Growth Hormone Regulation of SH2B1

描述（由申请人提供）：最近令人兴奋的遗传学研究表明支架蛋白SH2B1与人类疾病有关。人类SH2B1基因突变与严重的早发性肥胖、不成比例的严重胰岛素抵抗、大脑发育受损以及在某些情况下身材矮小有关。这种表型（与SH2B1-/-小鼠非常相似）与SH2B1是多种配体的信号分子一致，包括生长激素（GH）、瘦素和胰岛素。SH2B1被JAK2募集，是GH调控肌动蛋白细胞骨架的关键成分，是细胞增殖、分化和迁移的先决条件。然而，SH2B1促进gh诱导的肌动蛋白细胞骨架变化的机制在很大程度上仍然未知。长期目标是了解SH2B1促进GH和其他配体功能的机制，并深入了解GH未被识别的细胞作用；SH2B1的新功能；以及诸如SH2B1之类的支架蛋白如何将高度调控的动态复合物从膜结合受体移动到细胞中的多个（通常是远程的）位点（例如，局灶粘连、板足、细胞质、细胞核）。当前提案的目的是了解SH2B1如何调节“信号复合物”的组装和运动，以及这些功能的失调是否会导致细胞功能缺陷，最终导致人类肥胖、胰岛素抵抗、神经系统疾病和生长抑制。核心假设是SH2B1促进SH2B1“信号复合物”的形成和/或移动到细胞中的特定位置（例如，pm，板足，细胞质，细胞核）以响应GH。SH2B1的这些功能增强了GH对肌动蛋白细胞骨架、GH诱导的运动和GH在细胞核中的反应的调节。它们对正常的人体生理至关重要，因此由于突变导致的损伤有助于在人类患者中观察到的戏剧性表型。在强有力的初步数据的指导下，我们将通过三个具体目标来验证这一假设：1)阐明SH2B1可逆定位于质膜、细胞质溶胶和细胞核的机制。2)确定SH2B1在调节gh诱导的肌动蛋白细胞骨架变化、细胞运动和细胞间蛋白运输中的作用。3)确定与人类疾病相关的SH2B1基因突变的功能后果。实验将使用生化，免疫学，细胞生物学和成像技术在实验室标准的组合。还将使用最先进的显微镜、光激活探针、蛋白质组学、细胞系以及来自SH2B1-/-小鼠的原代巨噬细胞和mef。SH2B1的核定位信号是一个双重功能基元，介导核输入和质膜结合，并受附近丝氨酸磷酸化调节，这一概念是创新的。这项研究具有重要意义，因为它将深入了解SH2B1在GH和许多其他配体（如瘦素、胰岛素、神经生长因子）功能中的作用，这些配体利用SH2B1作为信号蛋白。

DESCRIPTION (provided by applicant): Exciting recent genetic studies implicate the scaffold protein SH2B1 in human disease. Human mutations in SH2B1 are associated with severe early onset obesity with disproportionately severe insulin resistance, impaired brain development, and, in some cases, short stature. This phenotype (strikingly similar to that of SH2B1-/- mice) is consistent with SH2B1 being a signaling molecule for multiple ligands, including growth hormone (GH), leptin, and insulin. SH2B1 is recruited to JAK2 and is a critical component in GH regulation of the actin cytoskeleton, a prerequisite for cellular proliferation, differentiation and migration. However, the mechanism by which SH2B1 facilitates GH-induced changes in the actin cytoskeleton remains largely unknown. The long-term goal is to understand the mechanism by which SH2B1 contributes to the function of GH and other ligands, and to gain insight into unrecognized cellular actions of GH; new functions of SH2B1; and how scaffold proteins such as SH2B1 move highly regulated and dynamic complexes from membrane-bound receptors to multiple, often remote, sites in the cell (e.g., focal adhesions, lamellipodia, cytoplasm, nucleus). The objective of the current proposal is to understand how SH2B1 regulates assembly and movement of "signaling complexes" and whether dysregulation of these functions contributes to defects in cellular function and ultimately human obesity, insulin-resistance, neurologic disorders, and suppressed growth. The central hypothesis is that SH2B1 facilitates the formation and/or movement of SH2B1 "signaling complexes" to specific locations in the cell (e.g., pm, lamellipodia, cytoplasm, nucleus) in response to GH. These functions of SH2B1 enhance GH regulation of the actin cytoskeleton, GH-induced motility, and GH responses in the nucleus. They are critical to normal human physiology, such that their impairment due to mutation contributes to the dramatic phenotype observed in human patients. Guided by strong preliminary data, this hypothesis will be tested by pursuing three specific aims: 1) Elucidate the mechanism by which SH2B1 reversibly localizes to the plasma membrane, cytosol and nucleus. 2) Determine the role of SH2B1 in regulating GH-induced changes in the actin cytoskeleton, cell motility, and trafficking of proteins between intracellular compartments. 3) Determine the functional consequences of mutations in SH2B1 associated with human disease. Experiments will use a combination of biochemical, immunologic, cell biology, and imaging techniques standard in the lab. State-of-the-art microscopes, photoactivable probes, proteomics, cell lines and both primary macrophages and MEFs from SH2B1-/- mice, will also be used. The concept that the nuclear localization signal of SH2B1 is a dual function motif that mediates both nuclear import and plasma membrane binding that is regulated by phosphorylation of nearby serines is innovative. The proposed research is significant because it will provide insight into the role of SH2B1 in the function of GH and the numerous other ligands (e.g., leptin, insulin, nerve growth factor) that utilize SH2B1 as a signaling protein. PUBLIC HEALTH RELEVANCE: These studies will provide insight into the role of a multifunctional adapter protein SH2B1 in the function of growth hormone and the many other cytokines, hormones and growth factors (e.g. leptin, insulin, nerve growth factor) that use SH2B1 to mediate their effects in cells. Such insight is critical to our understanding on how growth hormone brings about its well known diverse effects in the body, including body growth and metabolism, and other SH2B1 activating ligands contribute to, prevent and/or alleviate symptoms of a variety of diseases including obesity, various cancers, diabetes, multiple sclerosis, and diseases of the immune system.