Defining the ligand-binding site for vasopressin receptors: a peptide mimetic approach.

Introduction The diverse functions of the neurohypophysial peptide hormones [argininex]vasopressin (AVP) and oxytocin (OT) are mediated by a family of receptor proteins expressed in both the central nervous system and peripheral tissues [1,2]. cDNA clones encoding five vasopressin receptors (VPRs) and two oxytocin receptors (OTRs) have now been isolated from rodent, porcine and human sources [3-91. These proteins are members of the larger family of G-protein-coupled receptors (GPCRs), which have seven putative transmembrane domains. Sequence comparison of the neurohypophysial peptide hormone receptors reveals two highly conserved domains in the putative first (ECII) and second (ECIII) extracellular (EC) loops which we [9] and others [lo-121 have hypothesized to play a role in ligand binding. To test this hypothesis, we synthesized peptide mimics of the extracellular domains of neurohypophysial peptide hormone receptors. The rationale employed in this investigation was to determine whether molecular recognition occurs between isolated receptor domains and peptidelnon-peptide ligands. Using this approach, we have identified binding-site determinants in the extracellular domains of the rat V,, VPR (rV ,,R) which selectively ‘recognize’ neurohypophysial hormones. We propose that these domains are closely associated in the native receptor protein to form an extracellular ligandbinding site.