Adrenomedullary chromaffin cells are models of neuronal secretion, releasing adrenalin and noradrenalin in response to stimulation by acetylcholine. As with neurons, c-Src and several of its family members, Fyn and c-Yes, are expressed at high levels in chromaffin cells, suggesting that these kinases play important roles in chromaffin cell function (exocytosis). The goal of our studies is to investigate the role of Src family kinases, In particular c-Src, in regulating secretion from chromaffin cells through the neuronal nicotinic acetylcholine receptor (nAChR), a ligand- gated cation channel that initiates the secretory response. We have demonstrated that c-Src modulates the secretory process at or immediately downstream of the level of the receptor and is physically associated with the receptor and an unidentified protein tyrosine phosphatase (PTPase), associations that appear to take the form of a tri-partite complex. These findings suggest that c-Src regulates the receptor by binding to it and perhaps phosphorylating it and/or by transporting the PTPase to it. To elucidate the nature of the interactions between these three molecules and to determine If the interactions Influence either the activity of the receptor and)'or downstream secretory events, three aims are proposed. The first is to employ electrophysiological means to determine what effects c-Src and/or its associated PTPase have on receptor function. The approach we propose to take is to test the effect of pharmacological inhibitors of Src family kinases and PTPases as well as structural variants of c-Src or its associated PTPase on the various parameters of receptor channel activity. The effect of phosphorylation site mutations of the receptor on its own function will also be assessed. To facilitate this analysis, an HEK293 cell line that stably expresses a functional nAChR (alpha3alpha5[beta4]2) (and termed AChR293) was kindly provided by J. Lindstrom from U. Pennsylvania. AChR293 cells will be transiently transfected with expression plasmids encoding putative dominant negative forms of the kinase and PTPase and subjected to electrophysiological analysis. These studies will be corroborated in chromaffin cells by electrophysiological analysis of the endogenous chromaffin cell receptor, combined with amperometric measurements of secretory activity.
The second aim will utilize GST fusion proteins (in vitro) and transfected AChR293 cells (in vivo) to determine which domains or subunits of the three molecules are required for their association with one another, to investigate whether binding is direct or indirect, bi-partite or tri-partite, and to determine if the receptor is tyrosine phosphorylated/dephosphorylated upon ligand binding, and if so at which site(s) and by which kinase/PTPase. The results of these studies should reveal a number of important structural features of the complex and lend insights into potential mechanisms of regulation of the receptor by both c-Src and its associated PTPase. Finally, the purification and identification of c-Src- associated PTPase will be undertaken, utilizing conventional chromatographic and immunoprecipitation techniques, an in gel PTPase assay, and antibodies to known PTPases. Once identified, immunological and genetic probes will be obtained or generated and used in studies outlined above. Together these studies are designed to determine whether tyrosine kinases of the Src family and PTPases regulate the nAChR and if so, how such regulation affects the secretory process.
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