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Analyses of inhibitory effects induced by the loss-of-function mutant integrin cellular functions

功能丧失突变体整合素细胞功能诱导的抑制作用分析

基本信息

批准号：
14570979
负责人：
HONDA Shigenori
金额：
$ 2.24万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14570979/
关键词：
integrin αvβ3 ligand-binding site dominant negative effect αvβ3

项目摘要

Integrin αvβ3 is expressed in vascular and tumor tissues including endotherial cells, smooth muscle cells, and cancer cells. αvβ3 is now thought to play a key role in angiogenesis, tumor proliferation and metastasis. The primary goals of our study are to newly identify the ligand-binding sites on the αv subunit ofintegrin αvβ3, and elucidate the mechanisms of inhibitory effects induced by the loss-of-function αvβ3 mutant on cellular functions.Identification of the ligand-binding sites in the αv subunit of αvβ3: To evaluate functional significance of each residue within the RGD-contact loops of αv (Arg143-Phe154, Gly172-Gly18I, Asn2O6-Leu222) defined by crystallography, we have performed alanine-scanning mutagenesis. Each mutant αv was transiently co-transfected with WT β3 into 293T cells, and then the ligand-binding function of the mutants was assessed in flow cytometry employing WOW-1 Fab (a monovalent ligand-mimetic mAb specific for αvβ3) and fibrinogen. In agreement with crystallogr … More aphic analysis, asp2I8Ala as well as Tyr178Ala in αv abolished WOW-1 Fab and fibrinogen bindings. In contrast, Asp15OAla in αv did not impair the ligand-binding function. Interestingly, Ala215Tyr and Ala215Phe in αv increased ligand binding. Furthermore, Tyr178Pheαv did no disturb ligand binding, suggesting that aromatic structure of Tyr, rather than hydroxylic side chain is critical for ligand binding (cation-π interaction).Analyses of inhibitory effects induced by the loss-of-function mutant integrin on cellular functions: Overexpression of Tyr178Alaαvβ3 on 293T cells expressing αvβ3 (αvβ3-293T) inhibited the adhesion of the transfectants to immobilized fibrinogen, while the surface expression of endogenous αvβ3 was not decreased in the transfected cells. The data suggest that overexpression of Tyr178Alaαvβ3 may induce a dominant negative effect toward the function of endogenous αvβ3. To investigate a mechanism of the dominant negative effect, we constructed chimeric integrins in which the extracellular domains of integrins αv and β3 were replaced with the corresponding region of CD25. Overexpression of each chimera (CD25/αv, CD25/β3) into αvβ3-293T cells showed the inhibitory effects on the cell adhesion to immobilized fibrinogen, but the cells transfected with the same amount of mock DNA did not. The suppression of cell adhesion was more potent in the cells transfected with CD25/β3 as compared with CD25/αv. Thus, the data suggest that the dominant negative effect induced by Tyr178Alaαvβ3 may be associated with the cytoplasmic domains of both subunits. Less

整合素αvβ3在血管和肿瘤组织中表达，包括内皮细胞、平滑肌细胞和癌细胞。目前认为αvβ3在血管生成、肿瘤增殖和转移中起关键作用。本研究的主要目的是重新鉴定整合素αv β3 α v亚基上的配体结合位点，并阐明功能丧失的α v β3突变体对细胞功能的抑制作用的机制。为了评估晶体学定义的αv（Arg 143-Phe 154，Gly 172-Gly 18 I，Asn 2 O 6-Leu 222）的RGD接触环内每个残基的功能意义，我们进行了丙氨酸扫描诱变。将每个突变体αv与WT β3瞬时共转染到293 T细胞中，然后使用WOW-1 Fab（αvβ3特异性的单价配体模拟mAb）和纤维蛋白原在流式细胞术中评估突变体的配体结合功能。与crystallogr达成一致 ...更多信息免疫组化分析表明，αv中的Asp 2 I8 Ala和Tyr 178 Ala均能阻断WOW-1 Fab和纤维蛋白原的结合。相反，αv中的Asp 15 OAla不损害配体结合功能。有趣的是，αv中的Ala 215 Tyr和Ala 215 Phe增加配体结合。此外，Tyr 178 Phe αv不干扰配体结合，表明Tyr的芳香族结构而不是羟基侧链对配体结合至关重要功能丧失突变体整联蛋白对细胞功能诱导的抑制作用的分析：Tyr 178 Ala αvβ3在表达αvβ3的293 T细胞上的过表达（αvβ3- 293 T）抑制了转染细胞与固定化纤维蛋白原的粘附，而转染细胞内源性αvβ3的表面表达没有减少。这些数据表明，Tyr 178 Ala αvβ3的过表达可能对内源性αvβ3的功能产生显性负效应。为了研究显性负效应的机制，我们构建了嵌合整合素，其中整合素αv和β3的胞外结构域被CD 25的相应区域取代。在αvβ3- 293 T细胞中过表达CD 25/αv、CD 25/β3嵌合体均能抑制细胞与固定化纤维蛋白原的粘附，而转染等量mock DNA的细胞则无此作用。转染CD 25/β3的细胞粘附抑制作用强于转染CD 25/αv的细胞。因此，数据表明Tyr 178 Ala αvβ3诱导的显性负效应可能与两个亚基的胞质结构域有关。少