RESUMEN
A recently reported Y receptor that has been confusingly referred to as both Y5 and Y2b has now been designated as Y6 by the IUPHAR organization. Using random primed Y6 coding sequence as a hybridization probe we examined the mRNA expression pattern and gene distribution of the Y6 receptor in a variety of species. We detail the relative abundance of Y6 message in mouse and human tissues and report the apparent absence of message for this receptor in any rat tissues tested. We also document the presence of the Y6 gene in chicken, rabbit, cow, dog, mouse, monkey and human, but the complete absence of the Y6 gene in rat.
Asunto(s)
Eliminación de Gen , Hipotálamo/metabolismo , Ratas/genética , Receptores de Neuropéptido Y/biosíntesis , Receptores de Neuropéptido Y/genética , Animales , Bovinos , Pollos , Perros , Femenino , Haplorrinos , Humanos , Masculino , Ratones , Especificidad de Órganos , ARN Mensajero/biosíntesis , Conejos , Especificidad de la Especie , Transcripción GenéticaRESUMEN
The principal inhibitory neurotransmitter GABA (gamma-aminobutyric acid) exerts its effects through two ligand-gated channels, GABA(A) and GABA(C) receptors, and a third receptor, GABA(B) , which acts through G proteins to regulate potassium and calcium channels. Cells heterologously expressing the cloned DNA encoding the GABA(B)R1 protein exhibit high-affinity antagonist-binding sites, but they produce little of the functional activity expected from studies of endogenous GABA(B) receptors in the brain. Here we describe a new member of the GABA(B) polypeptide family, GABA(B)R2, that shows sequence homology to GABA(B)R1. Neither GABA(B)R1 nor GABA(B)R2, when expressed individually, activates GIRK-type potassium channels; however, the combination of GABA(B)R1 and GABA(B)R2 confers robust stimulation of channel activity. Both genes are co-expressed in individual neurons, and both proteins co-localize in transfected cells. Moreover, immunoprecipitation experiments indicate that the two polypeptides associate with each other, probably as heterodimers. Several G-protein-coupled receptors (GPCRs) exist as high-molecular-weight species, consistent with the formation of dimers by these receptors, but the relevance of these species for the functioning of GPCRs has not been established. We have now shown that co-expression of two GPCR structures, GABA(B)R1 and GABA(B)R2, belonging to the same subfamily is essential for signal transduction by GABA(B) receptors.
Asunto(s)
Receptores de GABA-B/metabolismo , Receptores de GABA , Secuencia de Aminoácidos , Animales , Western Blotting , Células CHO , Células COS , Línea Celular , Cricetinae , Agonistas de Receptores GABA-B , Antagonistas de Receptores de GABA-B , Hipotálamo/metabolismo , Masculino , Datos de Secuencia Molecular , Mutación , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de GABA-B/genética , Transfección , XenopusRESUMEN
Neuropeptide Y (NPY) receptors belong to the G-protein-coupled receptor (GPCR) superfamily and mediate several physiological responses, such as blood pressure, food intake, sedation and memory retention. To understand the interactions between the NPY Y1 receptor subtype and its ligands, computer modeling was applied to the natural peptide agonist, NPY and a small molecule antagonist, BIBP3226. An agonist and antagonist binding domain was elucidated using mutagenesis data for the Y1 receptor as well as for other GPCR families. The agonist and antagonist ligands which were investigated appear to share common residues for their interaction within the transmembrane regions of the Y1 receptor structure, including Gln120, Asn283 and His306. This is in contrast to findings with tachykinin receptors where the binding domains of the non-peptide antagonists have very little in common with the binding domains of the agonist, substance-P. In addition, a hydrogen bond between the hydroxyl group of Tyr36 of NPY and the side chain of Gln219, an interaction that is absent in the model complex between Y1 and the antagonist BIBP3226, is proposed as one of the potential interactions necessary for receptor activation.
Asunto(s)
Proteínas de Unión al GTP/química , Modelos Moleculares , Receptores de Neuropéptido Y/química , Secuencia de Aminoácidos , Animales , Arginina/análogos & derivados , Arginina/química , Sitios de Unión , Bovinos , Simulación por Computador , Humanos , Ligandos , Datos de Secuencia Molecular , Estructura Molecular , Mutagénesis Sitio-Dirigida , Neuropéptido Y/química , Neuropéptido Y/genética , Neuropéptido Y/metabolismo , Conformación Proteica , Ingeniería de Proteínas , Receptores de Neuropéptido Y/genética , Receptores de Neuropéptido Y/metabolismoRESUMEN
A 3-dimensional model of the seven transmembrane helical segments (TMs) of the human alpha 1a-adrenoceptor was initially built by analogy to the known structure of bacteriorhodopsin. However, the rotational orientation of TM V about its helical axis, and the roles of several TM V residues in ligand binding and receptor activation remained in question. Accordingly, we determined the effects of six site-specific mutations in TM V on binding affinity and functional potency of a structurally diverse series of agonists and antagonists. Mutation of Ser 192 and Phe 193 disrupted the binding of many of the tested ligands, as measured by displacement of [3H]prazosin. In addition, mutation of Ser 188, Ser 192, and Phe 193 disrupted receptor activation, as measured by [3H]inositol phosphate formation. On the basis of these results, a specific rotational orientation of TM V is proposed as part of a revised receptor model, which also takes into account more recently reported information about the structure of rhodopsin. This revised alpha 1a-adrenoceptor model accounts for direct interactions which are proposed between Ser 188 and Ser 192 and the meta and para hydroxyl groups of norepinephrine, respectively, in the G-protein coupled receptor state.
Asunto(s)
Mutagénesis Sitio-Dirigida , Estructura Secundaria de Proteína , Receptores Adrenérgicos alfa 1/química , Receptores Adrenérgicos alfa 1/genética , Secuencia de Aminoácidos , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Prazosina/farmacología , Estructura Secundaria de Proteína/efectos de los fármacos , Ensayo de Unión Radioligante , Receptores Adrenérgicos alfa 1/efectos de los fármacos , Receptores Adrenérgicos alfa 1/fisiologíaRESUMEN
The serotonin 5-HT1B and 5-HT1A receptors bind certain beta-adrenergic antagonists, such as propranolol and pindolol, with high affinity. Other 5-HT1 receptors that display very low affinity for beta-adrenergic antagonists, have either a threonine (T) (5-HT1D alpha, 5-HT1D beta and 5-HT1E) or an alanine (A) (5-HT1F) residue in the homologous position in the seventh transmembrane domain. In the case of the human 5-HT1D beta receptor, replacement of this T with asparagine (N), dramatically increases its ability to bind beta-adrenergic antagonists. To assess whether other 5-HT1 receptors would behave similarly, we have used site-directed mutagenesis to replace the T or A in 5-HT1D alpha, 5-HT1E and 5-HT1F receptors with N. Both the wild-type and mutant genes were expressed transiently in COS-7 cells and radioligand binding studies were performed by using [3H]5-HT and [125I]iodocyanopindolol. Using [3H]5-HT, we found that the affinities of all the mutant receptors for propranolol and pindolol were significantly increased by 100-1000 fold, 5-HT1D alpha and 5-HT1F receptors showing the highest and the 5-HT1E receptor displaying the lowest affinity. On the other hand, the affinities for 5-HT were essentially unchanged as compared to the wild-type receptors. All mutant receptors bound [125I]iodocyanopindolol with high affinity, KD values ranging between 0.04 nM (mutant 5-HT1D alpha) and 0.57 nM (mutant 5-HT1E), whereas the wild-type receptors failed to show any specific binding with this radioligand in the same concentration range used for the mutant receptors.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Antagonistas Adrenérgicos beta/farmacocinética , Mutación Puntual , Receptores de Serotonina/genética , Receptores de Serotonina/metabolismo , Secuencia de Aminoácidos , Animales , Humanos , Yodocianopindolol , Ligandos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Pindolol/análogos & derivados , Propranolol/farmacología , Ensayo de Unión Radioligante , Ratas , Receptores de Serotonina/efectos de los fármacos , Transfección/fisiologíaRESUMEN
The dopaminergic system implicated in human disorders such as Parkinson's disease, schizophrenia and prolactinomas, exerts its effects through several dopamine receptors. The diversity of the dopaminergic system has been revealed by the application of molecular biology techniques to this system, which allowed the identification of five different types of dopamine receptors to date. Even though the structure of these receptors has now been identified, their physiological roles are still under investigation. The coupling of the D1 and D2 dopamine receptor to second messengers has been investigated using cell lines transfected with the cDNAs of these receptors. However, until recently, there was no technique allowing non-invasive real-time measurement of the metabolic activity of cells after agonist stimulation. We present here real-time measurement of events induced by dopaminergic agents on either the D1 or the D2 dopamine receptors using a novel technique employing a silicon-based microphysiometer.