FUNCTION OF C-SRC AND RELATED KINASES IN CHROMAFFIN CELLS

肾上腺髓质染色质细胞是神经元分泌的模型，在乙酰胆碱刺激下释放肾上腺素和去甲肾上腺素。与神经元一样，c-Src及其家族成员Fyn和c-Yes在染色质细胞中高水平表达，这表明这些激酶在染色质细胞功能（胞吐）中发挥重要作用。我们的研究目的是研究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是一种配体门控的阳离子通道，可启动分泌反应。我们已经证明，c-Src调节受体水平或直接下游的分泌过程，并与受体和未识别的蛋白酪氨酸磷酸酶（PTPase）物理关联，这种关联似乎以三方复合物的形式存在。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PTPase转运到受体上来调节受体。为了阐明这三种分子之间相互作用的性质，并确定这种相互作用是否影响受体的活性或下游分泌事件，提出了三个目标。第一种是采用电生理手段确定c-Src和/或其相关PTPase对受体功能的影响。我们建议采用的方法是测试Src家族激酶和PTPase的药理学抑制剂以及c-Src或其相关PTPase的结构变体对受体通道活性的各种参数的影响。受体磷酸化位点突变对其自身功能的影响也将被评估。为了便于分析，宾夕法尼亚大学的J. Lindstrom提供了一种稳定表达功能性nAChR （alpha3alpha5[beta4]2）的HEK293细胞系（命名为AChR293）。AChR293细胞将瞬时转染编码推定的激酶和PTPase显性阴性形式的表达质粒，并进行电生理分析。这些研究将通过内源性染色质细胞受体的电生理分析，结合分泌活性的安培测量，在染色质细胞中得到证实。第二个目标将利用GST融合蛋白（体外）和转染的AChR293细胞（体内）来确定这三个分子的哪些结构域或亚基是它们相互结合所必需的，研究结合是直接的还是间接的，是双向的还是三方的，并确定受体在配体结合时是否被酪氨酸磷酸化/去磷酸化，如果是，在哪个位点，通过哪个激酶/PTPase。这些研究的结果将揭示该复合物的许多重要结构特征，并为c-Src及其相关PTPase调节受体的潜在机制提供见解。最后，c-Src相关PTPase的纯化和鉴定将进行，利用传统的色谱和免疫沉淀技术，在凝胶PTPase测定和抗体已知的PTPase。一旦确定，免疫和遗传探针将获得或生成并用于上述研究。这些研究旨在确定Src家族的酪氨酸激酶和PTPases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.