RESUMEN
GPCRs (G-protein-coupled receptors) are a large family of structurally related proteins which mediate their effects by coupling to G-proteins. The V(1a)R (V(1a) vasopressin receptor) is a member of a family of related GPCRs that are activated by vasopressin {AVP ([Arg(8)]vasopressin)}, OT (oxytocin) and related peptides. These receptors are members of a subfamily of Family A GPCRs called the neurohypophysial peptide hormone receptor family. GPCRs exhibit a conserved tertiary structure comprising a bundle of seven TM (transmembrane) helices linked by alternating ECLs (extracellular loops) and ICLs (intracellular loops). The cluster of TM helices is functionally important for ligand binding, and, furthermore, activation of GPCRs involves movement of these TM helices. Consequently, it might be assumed that the extracellular face of GPCRs is composed of peptide linkers that merely connect important TM helices. However, using a systematic mutagenesis approach and focusing on the N-terminus and the second ECL of the V(1a)R, we have established that these extracellular domains fulfil a range of important roles with respect to GPCR signalling, including agonist binding, ligand selectivity and receptor activation.
Asunto(s)
Ligandos , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Animales , Humanos , Unión Proteica/fisiología , Conformación ProteicaRESUMEN
A fundamental issue in molecular pharmacology is to define how agonist-receptor interaction differs from that of antagonist-receptor interaction. The V(1a) vasopressin receptor (V(1a)R) is a member of a family of related G-protein-coupled receptors (GPCRs) that are activated by vasopressin, oxytocin (OT) and related peptides. A segment of the N-terminus that was required for agonist binding, but not antagonist binding, was identified by characterizing truncated V(1a)R constructs. Site-directed mutagenesis revealed that a single residue (Arg(46)) was critical for agonist binding and receptor activation. The N-terminus of the related OT receptor (OTR) could recover agonist binding in a chimaeric OTR(N)-V(1a)R construct. Furthermore, Arg(34) of the human OTR, which corresponds to Arg(46) of the rat V(1a)R, provided agonist-specific binding epitopes in the OTR, indicating a conserved function of this locus throughout this GPCR subfamily. Mutation of Arg(46) revealed that high-affinity agonist binding had an absolute requirement for arginine at this position.
Asunto(s)
Receptores de Péptidos/agonistas , Receptores de Péptidos/metabolismo , Animales , Arginina/química , Sitios de Unión , Epítopos , Humanos , Ligandos , Membrana Dobles de Lípidos , Modelos Biológicos , Mutagénesis Sitio-Dirigida , Mutación , Unión Proteica , Estructura Terciaria de Proteína , Receptores de Vasopresinas/metabolismoRESUMEN
A fundamental issue in molecular pharmacology is to define how agonist:receptor interaction differs from that of antagonist:receptor. The V(1a) receptor (V(1a)R) is a member of a family of related G-protein-coupled receptors that are activated by the neurohypophysial peptide hormone arginine-vasopressin (AVP). Here we define a short subdomain of the N-terminus of the V(1a)R from Glu(37) to Asn(47) that is an absolute requirement for binding AVP and other agonists. In marked contrast to the situation for agonists, deleting this segment has little or no effect on the binding of either peptide or non-peptide antagonists. In addition, we established that this subdomain was crucial for receptor activation and second messenger generation. The oxytocin receptor (OTR) also binds AVP with high affinity but exhibits a different pharmacological profile to the V(1a)R. Substitution of the N-terminus of the V(1a)R with the corresponding sequence from the OTR generated a chimeric receptor (OTR(N)-V(1a)R). The presence of the OTR N-terminus recovered high affinity agonist binding such that the OTR(N)-V(1a)R possessed almost wild-type V(1a)R pharmacology and signaling. Consequently, a domain within the N-terminus is required for agonist binding but it does not provide the molecular discriminator for subtype-selective agonist recognition. Cotransfection and peptide mimetic studies demonstrated that this N-terminal subdomain had to be contiguous with the receptor polypeptide to be functional. This study establishes that a segment of the V(1a)R N-terminus has a pivotal role in the mechanism of agonist binding and provides molecular insight into key differences between the interaction of agonists and antagonists with a peptide receptor family.