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

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。αvβ3现在被认为在血管生成、肿瘤增殖和转移中起关键作用。本研究的主要目的是新发现蛋白素αvβ3 αv亚基上的配体结合位点，并阐明功能缺失αvβ3突变体对细胞功能的抑制作用机制。αvβ3 αv亚基的配体结合位点鉴定：为了评估αv RGD-contact环（Arg143-Phe154, Gly172-Gly18I, Asn2O6-Leu222）中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293T细胞中，利用WOW-1 Fab （αvβ3特异性的单价拟配单抗）和纤维蛋白原，流式细胞术检测突变体的配体结合功能。与晶体学分析一致，αv中的asp2I8Ala和Tyr178Ala消除了WOW-1 Fab和纤维蛋白原的结合。相反，αv中的Asp15OAla不影响配体结合功能。有趣的是，αv中的Ala215Tyr和Ala215Phe增加了配体结合。此外，tyr178phea αv没有干扰配体结合，这表明Tyr的芳香结构，而不是羟基侧链对配体结合（阳离子-π相互作用）至关重要。功能缺失突变体整合素对细胞功能的抑制作用分析：在表达αvβ3的293T细胞上过表达Tyr178Alaαvβ3 （αvβ3-293T）可抑制转染物对固定化纤维蛋白原的粘附，而转染细胞中内源性αvβ3的表面表达不降低。这些数据表明，过表达Tyr178Alaαvβ3可能对内源性αvβ3的功能产生显性的负作用。为了研究显性负作用的机制，我们构建了嵌合整合素，将整合素αv和β3的细胞外结构域替换为CD25的相应区域。在αv - β3- 293t细胞中过表达CD25/αv、CD25/β3嵌合体对固定纤维蛋白原的粘附均有抑制作用，而转染等量模拟DNA的细胞对固定纤维蛋白原的粘附无抑制作用。与转染CD25/αv相比，转染CD25/β3对细胞粘附的抑制作用更强。因此，这些数据表明Tyr178Alaαvβ3诱导的显性负作用可能与这两个亚基的细胞质结构域有关。少

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