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

ALPHA-E-BETA-7 整合素的结构和功能

• 批准号: 3469103
• 负责人: CHRISTINA M PARKER
• 金额: $ 1.85万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-05-01 至 1998-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3469103
• 关键词: SDS polyacrylamide gel electrophoresis; biological signal transduction; cell adhesion; cell adhesion molecules; electroporation; epithelium; flow cytometry; gene expression; human tissue; immunoaffinity chromatography; immunoprecipitation; integrins; laboratory mouse; laboratory rabbit; ligands; lymphocyte; molecular cloning; monoclonal antibody; northern blottings; nucleic acid hybridization; protein structure function; receptor binding; site directed mutagenesis; southern blotting; transfection

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.

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β 7亚基组成， 新发现的α链家族成员，这里称为α E。 α Ebeta 7复合物在>95%的IEL上表达，但在<2%的IEL上表达。 外周血淋巴细胞。 初步证据显示， α E β 7整合素是介导粘附的整合素之一 转化为上皮细胞。 我们建议研究 这种α E β 7整联蛋白的结构和功能， α Ebeta 7反受体被认为在上皮细胞上表达 细胞 首先，将通过以下方法克隆编码α E链的基因： IEL衍生的ZAP II的简并寡核苷酸筛选 文库中，并且编码全长β 7亚基的基因将被 得到了 接下来，上皮细胞的α-E β 7反受体 将通过产生抗上皮细胞单克隆抗体 阻断IEL和α E β 7转染子粘附的抗体 上皮细胞。 上皮细胞粘附分子（ECAM-1） 将被生物化学表征，编码它的基因将被 通过在COS细胞中表达上皮细胞来源的文库来克隆 用抗ECAM-1单克隆抗体筛选。 ECAM-1将 然后纯化并用于研究α E β 7与 这种受体在一个确定的系统，其中的能力， 可以评估干扰信号。 此外，其他有效的配体 将利用单克隆抗体 阻断IEL与细胞外基质粘附的抗体 proteins. 表达重组α E β 7 将产生ECAM-1的异二聚体并用于直接确认 alphaEbeta 7功能 最后，表达 突变的α E或β 7亚基将被开发出来， 用于评估结构变化的影响， α E或β 7亚基或α E β 7上的ECAM-1分子 功能 通过这种方式，我们希望获得有关 这种α-Ebeta 7整合素在发育和生理学中的作用 上皮内淋巴细胞