STRUCTURE AND FUNCTION OF THE ALPHA-E-BETA-7 INTEGRIN

最近我们在人上皮细胞内发现了一种新的整合素 淋巴细胞(IEL)，由β7亚基组成，与 新发现的阿尔法链家族成员，这里称为阿尔法E。 αEbeta7复合体在95%的IEL上表达，而在2%的IEL上表达 外周血淋巴细胞。初步证据表明，这 αEbeta7整合素是介导细胞黏附的整合素之一 上皮内淋巴细胞转化为上皮细胞。我们建议研究 该α-β7整合素及其受体的结构和功能 上皮细胞表达α-Ebeta7反式受体 细胞。首先，将通过以下方式克隆编码AlphaE链的基因 IEL衍生型lambda-ZAP II的简并寡核苷酸筛选 文库，编码全长Beta7亚基的基因将被 获得。接下来，上皮细胞αEbeta7的反受体 将通过产生抗上皮细胞的单抗来鉴定 阻断IEL和α-Ebeta7转染体黏附的抗体 到上皮细胞。这种上皮细胞黏附分子(ECAM-1) 将被描述为生化特征和编码它的基因 上皮细胞衍生文库在COS细胞中的表达克隆 用抗ECAM-1单抗进行筛选。ECAM-1将 然后被纯化并用于研究αEbeta7与 这种受体在一个特定的系统中，其中αβ7的能力 可以对换能器信号进行评估。此外，其他有效的配体 对于αEbeta7复合体，将利用单克隆进行鉴定 阻断IELs与细胞外基质黏附的抗体 蛋白质。表达重组αEbeta7的转化子 将产生ECAM-1的杂二聚体并利用其直接证实 定义的alphaEbeta7函数。最后，转染体表达 将开发突变的AlphaE或Beta7亚基，这将是 用来评估结构变化的影响 αE或β7亚基或αEβ7上的ECAM-1分子 功能。通过这种方式，我们希望获得关于 这个α-β7整合素在卵巢癌的发育和生理学中的作用 上皮内淋巴细胞。

Recently we identified a novel integrin on human intraepithelial lymphocytes (IEL) which is composed of a beta7 subunit associated with a newly identified member of the alpha chain family, here called alphaE. The alphaEbeta7 complex is expressed on >95% of IEL but on <2% of peripheral blood lymphocytes. Preliminary evidence suggests that this alphaEbeta7 integrin is one of the integrins which mediates the adhesion of intraepithelial lymphocytes to epithelial cells. We propose to study the structure and function of this alphaEbeta7 integrin and of the alphaEbeta7 counter-receptor thought to be expressed on epithelial cells. First, the gene encoding of the alphaE chain will be cloned by degenerate oligonucleotide screening of an IEL derived lambda-ZAP II library, and the gene encoding the full length beta7 subunit will be obtained. Next, the epithelial cell counter-receptor for alphaEbeta7 will be identified by producing anti-epithelial cell monoclonal antibodies which block the adhesion of IEL and alphaEbeta7 transfectants to epithelial cells. This epithelial cell adhesion molecule (ECAM-1) will be characterized biochemically an the gene encoding it will be cloned by expression of an epithelial cell derived library in COS cells and selection with the anti-ECAM-1 monoclonal antibody. ECAM-1 will then be purified and utilized to study the binding of alphaEbeta7 to this receptor in a defined system, where the ability of alphaEbeta7 to transduce signals can be assessed. In addition, other potent ligands for the alphaEbeta7 complex will be identified utilizing monoclonal antibodies to block the adhesion of IELs to extracellular matrix proteins. Transfectants expressing either reconstituted alphaEbeta7 heterodimer of ECAM-1 will be generated and utilized to directly confirm defined alphaEbeta7 functions. Finally, transfectants expressing mutated alphaE or beta7 subunits will be developed which will be utilized to evaluate the effect of changes in the structure of the alphaE or beta7 subunits or the ECAM-1 molecule on the alphaEbeta7 function. In this way we hope to gain information about the role of this alphaEbeta7 integrin in the development and physiology of intraepithelial lymphocytes.