Function of GFL-Dependent & Independent RET Activation

超出提供的空间。神经营养因子支持中枢和外周神经元的存活、生长和突触。神经营养因子作用的高度特异性和保真度部分是由于它们激活了高亲和力的细胞表面受体。神经营养因子是以神经生长因子(NGF)为代表的第一个神经营养因子家族，通过激活其受体酪氨酸激酶(RTK)Trks发挥作用。第二个营养因子家族，胶质细胞源性神经营养因子(GDNF)家族配体(GFL)，通过激活它们的RTK，Ret来发挥作用。我们最近发现了一种RTK间信号机制，通过激活NGF受体TrkA，在没有GFLS的情况下，在体外和体内都会导致成熟依赖的激活Ret。NGF介导的Ret激活增强了成熟的交感神经元的营养状态，但不能增加未成熟的交感神经元的营养状态。为了确定NGF促进Ret激活的机制，利用体外培养的成熟交感神经元，提出了系统检验最可能的假说的生化实验。确定NGF促进Ret激活的机制对于形成一个长期的项目至关重要，该项目旨在揭示哪些受体参与RTK间信号转导，以及这一过程具有哪些发育功能。为了在体内鉴定GFL依赖和GFL非依赖(即NGF依赖)的Ret功能，将产生全部Ret功能缺陷或仅GFL依赖Ret功能缺陷的转基因动物。对这些动物的中枢和周围神经系统的详细检查将首次揭示Ret的出生后功能，因为Ret缺乏的动物在围产期具有致命性，无法检查出生后的发育。作为首席研究员，我将学习一套新的重要实验技能，如在解剖、生理和行为水平上生产和分析转基因动物，以及生产用于在初级神经元中表达外源蛋白的慢病毒载体。这些实验的结果，以及我将要学习的技术，将为我追求我的职业目标提供坚实的基础，以学术实验室的首席研究员的身份建立一个独立的研究计划。表演网站========================================Section End===========================================

EXCEED THE SPACE PROVIDED. Neurotrophlc tactors support the survival, growth, and d]tterentxation of both central and peripheral neurons. The great specificity and fidelity of the effects ofneurotrophic factors are due in part to their activation of high affinity cell surface receptors. The neurotrophins, the first identified family of neurotrophic factors exemplified by the prototypical member nerve growth factor (NGF), function via activation of their receptor tyrosine kinases (RTKs), the Trks. A second family oftrophic factors, the glial cell-line derived nenrotrophic factor (GDNF) family ligands (GFLs), function via activation of their RTK, Ret. We have recently identified an inter-RTK signaling mechanism by which activation of the NGF receptor, TrkA, leads both in vitro and in vivo to the maturation-dependent activation Ret in the absence of GFLs. NGF-mediated Ret activation augments the trophic status of mature, but not immature, sympathetic neurons. In order to identify the mechanism by which NGF promotes Ret activation, biochemical experiments designed to systematically test the most likely hypotheses are proposed, making use of mature sympathetic neurons maintained in vitro. The identification of the mechanism by which NGF promotes Ret activation is critically important for the formation of a long-term project to reveal which receptors participate in inter-RTK signaling, and what developmental functions this process has. In order to identify both the GFL-dependent and GFL-independent (i.e. NGF-dependent) Ret functions in vivo transgenie animals will be produced that are deficient in either all Ret functions, or in only GFL-dependent Ret functions. A detailed examination of the central and peripheral nervous systems of these animals will reveal, for the first time, the postnatal functions of Ret, given the perinatal lethality of Ret deficient animals that has not allowed examination of postnatal development. As the principle investigator I will leam a new repertoire of important experimental skills, such as the production and analysis oftransgenic animals at the anatomic, physiologic, and behavioral levels, as well as the production of lentiviral vectors for the expression of foreign proteins in primary neurons. The results from these proposed experiments, as well as the techniques I will learn, will provide a solid basis for me to pursue my career objective to establish an independent research program as the principle investigator of an academic laboratory. PERFORMANCE SITE ========================================Section End===========================================