Vasopressin (AVP) modulates body fluid osmolality, blood volume, blood pressure, vascular tone, and cell proliferation via its potent antidiuretic, vasoconstrictor, and mitogenic actions which may contribute to the pathogenesis of arterial hypertension, heart failure, and atherosclerosis. The development of AVP antagonists to be used in human diseases has been hampered by the existence of an unexpected species-related heterogeneity despite the high degree of homology between the amino acid sequences of AVP receptors from different species. The applicant has cloned, sequenced, and expressed a cDNA encoding of the human Vla AVP receptor (VlaR) whose amino acid sequence and hydropathy profile suggest that four categories of key amino acids and discrete regions within the human VlaR structure determine ligand binding characteristics and signal transduction specificity: the first category is shared by all members of 7-TMS receptor superfamily, the second category is common to the AVP/Oxytocin family of receptors, the third category is specific to the VlaR subtype across species, and the fourth category is unique to the human VlaR. It is hypothesized that interspecies differences in AVP antagonist binding properties are related to key amino acids and unique regions of AVP receptors. The establishment of the functional map of the human VlaR represents the first logical step in the rational development of specific human VlaR antagonists. The goal of the present project is to construct the functional map of the distinct molecular determinants of the human VlaR sequence involved in: 1. The formation of the ligand binding pocket specific for AVP, 2. Specific peptide agonist and antagonist binding of the VlaR subtype, 3. Specific nonpeptide antagonist binding of the VlaR subtype. These studies should provide important clues on the human VlaR epitopes involved in the aforementioned processes, help unravel the 3-D structure of the receptor, and eventually lead to the development of specific and potent compounds targeted at the ligand binding pocket of the human VlaR. These studies will be done by carrying out targeted point mutation, deletion, and chimeric alterations of the human VlaR sequence, overexpression of the cloned receptor, and induction of antibodies directed against specific regions of the VlaR sequence. Such studies should help reach the long term goals: 1. To decipher the complete molecular structure of the human VlaR in an effort to obtain a global scope of its structure and functions. 2. To help develop orally active specific and potent AVP analogs to be used in human diseases.
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