HEPARIN BINDING EGF--STRUCTURE AND FUNCTION

肝素结合的表皮生长因子样生长因子(HB-EGF)是一种 不溶于膜的锚定蛋白质，加工后释放可溶的蛋白质 成熟的HB-EGF。这两种形式都具有生物活性。HB-EGF是 多功能是因为成熟的HB-EGF是一种强大的有丝分裂原和趋化作用 平滑肌细胞、成纤维细胞和角质形成细胞的因子， 而跨膜型HB-EGF(HB-EGF/TM)作为一种 通过EGF受体发挥作用的旁分泌信号和黏附分子。 HB-EGF/TM也是白喉毒素(DT)的受体。的表达 Hb_EGF与创伤等正常生理过程有关 愈合、胚泡植入和肌肉发生，以及病理 SMC增生等过程。有研究认为HB-EGF的表达 活动受到几种方式的监管。一个是监管岗位-- 翻译加工机制，将旁分泌因子转化为 一种有效的旁分泌有丝分裂原。正常的处理可以实现快速 例如，作为对伤害的反应，动员成熟的HGB-EGF， 而异常处理可能会导致不希望的细胞增殖，如 表现为SMC增生症。Hb-EGF的活性也在 结合HB-EGF的特异性转录因子的基因表达 MyoD和NFkB等启动子以及HB-EGF的外源诱导子 转录，如凝血酶、佛波酯和溶血磷脂酶。少校 该提案的重点是深入了解HB-EGF的监管 深入分析HB-EGF/TM的生物学活性 相互作用，HB-EGF处理的机制， HB-EGF的异常释放及HB-EGF基因的调控 表情。这项建议的具体目的是：1.分析 HB-EGF/TM体外和体外的结构性质及旁分泌活性 包括：A)HB-EGF/TM在体内的表达和定位 培养和体内分化系统：B)HB-EGF/TM的分析 旁分泌信号和黏附活性，以及 调节这些活动，如HSPG和CD9的调节；以及C) HB-EGF/TM的内化及其与膜和膜的相互作用分析 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的处理机制， HB-EGF/TM C-末端裂解位点的鉴定(S)和 制备不可切割的HB-EGF/TM突变体；以及B)检测 表达成熟HB-EGF的转基因小鼠的表型后果 在HB-EGF启动子的控制下，胞质的去除 和跨膜结构域的同源重组；3.分析 HB-EGF基因表达及生长因子活性的调控 体外和体内，重点是HB-EGF在增殖期的活性 SMC病，包括：A)细胞内HB-EGF转录的调节 与血管SMC增生有关；以及B)表达和 HB-EGF在正常子宫肌层和子宫肌瘤中的定位 肌瘤)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.