ProTRH sorting to the regulated secretory pathway

描述(由申请人提供)： 了解神经肽原激素的加工过程，可以导致发现具有生物功能的新多肽，以及这些产物在细胞外传递的方式，对于这些分子在动物生理学中扮演的与沟通和神经内分泌功能有关的基础角色至关重要。促甲状腺激素释放激素(ProTRH)是TRH和其他非TRH多肽的蛋白质前体。TRH产生于下丘脑室旁核，刺激垂体前叶促甲状腺激素的生物合成和分泌。TSH反过来刺激甲状腺激素的生物合成和释放。TRH是调节下丘脑-垂体甲状腺(HPT)轴的中枢。TRH还通过HPT轴增加细胞的基本代谢率，在维持能量平衡和冷应激过程中发挥关键作用。因此，在这项提议中，我们将检验这样的假设，即TRH前激素到受调节的分泌途径(RSP)的分选涉及由前激素转换酶1(PC1)在高尔基复合体中的一个初始翻译后处理步骤，随后是每个加工中间产品的两条不同的分选路径。然后，这些中间形式通过两个潜在的序列基序被定向到RSP：N-末端中间体中的两个RGD(Arg-Gly-Asp)和在C-末端中间体形成的潜在二硫键结构。因此，我们将瞬时转染AtT-20细胞，并通过与神经元GT-1细胞系的比较来证明以下假设：目的1.ProTRH向RSP的分选需要初始的PC活性，该活性受proTRH的二级结构控制。目的2.N端和C端中间产物proTRH的分选分别由RGD基序和一个可能的二硫键基序指导；3.ProTRH N-端和C-端片段的分选分别通过两条不同的途径进行。目的4.ProTRH的三级结构有助于其被PC1和PC2处理并进一步分选到RSP。

DESCRIPTION (provided by applicant): Understanding pro-neuropeptide hormone processing, which can lead to the discovery of novel peptides with biologic function, and the way these products are delivered outside the cell is paramount to the fundamental role that these molecules play in animal physiology that relates to communication, and neuroendocrine function. ProThyrotropin Releasing Hormone (proTRH) is the protein precursor to TRH and other non-TRH peptides. TRH, produced in the paraventricular nucleus of the hypothalamus PVN, stimulates the biosynthesis and secretion of thyroid-stimulating hormone from the anterior pituitary. TSH, in turn, stimulates thyroid hormone biosynthesis and release. TRH is central in regulating the hypothalamic-pituitary thyroid (HPT) axis. TRH also plays a key role in maintaining energy balance and during cold stress by increasing the basic metabolic rate of the cells through the HPT axis. Therefore, in this proposal we will test the hypothesis that the sorting of the TRH prohormone to the regulated secretory pathway (RSP) involves one initial post-translational processing step in the Golgi complex by the prohormone convertase 1 (PC1), followed by two different pathways of sorting for each intermediate product of processing. These intermediate forms are then directed to the RSP by two potential sequence motifs: two RGDs (Arg-Gly-Asp) in the N-terminal intermediate and a potential disulfide bond structure formed at the C-terminal intermediate. Therefore, we will transiently transfect AtT-20 cells, and by comparison with the neuronal GT-1 cell line to prove the following hypotheses: Aim 1. The sorting of proTRH to the RSP requires initial PC activity that is governed by the secondary structure of proTRH. Aim 2. The sorting of the N- and C-terminal intermediate proTRH forms are directed by the RGD and a possible disulfide motifs respectively Aim 3. The sorting of the proTRH N- and C-terminal fragments is routed to two different pathways. Aim 4. The tertiary structure of proTRH contributes to its processing by PC1 and PC2 and further sorting to the RSP.