HEPARIN BINDING EGF--STRUCTURE AND FUNCTION

肝素结合EGF样生长因子（HB-EGF），是一种合成 一种不溶性膜锚定蛋白，经加工后释放出可溶性 成熟HB-EGF 这两种形式都具有生物活性。 EGF是 多功能的成熟HB-EGF是一种有效的有丝分裂原和趋化性 平滑肌细胞（SMC）、成纤维细胞和角质形成细胞的因子， 而跨膜HB-EGF（HB-EGF/TM）介导细胞-细胞接触， 表皮生长因子受体介导的表皮生长因子受体信号传导和粘附分子。 HB-EGF/TM也是白喉毒素（DT）的受体。 表达 HB_EGF与正常的生理过程有关，如伤口 愈合、胚泡植入和肌生成，以及病理性 如SMC增生的过程。 提示HB-EGF表达 这些活动以几种方式受到管制。 一个是监管岗位-- 一种翻译加工机制，将阿曲他克林因子转化为 一种有效的旁分泌促分裂剂 正常处理可以允许快速 动员成熟的HGB-EGF，例如，作为对损伤的反应， 尽管异常处理可能导致不希望的细胞增殖 平滑肌细胞增生。 HB-EGF的活性也受到调节， 通过与HB-EGF结合的特异性转录因子的基因表达 启动子如MyoD和NFkB，以及HB-EGF的外部诱导剂 转录，如凝血酶、佛波酯和lysoPC。 主要 该提案的重点是深入了解HB-EGF的监管 通过深入分析HB-EGF/TM的生物学活性， HB-EGF处理的机制， 成熟HB-EGF的异常释放和HB-EGF基因的调控 表情 该提案的具体目标是：1。 分析 HB-EGF/TM的结构特性和体外抗肿瘤活性， 体内;包括：A）HB-EGF/TM的表达和定位， 培养和体内分化系统：B）HB-EGF/TM的分析 阿曲他克林信号传导和粘附活性，以及 调节这些活性，例如通过HSPG和CD 9的调节;和C） 分析HB-EGF/TM内化和与膜的相互作用， 细胞蛋白; 2. 分析HB-EGF/TM的体外加工过程 和体内;包括：A）HB-EGF/TM加工的机制， HB-EGF/TM C-末端切割位点的鉴定和 制备不可切割的HB-EGF/TM突变体;和B）检测 表达成熟HB-EGF的转基因小鼠的表型结果 在HB-EGF启动子的控制下， 和跨膜结构域; 3. 分析 肝细胞HB-EGF基因表达及生长因子活性调节 体外和体内，重点是HB-EGF在增殖细胞中的活性， SMC疾病;包括：A）细胞中HB-EGF转录的调节 与血管SMC增生相关;和B）表达和 HB-EGF在正常子宫肌层和子宫平滑肌瘤中的定位 纤维瘤）SMC。

Heparin-binding EGF-like growth factor (HB-EGF), is synthesized as an insoluble membrane-anchored protein that is processed to release soluble mature HB-EGF. Both forms are biologically active. HB-EGF is multifunctional in that mature HB-EGF is a potent mitogen and chemotactic factor for smooth muscle cells (SMC), fibroblasts and keratinocytes, while transmembrane HB-EGF (HB-EGF/TM) mediates cell-cell contact as a juxtacrine signaling and adhesion molecule acting via the EGF receptor. HB-EGF/TM is also the receptor for diphtheria toxin (DT). Expression of HB_EGF has been linked to normal physiological processes such as wound healing, blastocyst implantation and myogenesis, and to pathological processes such as SMC hyperplasia. It is proposed that HB-EGF expression and activities are regulated in several ways. One is a regulatory post- translational processing mechanism that converts a juxtacrine factor into a potent paracrine mitogen. Normal processing could allow for rapid mobilization of mature HGB-EGF, for example, as a response to injury, while abnormal processing could lead to undesired cell proliferation such as SMC hyperplasia. HB-EGF activity is also regulated at the level of gene expression by specific transcription factors that bind to the HB-EGF promoter such as MyoD and NFkB, and by external inducers of HB-EGF transcription such as thrombin, phorbol ester and lysoPC. The major focus of the proposal is to gain insights into the regulation of HB-EGF activity by analyzing in depth the biology of HB-EGF/TM, juxtacrine interactions, the mechanisms of HB-EGF processing, the consequences of aberrant release of mature HB-EGF, and the control of HB-EGF gene expression. The Specific Aims of this proposal are: 1. To analyze the structural properties and juxtacrine activities of HB-EGF/TM in vitro and in vivo; including: A) expression and localization of HB-EGF/TM in differentiating systems in culture and in vivo: B) analysis of HB-EGF/TM juxtacrine signaling and adhesion activities, and the mechanisms that regulate these activities such as modulation by HSPG and CD9; and C) analysis of HB-EGF/TM internalization and interactions with membrane and cellular proteins; 2. To analyze the processing of HB-EGF/TM in vitro and in vivo; including: A) mechanisms of HB-EGF/TM processing, identification of the HB-EGF/TM C-terminal cleavage site(s) and preparation of non-cleavable HB-EGF/TM mutants; and B) examining the phenotypic consequences in transgenic mice of expressing mature HB-EGF under control of the HB-EGF promoter, and of removal of the cytoplasmic and transmembrane domains by homologous recombination; 3. To analyze the regulation of HB-EGF gene expression and growth factor activity in vitro and in vivo with an emphasis on HB-EGF activity in proliferative SMC disease; including: A) regulation of HB-EGF transcription in cells associated with vascular SMC hyperplasia; and B) expression and localization of HB-EGF in normal myometrial and leiomyoma (uterine fibroids) SMC.