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

点击翻译按钮获取中文摘要

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.