An arginyl in the N‐terminus of the V_1a vasopressin receptor is part of the conformational switch controlling activation by agonist

Abstract

Defining how the agonist-receptor interaction differs from that of the antagonist-receptor and understanding the mechanisms of receptor activation are fundamental issues in cell signalling. The V 1a vasopressin receptor (V 1a R) is a member of a family of related G-protein coupled receptors that are activated by neurohypophysial peptide hormones, including vasopressin (AVP). It has recently been reported that an arginyl in the distal N-terminus of the V 1a R is critical for binding agonists but not antagonists. To determine specific features required at this locus to support high affinity agonist binding and second messenger generation, Arg46 was substituted by all other 19 encoded amino acids. Our data establish that there is an absolute requirement for arginyl, as none of the [R46X]V 1a R mutant constructs supported high affinity agonist binding and all 19 had defective signalling. In contrast, all of the mutant receptors possessed wildtype binding for both peptide and nonpeptide antagonists. The ratio of K i to EC 50 , an indicator of efficacy, was increased for all substitutions. Consequently, although [R46X]V 1a R constructs have a lower affinity for agonist, once AVP has bound all 19 are more likely than the wildtype V 1a R to become activated. Therefore, in the wildtype V 1a R, Arg46 constrains the inactive conformation of the receptor. On binding AVP this constraint is alleviated, promoting the transition to active V 1a R. Our findings explain why arginyl is conserved at this locus throughout the evolutionary lineage of the neurohypophysial peptide hormone receptor family of G-protein coupled receptors. . The receptors that mediate the effects of these hormones are G-protein coupled receptors (GPCRs) and include the isotocin receptor, the mesotocin receptor, the oxytocin receptor (OTR), the vasotocin receptor and three subtypes of vasopressin receptor: V 1a , V 1b and V 2 (V 1a R, V 1b R and V 2 R respectively). In addition to the characteristic architecture of GPCRs [2], the neurohypophysial peptide hormone receptors exhibit certain conserved sequence motifs and share related pharmacologies (reviewed in [3][4][5][6]). This has allowed these receptors to be classified as a subfamily of GPCRs.The V 1a R mediates a plethora of responses to AVP in addition to the well-characterized vasopressor effect [3]. Consequently, this receptor subtype is widely distributed and generates nearly all of the physiological actions of AVP with the notable exceptions of antidiuresis (V 2 R) and adrenocorticotropic hormone secretion (V 1b R). This has been the stimulus for the development of a range of V 1a R antagonists, initially peptides [7] and more recently nonpeptides [8,9]. Although agonists and antagonists exhibit competitive binding to the V 1a R, only agonists promote the active receptor conformation with subsequent second messenger generation. Understanding how the agonistreceptor interaction differs from that of the antagonistreceptor and defining the mechanisms of receptor activation are fundamental issues in cel...

The N-Terminal Juxtamembrane Segment of the V1a Vasopressin Receptor Provides Two Independent Epitopes Required for High-Affinity Agonist Binding and Signaling

“…Furthermore, the structural requirements at both loci are very specific. The construct [E54D]V 1a R did not display wildtype pharmacology and, likewise, none of the 19 encoded amino acids could replace arginyl at position 46, including lysyl (23). These observations raised the possibility that Glu 54 and Arg 46 form a mutual ionic interaction, which is required for the V 1a R to adopt a highaffinity conformation for AVP.…”

“…This indicated that Glu 54 was one of several residues delimiting the ligand-binding cavity; therefore it is plausible that Glu 54 may interact directly with AVP. In contrast, Arg 46 is positioned where it can interact with other extracellular structures in the receptor, which apparently allows Arg 46 to constrain the ground state of the receptor and form part of the conformational switch controlling activation by agonist (23).…”

“…Although the ligand-binding pocket of ␤-adrenergic receptors and bRho is contained within the TM domain, the N-terminal domain of these receptors is not functionally inert. The crystal structure of bRho revealed that the N-terminal domain is actually very structured (1) and, furthermore, point mutations to Pro 23 and Gln 28 in this domain have been linked to the degenerative disease, autosomal dominant retinitis pigmentosa (4). Likewise, polymorphisms in the N terminus of the human ␤ 2 -adrenergic receptor at residue 16 (Gly or Arg) and residue 27 (Gln or Glu) have also been reported to affect receptor function.…”

Charged Extracellular Residues, Conserved throughout a G-protein-coupled Receptor Family, Are Required for Ligand Binding, Receptor Activation, and Cell-surface Expression

“…In particular, two charged residues (Arg 46 and Glu 54 ) in the V 1a R N terminus are required for high affinity agonist binding but not antagonist binding (14,15). Likewise, Arg 34 in the N terminus of the OTR is required for agonist binding (16).…”

Trapping of a Nonpeptide Ligand by the Extracellular Domains of the Gonadotropin-Releasing Hormone Receptor Results in Insurmountable Antagonism

“…6C) (Palczewski et al, 2000). In addition, interactions between the extracellular domains and bound ligands have been shown for the dopamine D 2 (Shi and Javitch, 2004) and the V 1a vasopressin (Hawtin et al, 2003) receptors. Davidson et al (1997) showed that the two disulfide bonds in the GnRHR (between TM3 and ECL2 and between ECL2 and the NH 2 terminus) introduce covalent constraints holding these regions together in the proximity of the transmembrane helical bundle.…”