RESUMEN
In this work, we studied a novel chimeric peptide, M242, galanin(1-13)-[D-Trp(32)]-neuropeptide Y(25-36)amide, and examined its properties in comparison with its parent peptide, M32, galanin(1-13)-neuropeptide Y(25-36)amide, a previously known high-affinity ligand for galanin receptors, and galanin itself. Binding assays performed in Bowes cells known to express human galanin receptor type 1 (hGalR1) and in Chinese hamster ovary cells overexpressing human galanin receptor type 2 (hGalR2) revealed that all three ligands had comparable affinities: at hGalR1<1 nM and at hGalR2<10 nM. However, in rat hippocampal membranes M242 had a 24-fold lower affinity than galanin (9.4 vs. 0.4 nM) and 134-fold lower affinity than M32 (9.4 vs. 0.07 nM). In the same tissue, we also examined the effects of these peptides on adenylate cyclase activity. M32 showed a weak antagonistic behaviour but M242 acted as a potent biphasic regulator of adenylate cyclase. In conclusion, we present and characterise a new peptide M242, which could be a useful tool in studies of galaninergic signalling.
Asunto(s)
Galanina/química , Neuropéptido Y/análogos & derivados , Neuropéptido Y/química , Fragmentos de Péptidos/química , Receptores de Neuropéptido/metabolismo , Adenilil Ciclasas/metabolismo , Animales , Células CHO , Cricetinae , Galanina/antagonistas & inhibidores , Galanina/metabolismo , Hipocampo/metabolismo , Humanos , Ligandos , Neuropéptido Y/antagonistas & inhibidores , Neuropéptido Y/metabolismo , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/metabolismo , Unión Proteica , Ratas , Receptor de Galanina Tipo 1 , Receptor de Galanina Tipo 2 , Receptores de Galanina , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
To determine the domains essential for G-protein coupling of the human galanin receptor type 1 (GalR1), we have used both GalR1 mutants and synthetic receptor-derived peptides in(125)I-galanin and [(35)S]-GTPgammaS binding studies. Replacement of potential phosphorylation sites by Leu in the third intracellular loop (IC3) of GalR1 did not affect K(D)values for the receptor. Peptides derived form the IC3 loop, and especially the N-terminal part of it were able to increase the rate of [(35)S]-GTPgammaS binding to the trimeric Gialpha1beta1gamma2, but not to Gsalphabeta1gamma2, whereas the peptides corresponding to the IC1 and IC2 loops had no such effect. IC3 loop peptides also inhibited the binding of(125)I-galanin to GalR1 in membranes from Rin m5F cells. Our results suggest that the IC3 loop of GalR1, especially its N-terminal part, defines the coupling of the receptor to the Gialpha1beta1gamma2 protein and consequently, to the signal transduction cascade.
Asunto(s)
Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Receptores de Neuropéptido/química , Receptores de Neuropéptido/metabolismo , Transducción de Señal/fisiología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Sitios de Unión , Línea Celular , Membrana Celular/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Galanina/metabolismo , Humanos , Cinética , Leucina , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacología , Fosforilación , Estructura Secundaria de Proteína , Receptor de Galanina Tipo 1 , Receptores de Galanina , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Spodoptera , TransfecciónRESUMEN
Peptide nucleic acids (PNAs) form stable and tight complexes with complementary DNA and/or RNA and would be promising antisense reagents if their cellular delivery could be improved. We show that a 21-mer PNA, complementary to the human galanin receptor type 1 mRNA, coupled to the cellular transporter peptides, transportan or pAntennapedia(43-58), is efficiently taken up into Bowes cells where they block the expression of galanin receptors. In rat, the intrathecal administration of the peptide-PNA construct results in a decrease in galanin binding in the dorsal horn. The decrease in binding results in the inability of galanin to inhibit the C fibers stimulation-induced facilitation of the rat flexor reflex, demonstrating that peptide-PNA constructs act in vivo to suppress expression of functional galanin receptors.
Asunto(s)
Proteínas Nucleares , Dolor/fisiopatología , Ácidos Nucleicos de Péptidos/metabolismo , Receptores de Neuropéptido/metabolismo , Transducción de Señal , Factores de Transcripción , Secuencia de Aminoácidos , Animales , Proteína con Homeodominio Antennapedia , Secuencia de Bases , Regulación hacia Abajo , Femenino , Galanina , Proteínas de Homeodominio/química , Proteínas de Homeodominio/metabolismo , Humanos , Melanoma/metabolismo , Melanoma/patología , Melanoma/fisiopatología , Datos de Secuencia Molecular , Dolor/metabolismo , Fragmentos de Péptidos/metabolismo , Ácidos Nucleicos de Péptidos/química , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Receptor de Galanina Tipo 1 , Receptores de Galanina , Receptores de Neuropéptido/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Médula Espinal/metabolismo , Células Tumorales Cultivadas , Venenos de AvispasAsunto(s)
Proteínas de Unión al GTP/metabolismo , Galanina/metabolismo , Receptores de la Hormona Gastrointestinal/química , Receptores de la Hormona Gastrointestinal/fisiología , Transducción de Señal , Secuencia de Aminoácidos , Animales , Humanos , Cinética , Datos de Secuencia Molecular , Fragmentos de Péptidos/farmacología , Receptores de GalaninaRESUMEN
The aim of this study was to evaluate a variety of synthetic strategies pertinent to the development of chimeric analogs of the structurally divergent nonapeptide hormones arginine vasopressin (AVP) and bradykinin (BK). Single-chain peptides combining AVP and BK directly, AVP(1-9)-BK(1-9) or via a flexible aminohexanoic acid (epsilonAhx) linker, AVP(1-9)-epsilonAhx-BK(1-9), bind with relatively high affinity to the bovine kidney medulla B2a bradykinin receptor (B2a BKR). Significantly, amino-terminal extended chimeric analogs of BK, including AVP(1-9)-BK(1-9) and galanin(1-13)-BK(1-9), are functional B2 BKR agonists. These findings illustrate that chimeric peptides can activate G-protein-coupled receptors (GPCRs) in a manner analogous to that of endogenous monomeric agonists. Further development, combining the sequences of receptor subtype-selective antagonists, produced high-affinity chimeric antagonists of the V1a vasopressin receptor (V1a VPR) and the B2a BKR. We also determined the pharmacological characteristics of high-affinity chimeric hormone analogs derivatized with the membrane targeting function of mastoparan. Homodimers of an amino-terminal extended BK analog and a V1a-selective antagonist represent the first examples of new classes of B2 BKR and V1a VPR antagonists, respectively. These findings are discussed in relation to the GPCR binding site for small peptides and the development of novel biological probes and therapeutic agents.
Asunto(s)
Arginina Vasopresina/síntesis química , Bradiquinina/síntesis química , Secuencia de Aminoácidos , Animales , Arginina Vasopresina/farmacología , Bradiquinina/farmacología , Bovinos , Línea Celular , Membrana Celular/metabolismo , Dimerización , Diseño de Fármacos , Proteínas de Unión al GTP/metabolismo , Ligandos , Datos de Secuencia Molecular , Péptidos/síntesis química , Péptidos/farmacología , Ratas , Receptores de Bradiquinina/metabolismo , Receptores de Vasopresinas/metabolismo , Proteínas Recombinantes de Fusión/síntesis química , Proteínas Recombinantes de Fusión/farmacología , Relación Estructura-ActividadRESUMEN
Rat galanin inhibits basal as well as forskolin-stimulated adenylate cyclase activity in rat ventral and dorsal hippocampus. The inhibition of adenylate cyclase activity, both basal and forskolin-stimulated, is characterised by IC50 values being 250-fold lower in ventral hippocampus (IC50 = 1.1 nM) compared to the dorsal hippocampus (IC50 = 270 nM). The maximal inhibition of basal and forskolin-stimulated adenylate cyclase activity in both ventral and dorsal hippocampus in the presence of 10 microM rat galanin is 34-45%. The analysis of the binding data obtained with 125I-labelled Tyr26-porcine galanin as a tracer reveals similar binding constants for rat galanin in both ventral and dorsal hippocampus with 4.8-fold higher concentration of galanin receptors in the ventral hippocampus. Putative galanin receptor subtype differences between the ventral and dorsal hippocampus have been noted by Hedlund et al. (Eur. J. Pharmacol., 224 (1992) 203-205). This study yields further confirmation for the existence of different galanin receptor subtypes or for differential coupling of galanin receptors to the adenylate cyclase in the dorsal versus ventral hippohampus.