Abstract
To study the vasopressin receptor domains involved in the hormonal binding, we synthesized natural and modified fragments of V1a 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 V1a receptor. In contrast, the natural N-terminal part of the V1a 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 V1a vasopressin receptor and the vasopressin-sensitive phospholipase C of WRK1 cells. The peptide-mediated inhibition involved specific interactions between the V1a receptor and synthetic V1a vasopressin receptor fragment since 1) it was dependent upon the vasopressin receptor subtype tested (Ki(app) for the peptide: 3.7, 14.6, and 64.5 microM for displacing [3H]vasopressin from rat V1a, V1b, and V2 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 [3H]vasopressin and synthetic peptide linked to an affinity chromatography column could be observed. Such an inhibition affected both the maximal binding capacity of the V1a 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 V1a vasopressin receptor may interact with the vasopressin receptor itself and modify its coupling with phospholipase C.
Highlights
Hormone, exerts different biological effects in mammals
Three-dimensional computer modeling of rat V1a vasopressin receptor structure, combined with directed sitemutagenesis experiments, has indicated that the transmembrane domains of the receptor are involved in the vasopressin binding site (13)
Vasopressin receptors belong to the G-protein-coupled receptor family exhibiting an N-terminal part, seven transmembrane domains connected by three extracellular loops and three intracellular loops, and possessing a C-terminal intracellular tail (38)
Summary
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. The precise location of vasopressin binding to the three receptor isoforms remains incompletely characterized Another approach to the study of hormone-receptor interactions involves the use of small synthetic peptides mimicking the sequence of the supposed active region of the receptor. This approach allows the use of pure and well characterized fragments, available in large amounts, to determine their possible interaction with the specific ligand or with the receptor itself. This approach has been successfully used in the case of the thyrotropin-stimulating hormone receptor and luteotropin human choriogonadotropin receptor, where synthetic peptides mimicking the N-terminal extracellular sequence of the receptor were able to bind the hormone (14, 15).
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