The quanine-nucleotide binding regulatory proteins (G-proteins) are alpha1beta1gamma1 heterotrimers which function as transmembrane signal transducers by coupling receptors for extracellular stimuli to intracellular effectors (enzymes, ion channels). G-proteins constitute a diverse family distinguished by specific receptor and effector interactions which in turn are determined by the structure of the three constituent subunits. The alpha subunit binds quanine nucleotides and has a well established role in effector modulation. The beta and gamma subunits are tightly associated as a beta gamma complex, comprising a single functional entity which, like the alpha subunit, is absolutely required for G-protein interaction with receptor. An effector modulatory role for the beta gamma complex is becoming increasingly apparent in several systems. The present research emphasizes the role of the beta gamma complex in G-protein-mediated signal transduction. We have used subunit specific peptide antibodies to probe regions of the beta gamma complex important for functional interaction with the alpha subunit and to monitor expression of recombinant subunits. Site directed mutagenesis has been used to study the assembly, processing and effector function of the beta gamma complex in both transient and stable transfected cell systems. These studies may elucidate the contribution of the beta gamma subunit complex to the receptor and effector selectivity characteristic of G-proteins and to the adaptive responses pursuant to agonist stimulation. GRANTS=Z01DK59005 Nephrogenic diabetes insipidus (NDI) is an inherited X-lined disorder in which affected subjects are resistant ot the actions of vasopressin (AVP) on renal medullary cells responsible for water concentration. Clinical manifestations include severe polydipsia and polyuria, and resultant severe dehydration can lead to cerebral swelling and death. Treatment with a potent AVP analog (DDAVP, useful in other forms of DI, is in-effective in NDI because of end-organ resistance to tahe hormone. The renal actions of AVP are mediated through a V2 type receptor linked via the Gs protein to stimulation of the 2nd messenger CAMP. In theory, the inherited gene defect could be located anywhere along the signal transduction path, but indirect evidence suggested a likely receptor defect. The recent cloning of a human V2 receptor permitted chromosomal localization studies which showed that the receptor is localized to Xq28, the site of the gene defect as determined by family linkage studies. This strongly suggested but did not prove that a receptor gene mutation is the underlying defect in NDI. We have obtained genomic DNA samples on multiple families with NDI, and in three families thus far have identified mutations predicted to disrupt formation of a normal V2 receptor. These findings have important implications for our under- standing of the pathogenesis of NDI and of normal V2 receptor structure and function, for identification of affected subjects and carriers, and eventually for gene therapy of the disease.
