FUNCTION OF C-SRC AND RELATED KINASES IN CHROMAFFIN CELLS

肾上腺髓质嗜铬细胞是神经元分泌的模型，其响应于乙酰胆碱的刺激而释放肾上腺素和去甲肾上腺素。与神经元一样，c-Src及其几个家族成员Fyn和c-Yes在嗜铬细胞中以高水平表达，表明这些激酶在嗜铬细胞功能（胞吐）中发挥重要作用。我们研究的目的是研究Src家族激酶，特别是c-Src，在通过神经元烟碱乙酰胆碱受体（nAChR）调节嗜铬细胞分泌中的作用，nAChR是一种启动分泌反应的配体门控阳离子通道。 我们已经证明，c-Src调节分泌过程中或直接下游的受体的水平，是物理上与受体和一个身份不明的蛋白酪氨酸磷酸酶（PTP 3），协会似乎采取的形式的三方复合物。这些发现表明c-Src通过与受体结合并可能使其磷酸化和/或通过将PTB转运至受体来调节受体。为了阐明这三种分子之间相互作用的性质并确定相互作用是否影响受体的活性和β ′或下游分泌事件，提出了三个目的。 第一个是采用电生理手段来确定c-Src和/或其相关的PTK对受体功能的影响。我们建议采取的方法是测试Src家族激酶和PTPases的药理学抑制剂以及c-Src或其相关PTPases的结构变体对受体通道活性的各种参数的影响。还将评估受体磷酸化位点突变对其自身功能的影响。为了便于该分析，稳定表达功能性nAChR（α 3 α 5 [β 4]2）（并称为AChR 293）的HEK 293细胞系由J. Lindstrom从U.宾夕法尼亚 AChR 293细胞将用编码激酶和PTK的推定显性负性形式的表达质粒瞬时转染，并进行电生理学分析。 这些研究将证实在嗜铬细胞的内源性嗜铬细胞受体的电生理分析，结合安培测量分泌活性。第二个目标是利用GST融合蛋白（体外）和转染的AChR 293细胞（体内），以确定这三种分子的哪些结构域或亚基是它们彼此缔合所必需的，以研究结合是直接的还是间接的、二分的还是三分的，并确定受体在配体结合后是否被酪氨酸磷酸化/去磷酸化，以及如果是，在哪个位点和通过哪个激酶/PTB。这些研究的结果应该揭示了一些重要的复杂的结构特征，并提供洞察到潜在的机制，调节受体的c-Src和其相关的PTlets。 最后，纯化和鉴定的c-Src相关的PTPases将进行，利用传统的色谱和免疫沉淀技术，在凝胶PTPases测定，和抗体已知的PTPases。 一旦确定，将获得或生成免疫学和遗传探针，并用于上述研究。这些研究旨在确定Src家族和PTPases的酪氨酸激酶是否调节nAChR，如果是，这种调节如何影响分泌过程。

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.