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

申请者是研究产生促红细胞生成素的肝细胞激酶(EPHs)的先驱。这些研究代表了自2000年以来的一项重大的长期计划(由NSERC和其他来源提供资金)。这一长期计划的目标是在细胞和分子水平上阐明不同的EPH及其配体肾上腺素(EFN)如何调节血压(BP)和免疫系统的潜在机制。该项目将在未来10年内培养3至6名博士生。*本提案的短期目标是研究EPHB6与睾酮共同调节肾上腺嗜铬细胞(AGCC)中儿茶酚胺(CAT)释放的机制。两名博士生将在未来5年内接受培训。EPH是最大的受体酪氨酸激酶家族。我们已经报道了雄性Ephb6基因敲除(KO)小鼠去势后血压升高。EPHB6至少针对两个组织进行血压调节：血管平滑肌细胞(VSMCs)和AGCCs。我们已经确定在雄性EPHB6KO小鼠的AGCC中CAT的分泌减少。在分子水平上，雄性KO AGCC表现出由于大钾(BK)通道增加而导致的钙内流减少。所有这些缺陷都需要睾丸激素的存在。潜在的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，导致电压门控钙通道(VGCC)的早期开放，从而关闭VGCC；2)EPHB6和睾酮共同调节Rho GTP酶和/或MAPK活性，这在AGCC的CAT胞吐中起关键作用。*具体目的**i.研究EPHB6和睾酮如何调制BK通道电流*a.研究EPHB6是否与电压门控钙通道(VGCC)直接相互作用。*b.评估EPHB6是否影响睾酮和BK通道之间的相互作用。*II.研究EPHB6和睾酮如何控制AGCC的胞吐作用*a.研究EPHB6如何影响AGCC中Rho GTPase的活性。*b.找到睾酮和EPHB6信号通路相遇的联系。*意义**嗜铬细胞在内分泌和中枢神经系统中至关重要。胞吐作用不仅对AGCC，而且对许多其他类型的细胞，如胰岛β细胞、细胞毒性T细胞和中性粒细胞，都是一个至关重要的细胞过程。这项研究将极大地提高我们对新近发现的性激素依赖性胞吐调节因子EPHB6在胞吐作用中的认识，并将对内分泌、中枢神经系统和免疫系统产生重大影响。

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.