The neurohypohyseal hormone, vasopressin, plays a major role in the regulation of salt and water balance through its action on the mammalian nephron. Important to our understanding of these processes has been our development over the last decade of cell culture models for vasopressin action, particularly the renal epithelial cell, LLC-PK1. Our previous work with this cell line has resulted in: a) the development of a """"""""cyclic-dissociation"""""""" model for vasopressin signal transduction that predicts the interactions between guanine nucleotide-regulatory (G) proteins involved in the stimulation (Gs) and inhibition (Gi) of adenylyl cyclase, and b) the demonstration of the inhibition of Na+ transport by pertussis toxin, presumably mediated by the ADP- ribosylation of a member of the Gi family of proteins. These G- proteins are heterotrimers composed of alpha beta gamma subunits with differences in the 'alpha' subunit providing for their functional identity. Our goals are to further probe G-protein function as it relates to vasopressin action. We will utilize a) developing LLC-PK1 cells that demonstrate an induction of Gi proteins during growth, b) a stable LLC-PK1 cell line transfected with cDNAs for one or more 'alpha 1' proteins and abundantly expressing these proteins, and c) the amphibian cell line, A6.
Our specific aims are: 1) to address the predictions of the """"""""cyclic- dissociation"""""""" model for adenylyl cyclase dealing with the nature of Gi and Gs interactions with the vasopressin receptor, the role of alpha 1 in the inhibition of adenylyl cyclase, and the mechanism by which C1- and Mg++ generate """"""""active"""""""" states of Gs. These studies will be performed by utilizing real-time fluorescent vasopressin binding, biochemical characterization of G-protein activity states and immunocytochemistry for the localization and quantitation of Gi proteins; 2) define the role of pertussis toxin- sensitive G-proteins and their interaction with protein kinase C in the regulation of the amiloride-sensitive Na+ channel. The electrophysiological characteristics of Na+ transport will be determined by the patch clamp technique. The identity of G- proteins involved in Na+ transport will be determined by immunolocalization of antibodies specific for alpha 1 proteins. Although these studies will focus on G-proteins and vasopressin action, it has become clear that G-proteins are """"""""universal transducers"""""""" of a wide variety of biological processes. A better understanding of G-protein function will provide insights that can be broadly applied to signal transduction and membrane transport.
Showing the most recent 10 out of 27 publications
