To roles of EPH kinases in regulating catecholamine secretion in chromaffin cells

申请人是促红细胞生成素产生肝细胞激酶（EPHs）研究的先驱。这些研究代表了自2000年以来的一个主要的长期项目（由NSERC和其他来源资助）。这个长期项目的目标是在细胞和分子水平上阐明关于不同的EPHs及其配体ephrins （efn）如何调节血压（BP）和免疫系统的潜在机制。未来10年将培养3 - 6名博士研究生。******当前提案的短期目标是研究EPHB6与睾酮协同调节肾上腺嗜铬细胞（agcc）中儿茶酚胺（CAT）释放的机制。未来5年将培养2名博士研究生。******原理***EPH是最大的受体酪氨酸激酶家族。我们曾报道过雄性Ephb6基因敲除（KO）小鼠去势后血压升高。EPHB6至少针对2种组织进行血压调节：血管平滑肌细胞（VSMCs）和agcc。我们已经确定，在雄性EPHB6 KO小鼠的agcc中，CAT分泌减少。在分子水平上，男性KO agcc表现出Ca2+内流减少，这是由于大钾（BK）通道增加引起的。所有这些缺陷都需要睾丸激素的存在。其潜在机制将是本研究的重点。******我们假设：1)EPHB6和睾酮直接与BK通道相互作用，导致其提前打开，从而关闭电压门控钙通道（VGCCs）；2) EPHB6和睾酮共同调节Rho GTPase和/或MAPK活性，这在agcc的CAT分泌中是至关重要的。特定目标* * * * * * * * *我。目的探讨EPHB6和睾酮对BK通道电流的调节作用。研究在agcc中EPHB6是否直接与BK通道相互作用。探讨EPHB6是否影响睾酮与BK通道的相互作用。* * * * * *。目的探讨EPHB6和睾酮对agcc胞外分泌的调控作用。研究EPHB6对AGCC中Rho GTPase活性的影响。寻找睾酮和EPHB6信号通路交汇的地方。******意义***嗜铬细胞在内分泌和中枢神经系统中起关键作用。胞吐作用不仅是agcc的重要细胞过程，也是许多其他类型的细胞，如胰腺β细胞、细胞毒性T细胞和中性粒细胞的重要细胞过程。这项研究将大大提高我们对新发现的性激素依赖性胞吐调节因子EPHB6在胞吐中的认识，并将在内分泌、中枢神经和免疫系统中具有重要意义。

The applicant is a pioneer in research on erythropoietin-producing hepatocyte kinases (EPHs). These studies represent a major, long-term program since 2000 (with funding from NSERC and other sources). The objectives of this long-term program focus on elucidating underlying mechanisms at the cellular and molecular levels with regard to how different EPHs and their ligand ephrins (EFNs) modulate blood pressure (BP) and the immune system. Three to 6 Ph.D. students will be trained in the next 10 years in this program. ******The short-term objective of the current proposal is to investigate the mechanisms by which EPHB6 in concert with testosterone regulates catecholamine (CAT) release in adrenal gland chromaffin cells (AGCCs). Two Ph.D. students will be trained in the next 5 years.******Rationale***EPH are the largest family of receptor tyrosine kinases. We have reported that BP is elevated in male Ephb6 gene knockout (KO) mice after castration. EPHB6 targets at least 2 tissues for BP regulation: vascular smooth muscle cells (VSMCs) and AGCCs. We have determined that CAT secretion is reduced in the AGCCs of male EPHB6 KO mice. At the molecular level, male KO AGCCs show decreased Ca2+ influx caused by increased big potassium (BK) channels. All these defects need the presence of testosterone. The underlying mechanism will be the focus of this study. ******We hypothesize that: 1) EPHB6 and testosterone interact directly with BK channels, leading to their early opening and, hence, shut down of voltage-gated calcium channels (VGCCs); 2) EPHB6 and testosterone jointly regulate Rho GTPase and/or MAPK activity, which are critical in CAT exocytosis in AGCCs.******Specific aims***I. To investigate how EPHB6 and testosterone modulate BK channel currents***a. To study whether EPHB6 interacts directly with BK channels in AGCCs.***b. To assess whether EPHB6 affects the interaction between testosterone and BK channels. ******II. To investigate how EPHB6 and testosterone control exocytosis in AGCCs***a. To study how EPHB6 affects Rho GTPase activity in AGCC.***b. To find the link where testosterone and EPHB6 signalling pathways meet.******Significance***Chromaffin cells are critical in the endocrine and central nervous systems. Exocytosis is a crucial cellular process not only for AGCCs but also for many other types such as pancreatic beta-cell, cytotoxic T cells and neutrophils. This study will greatly enhance our knowledge of a newly found sex hormone-dependent exocytosis regulator, EPHB6, in exocytosis, and will have significant implications in the endocrine, central nervous and immune systems.