Study of the activity of proHB-EGF complex.

我们研究了肝素结合型EGF样生长因子(proHB-EGF)的膜锚定型及其生理功能。最近，我们发现在Vero细胞的细胞接触部位，proHB-EGF和CD9与整合素α3β形成了一个复合体。为了研究proHB-EGF复合体的功能，本课题研究了proHB-EGF的生物学活性，2)proHB-EGF胞外结构域脱落的机制，3)proHB-EGF复合体的新成分的鉴定和鉴定1)proHB-EGF的生物活性分析我们通过将表达proHJB-EGF的效应细胞与表达EGFR的靶细胞共同培养的模型来研究proHB-EGF的生物学活性。在这个实验系统中，我们发现proHB-EGF诱导表达Eger的靶细胞的生长抑制和随后的凋亡。此外，我们还发现，proHB-egf诱导的抑制信号是通过egfr介导的，并且egfr…的胞质结构域在proHB-EGF诱导的细胞凋亡中，更多是必不可少的。根据这些结果，我们得出结论：proHB-EGF通过细胞间接触具有独特的生物活性，这与SHB-EGF的活性不同(提交时)。2)proHB-EGF胞外结构域脱落机制的分析位于细胞表面的许多蛋白质在细胞刺激下胞外结构域被脱落。其中一种蛋白质是HB-EGF，它以膜锚定的形式存在，在PKC激活剂TPA的刺激下转化为可溶性形式。我们发现PKCDelta在体内和体外都与金属蛋白酶去整合素家族成员MDC9/Meltrin-Gamma/ADAM9的细胞质结构域结合。此外，结构活性PKCDelta或MDC9的存在会导致proHB-EGF胞外结构域的脱落，而缺乏金属蛋白酶结构域的MDC9突变体以及激酶阴性的PKCDelta则抑制TPA诱导的胞外结构域的脱落。这些结果表明MDC9和PKCDelta参与了刺激偶联的前HB-EGF胞外结构域的脱落(EMBO J.，17,7260-，1998)。3)ProHB-EGF复合体新成分的鉴定为了鉴定与ProHB-EGF相关的新蛋白(S)，我们制备了几种单抗来识别由白喉毒素(DT)与前HB-EGF共沉淀的分子。其中mAblC9-2能识别dT特异性地与proHB-EGF和CD9共沉淀的抗原，但不能被抗HB-EGF抗体识别，提示proHB-EGF与1C9-2抗原分子的结合是生理性的，因为dT只能与proHB-EGF结合而与CD9形成复合物，而抗HB-EGF抗体可以结合所有proHB-EGF群体。目前，我们正在进行1C9-2抗原分子的鉴定和纯化。较少

We have studied the membrane-anchored form of heparin-binding EGF-like growth factor (proHB-EGF) and its physiological function. Recently we demonstrated that proHB-EGF and CD9 form a complex with integrin alpha3betal at cell-cell contact sites of Vero cells. To study the function of proHB-EGF complex, in this project, we studied 1) the biological activity of proHB-EGF, 2) the mechanism of the ectodomain shedding of proHB-EGF, and 3) identification and characterization of a novel component of proHB-EGF complex.1)Analysis of the biological activity of proHB-EGF We studied the biological activity of proHB-EGF by using a model in which proHJB-EGF-expressing effector cells were co-cultured with EGFR-expressing target cells. From this experimental system, we found that proHB-EGF induces growth inhibition and subsequent apoptosis of the EGER-expressing target cells. Moreover, we found that the inhibitory signal induced by proHB-EGF is mediated via EGFR and that the cytoplasmic domain of EGFR … More is essential for proHB-EGF-induced apoptosis. From these results, we concluded that proHB-EGF has unique biological activity through cell-cell contact which is distinct from the activity of sHB-EGF (on submitting).2)Analysis of the mechanism of the ectodomain shedding of proHB-EGF The ectodomains of many proteins located at the cell surface are shed upon cell stimulation. One such protein is HB-EGF that exists in a membrane-anchored form which is converted to a soluble form upon cellstimulation with TPA, an activator of PKC.We found that PKCdelta binds in vivo and in vitro to the cytoplasmic domain of MDC9/meltrin-gamma/ADAM9, a member of the metalloprotease-disintegrin family. Furthermore, the presence of constitutively active PKCdelta or MDC9 results in the shedding of the ectodomain of proHB-EGF, whereas MDC9 mutants lacking the metalloprotease domain, as well as kinase-negative PKCdelta suppress the TPA-induced shedding of the ectodomain. These results suggest that MDC9 and PKCdelta are involved in the stimulus-coupled shedding of the ptoFLB-EGF ectodomain (EMBO J., 17, 7260-, 1998).3)Identification of novel components of proHB-EGF complex To identify the novel protein(s) associated with proHB-EGF, we preapred several monoclonal antibodies that recognize molecule which is co-precipitated with proHB-EGF by diphtheria toxin (DT). Among them, mAblC9-2 recognizes its antigen that is co-precipitated with proHB-EGF and CD9 specifically by DT, but not by anti-HB-EGF antibody, suggesting that association of proHB-EGF and 1C9-2 antigen molecule is physiological because DT can bind to only proHB-EGF which forms a complex with CD9 while anti-HB-EGF antibody can bind all population of proHB-EGF.Now we are undergoing identification and purification of the 1C9-2 antigen molecule. Less