Synthetic Rat V1a Vasopressin Receptor Fragments Interfere with Vasopressin Binding via Specific Interaction with the Receptor

Abstract

To study the vasopressin receptor domains involved in the hormonal binding, we synthesized natural and modified fragments of V 1a vasopressin receptor and tested their abilities to affect hormone-receptor interactions. Natural fragments mimicking the external loops one, two, and three were able to inhibit specific vasopressin binding to V 1a receptor. In contrast, the natural N-terminal part of the V 1a vasopressin receptor was found inactive. One fragment, derived from the external second loop and containing an additional C-terminal cysteine amide, was able to fully inhibit the specific binding of both labeled vasopressin agonist and antagonist to rat liver V 1a vasopressin receptor and the vasopressin-sensitive phospholipase C of WRK1 cells. The peptide-mediated inhibition involved specific interactions between the V 1a receptor and synthetic V 1a vasopressin receptor fragment since 1) it was dependent upon the vasopressin receptor subtype tested (K i(app) for the peptide: 3.7, 14.6, and 64.5 M for displacing [ 3 H]vasopressin from rat V 1a , V 1b , and V 2 receptors, respectively; 2) it was specific and did not affect sarcosin 1-angiotensin II binding to rat liver membranes; 3) it was not mimicked by vasopressin receptor unrelated peptides exhibiting putative detergent properties; and 4) no direct interaction between [ 3 H]vasopressin and synthetic peptide linked to an affinity chromatography column could be observed. Such an inhibition affected both the maximal binding capacity of the V 1a vasopressin receptor and its affinity for the labeled hormone, depending upon the dose of synthetic peptide used and was partially irreversible. Structure-activity studies using a serie of synthetic fragments revealed the importance of their size and cysteinyl composition. These data indicate that some peptides mimicking extracellular loops of the V 1a vasopressin receptor may interact with the vasopressin receptor itself and modify its coupling with phospholipase C.Vasopressin (AVP), 1 a small polypeptidic neurohypophysial hormone, exerts different biological effects in mammals. At the periphery, its major physiological role is played in regulating water and solute excretion by the kidney. This hormone is also involved in blood pressure control, platelet aggregation, corticotropin and aldosterone secretion (by the adenohypophysis and the adrenals, respectively), hepatic glycogenolysis, and uterine motility (for review, see Refs. 1 and 2). In the central nervous system, AVP is also involved in interneuronal communication (3). These distinct biological functions are mediated, in mammals, by at least three distinct receptor subtypes: V 2 , V 1a , and V 1b . V 2 receptors, involved in the antidiuretic response, are positively coupled to adenylyl cyclase (4). V 1a and V 1b receptors, involved in multiple peripheral responses and in corticotropin release, stimulate phospholipase C and activate protein kinase C (5, 6). Cloning the different subtypes of receptors (7-9) confirmed that these peptidic receptors belong to the family ...

Active Peptidic Mimics of the Second Intracellular Loop of the V1AVasopressin Receptor Are Structurally Related to the Second Intracellular Rhodopsin Loop: A Combined1H NMR and Biochemical Study

“…Maximal inhibitions observed at 10 µM were 62 ( 2 and 50 ( 4% for i2 cyc and i2 lin, respectively. This micromolar concentration range is consistent with values obtained with peptide segments derived from distinct receptors such as rhodopsin, R2A adrenergic, D1 dopamine, 5-HT-1A, V 1a vasopressin, or β2 adrenergic receptors (24)(25)(26)(27)(28)(29). In contrast, used at the same concentration, these peptides were not able to inhibit [ 125 I]OH-LVA specific binding (Figure 3A).…”

Vasopressin Receptors, the Signalling Cascade and Mechanisms of Action

“…A twelve amino acid synthetic peptide from the sequence of the first extracellular loop of the V1a receptor inhibited both the binding of agonist and antagonist radioligands to the receptor and the activation of glycogen phosphorylase in isolated hepatocytes (Howl and Wheatley, 1996). Another synthetic peptide, 25 amino-acids long, analogous to the second extra-cellular loop of the V1a receptor, also inhibited ligand binding and production of second messenger (Mendre et al, 1997). These results may indicate that the extracellular part of the receptor facilitates the initial capture of the ligand, whether agonist or antagonist .…”