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

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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.