Arginine vasopressin (AVP) hormone has a broad range of effects on mammalian tissues and is a major hormone regulating water and solute reabsorption. AVP also regulates blood pressure, acts as a mitogenic agent, and regulates a variety of biochemical and physiological functions in the brain. These actions of AVP appear to involve two well-characterized receptors, V1 and V2, that have recently been cloned and shown to belong to a superfamily of receptors characterized by the presence of seven highly conserved transmembrane regions, and to be regulated by """"""""G-proteins"""""""". Although there is no direct evidence for additional AVP receptors, several studies support this possibility. The investigators have recently identified and cloned, from rabbit kidney, what appears to be a new AVP binding protein which mediates AVP-activated cytosolic free calcium (Ca2+) mobilization (VACM-1 protein). VACM-1 shows no homology with the two known AVP receptors, or any other protein sequence presently stored in the DATABASES, and thus may represent a novel family of G-protein linked receptors. Present work is directed at characterizing VACM-1. The proposed studies will further characterize VACM-1 and are intended to help to understand the molecular mechanism of AVP action. Specifically, they may explain """"""""anomalies"""""""" in AVP actions in tissues where the presence of either V1 and V2 receptors cannot fully explain the action of AVP. Ultimately, a better understanding of AVP action at the molecular level may help in the development of specific drugs for the treatment of water imbalance and regulation of blood pressure disorders. As a consequence, the present experimental plan proposes to: 1) determine the second messenger system that couples to VACM-1 and to determine which G- protein(s) are involved in this coupling that leads to increases in the cytosolic free calcium concentration; 2) use site-directed mutagenesis and antibody probes to elucidate the structure-function characteristics of VACM-1. Specifically, the investigators will determine the glycosylation sites that may be important in the activity of VACM-1 and identify domains involved in ligand binding and in the interaction with G-proteins; and 3) examine whether VACM-1 is expressed in certain tissues where it may be involved in specific AVP-regulated biochemical and physiological responses.
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