor mutant exhibited an IC50 for BK of 500 nM. These mutations had little effect on binding affinity when NPC 17761, a BK antagonist, was used to compete [3H] NPC 17731 binding. Electrophysiological examination of Xenopus oocytes expressing wild type or Ala268 Ala286 receptors confirmed the importance of the Asp268 and Asp286 residues for BK recognition. BK activated the mutant receptor with comparable efficacy relative to the wild type receptor, but a 1750-fold reduction in potency was observed.
Asunto(s)
Ácido Aspártico , Bradiquinina/metabolismo , Mutagénesis Sitio-Dirigida , Estructura Secundaria de Proteína , Receptores de Bradiquinina/química , Receptores de Bradiquinina/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Unión Competitiva , Membrana Celular/fisiología , Cartilla de ADN , Femenino , Cinética , Potenciales de la Membrana/fisiología , Datos de Secuencia Molecular , Oligopéptidos/metabolismo , Oocitos/fisiología , Reacción en Cadena de la Polimerasa/métodos , Ensayo de Unión Radioligante , Ratas , Receptores de Bradiquinina/fisiología , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Mapeo Restrictivo , Tritio , Xenopus laevisRESUMEN
A putative model of bradykinin bound to the rat B2 receptor was generated using a combination of homology modeling (from the known transmembrane structure of bacteriorhodopsin), energy minimization, molecular dynamics, and a two-stage conformational search as a docking simulation. Overall, the proposed bound ligand adopts a twisted "S" shape, wherein a C-terminal beta-turn is buried in the receptor just below the extracellular boundary of the cell membrane and the N-terminus is interacting with negatively charged residues in extracellular loop 3 of the receptor (most notably Asp268 and Asp286). Mutagenesis experiments describing mutations which result in both a loss of bradykinin affinity as well as those which have no effect on bradykinin affinity are in good agreement with the proposed structure. In short, the mutagenesis results and the computational simulations each point to the same region of the receptor as likely to bind bradykinin. A double mutation, predicted as being likely to have a dramatic effect on bradykinin binding affinity, was confirmed experimentally, adding some validation to the proposed complex. Moreover, a new pseudopeptide bradykinin receptor antagonist (D-Arg0-Arg1-[12-aminododecanoyl]2- Ser3-D-Tic4-Oic5-Arg6) was designed on the basis of the model, and found to have good receptor affinity. Speculation regarding other possible sites for mutagenesis are also described.
Asunto(s)
Bradiquinina/química , Bradiquinina/metabolismo , Diseño de Fármacos , Modelos Moleculares , Receptores de Bradiquinina/química , Receptores de Bradiquinina/metabolismo , Alanina , Secuencia de Aminoácidos , Animales , Membrana Celular/química , Simulación por Computador , Electroquímica , Glutamatos , Ácido Glutámico , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación Puntual , Conformación Proteica , Estructura Secundaria de Proteína , Ratas , TermodinámicaRESUMEN
A family of five muscarinic acetylcholine receptor genes (m1-m5) encode highly related proteins; however, for methodological reasons it has not been possible to detect the gene products individually. To develop antibody probes specific for the receptor subtypes, unique regions of m1-m5 cDNAs, corresponding to the third cytoplasmic (i3) loops, were subcloned into bacterial expression vectors and the fusion proteins expressed in E. coli were used to generate rabbit antisera. These antisera react specifically with the respective fusion proteins on immunoblots and selectively immunoprecipitate each of the native cloned receptors. Since the i3 loops are immunogenic and the epitopes in the cloned receptors are accessible to antibodies, this approach should be valuable for immunological studies of the native receptors.
Asunto(s)
Receptores Muscarínicos/genética , Secuencia de Aminoácidos , Secuencia de Bases , Western Blotting , Escherichia coli/genética , Expresión Génica , Humanos , Datos de Secuencia Molecular , Peso Molecular , Oligonucleótidos , Fragmentos de Péptidos/genética , Reacción en Cadena de la Polimerasa , Receptores Muscarínicos/clasificación , Receptores Muscarínicos/inmunología , Proteínas Recombinantes de FusiónRESUMEN
Receptors for dopamine have been classified into two functional types, D1 and D2. They belong to the family of receptors acting through G (or guanine nucleotide-binding) proteins. D2 receptors inhibit adenylyl cyclase, but D1 receptors stimulate adenylyl cyclase and activate cyclic AMP-dependent protein kinases. Dopamine D1 and D2 receptors are targets of drug therapy in many psychomotor disorders, including Parkinson's disease and schizophrenia, and may also have a role in drug addiction and alcoholism. D1 receptors regulate neuron growth and differentiation, influence behaviour and modify dopamine D2 receptor-mediated events. We report here the cloning of the D1 receptor gene, which resides on an intronless region on the long arm of chromosome 5, near two other members of the G-linked receptor family. The expressed protein, encoded by 446 amino acids, binds drugs with affinities identical to the native human D1 receptor. The presence of a D1 receptor gene restriction fragment length polymorphism will be helpful for future disease linkage studies.
Asunto(s)
Cromosomas Humanos Par 5 , Receptores Dopaminérgicos/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Benzazepinas/metabolismo , Química Encefálica , Bovinos , Clonación Molecular , Glicosilación , Humanos , Intrones , Masculino , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Fosforilación , Polimorfismo de Longitud del Fragmento de Restricción , ARN Mensajero/análisis , Ratas , Ratas Endogámicas , Receptores de Dopamina D1 , Mapeo Restrictivo , Distribución Tisular , TransfecciónRESUMEN
Based on the sequence of a dopamine D2 receptor cloned from rat brain, we prepared a series of oligodeoxynucleotide probes. A mixture of these probes hybridized with a 2.6-kilobase species of mRNA extracted from several rat tissues including retina and, using in situ hybridization of these probes to cryostat sections of rat retina, they densely label the inner nuclear and outer plexiform layers. Labeling was also observed in the inner plexiform and ganglion cell layers. No hybridization was observed to the photoreceptor layers. A similar pattern of labeling was observed in monkey retina, indicating that the probes also hybridize with a homologous primate mRNA. The probes were used to screen a lambda gt10 library of human retina. A 2.5-kilobase clone was isolated, which encodes a protein that differs from the rat brain protein by 18 amino acids. The 5' and 3' untranslated regions of the human retinal cDNA were also strongly homologous with the rat brain cDNA. The clone was subcloned into the pCD-PS expression vector and transfected into COS-7 cells. The transfected cells bound [3H]-raclopride with a pharmacology expected of dopamine D2 receptors. These data indicate that D2 receptors expressed in the inner retina and outer plexiform layer have genetic identity with those expressed by brain and that the human and rat D2 receptors are derived from highly related genes.
Asunto(s)
Receptores Dopaminérgicos/genética , Retina/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Chlorocebus aethiops , Clonación Molecular , ADN/genética , Humanos , Datos de Secuencia Molecular , Sondas de Oligonucleótidos , ARN Mensajero/metabolismo , Ratas , Receptores Dopaminérgicos/fisiología , Receptores de Dopamina D2 , Distribución Tisular , TransfecciónRESUMEN
A glutamate-binding protein from rat brain synaptic plasma membranes has been purified to apparent homogeneity. This protein has a Mr of 14,300 based on amino acid and carbohydrate analyses. The protein is enriched with tryptophan residues, which contribute substantially to its hydrophobic nature. It also has a relatively high content of acidic amino acids, which determine is low isoelectric point (4.82). The protein exhibits either a single, high-affinity class of sites for L-[3H]glutamate binding (KD = 0.13 microM) when binding is measured at low protein concentrations, or two classes of sites with high (KD = 0.17 microM) and low affinities (KD = 0.8 microM) when binding is measured at high protein concentrations. These observations suggest preferential binding of L-glutamate to a self-associating form of the protein. The displacement of protein-bound L-[3H]glutamic acid by other neuroactive amino acids has characteristics similar to those observed for displacement of L-glutamate from membrane binding sites. Chemical modification of the cysteine and arginine residues results in an inhibition of glutamate binding activity. The possible function of this protein in the physiologic glutamate receptor complex of neuronal membranes is discussed.