Structural and functional analyzes of integrin αvβ3 : identification of ligand-binding sites in the αv subunit of αvβ3 and development of inhibitor for αvβ3

整合素 αvβ3 的结构和功能巢：鉴定 αvβ3 αv 亚基中的配体结合位点并开发 αvβ3 抑制剂

• 批准号: 12670989
• 负责人: HONDA Shigenori
• 金额: $ 2.11万
• 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: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12670989/
• 关键词: integrin; αvβ3; ligand-binding site; dominant negative effect; αVβ3; リガンド結合部位

Integrin αvβ3, originally identified as the vitronectin receptor, is mainly expressed in the vascular or 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. For these reasons, it is important in developing a new therapy to clarify the precise role of αvβ3 and control it.The primary aims of this study are to identify the ligand-binding sites of integrin αvβ3, and elucidate the functional roles of this integrin, and develop inhibitors specific for αvβ3.Identification of the ligand-binding sites in the αv subunit of αvβ3 and establishment of the cell lines expressing function-deficit αvβ3 : We succeeded to identify the ligand-binding site in the av subunit by performing alanine-scanning mutagenesis of recombinant αvβ3 expressed in 293 cells (a human embryonic kidney cell). For example, Tyr178Alaαvβ3 in which Tyr178 in the αv subunit is substituted with alanine, failed … More to bind soluble ligands including fibrinogen and a ligand-mimetic monoclonal antibody (WOW-1 Fab). Next, we established the cell line expressing Tyr178Alaαvβ3 by introducing mutant αv into the αv-deficit melanoma cell. The cell expressing Tyr178Alaαvβ3 markedly impaired cell adhesion to immobilized fibrinogen as well as vitronectin. Thus, the data confirm that Tyr178 is critical for receptor-ligand interaction.Functional analyzes of mutant αvβ3 and development of inhibitors of αvβ3 : To investigate whether Tyr178Alaαvβ3 inhibit the function of endogenous integrins, we examined the effect of Tyr178Alaαvβ3 transiently transfected in the β3-293 cells that constitutively express wild-type αvβ3, on cell adhesive function. The β3-293 cell expressing Tyr178Alaαvβ3 markedly impaired cell adhesion to immobilized fibrinogen as compared with mock-transfected β3-293 cells, while the surface expression of endogenous αvβ3 was not suppressed in those cells. Thus, the data suggest that overexpression of Tyr178Alaαvβ3 may induce a dominant negative effect toward the function of endogenous αvβ3. Less

整合素αvβ3最初被鉴定为玻连蛋白受体，主要在血管或肿瘤组织中表达，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。因此，阐明αvβ3的确切作用并对其进行控制，对于开发新的治疗方法具有重要意义。本研究的主要目的是鉴定整合素αvβ3的配体结合位点，并阐明该整合素的功能作用，α v β 3亚基配体结合位点的鉴定及功能表达细胞系的建立缺陷αvβ3：我们通过对293细胞（一种人胚肾细胞）中表达的重组αvβ3进行丙氨酸扫描诱变，成功鉴定了av亚基中的配体结合位点。例如，Tyr 178 Ala αvβ3，其中αv亚基中的Tyr 178被丙氨酸取代， ...更多信息 结合包括纤维蛋白原和配体模拟单克隆抗体（WOW-1 Fab）的可溶性配体。接下来，我们通过将突变体αv导入α v缺陷型黑素瘤细胞中，建立了表达Tyr 178 Ala αvβ3的细胞系。表达Tyr 178 Ala αvβ3的细胞显著损害细胞与固定化纤维蛋白原以及玻连蛋白的粘附。突变体αvβ3的功能分析和αvβ3抑制剂的开发：为了研究Tyr 178 Ala αvβ3是否抑制内源性整合素的功能，我们检测了Tyr 178 Ala α v β3瞬时转染组成型表达野生型αvβ3的β3-293细胞对细胞粘附功能的影响。与模拟转染的β3- 293细胞相比，表达Tyr 178 Ala αvβ3的β3 - 293细胞显著损害细胞与固定化纤维蛋白原的粘附，而在这些细胞中内源性αvβ3的表面表达没有受到抑制。因此，数据表明Tyr 178 Ala αvβ3的过表达可能诱导对内源性αvβ3功能的显性负效应。少

项目成果

Y.Tomiyama: "Glanzmann thrombasthenia : integrin αIIbβ3 deficiency"Int J Hematol. 72(4). 448-454 (2000)

Y. Tomiyama：“Glanzmann 血小板无力症：整合素 αIIbβ3 缺乏”Int J Hematol 72(4) (2000)。

S.Kosugi, Y.Tomiyama, S.Honda. et al.: "Anti-avβ3 antibodies in chronic immune thrombocytopenic purpura"Thromb Haemost. 85(1). 36-41 (2001)

S.Kosugi、Y.Tomiyama、S.Honda 等人：“慢性免疫性血小板减少性紫癜中的抗 avβ3 抗体”，Thromb Haemost 85(1)。

S. Kosugi. Y. Tomiyama, S. Honda, et al: "Platelet-associated anti-GPIIb-IIIa autoantibodies in chronic immune thrombocytopenic purpura recognizing epitopes close to the Hgand-binding site of glycoprotein (GP) IIb"Blood. 98(6). 1819-1827 (2001)

S.小杉。

S.Kosugi, Y.Tomiyama, S.Honda, et al.: "Anti-αvβ3 antibodies in chronic immune thrombocytopenic purpura"Thromb Haemost. 85(1). 36-41 (2001)

S.Kosugi、Y.Tomiyama、S.Honda 等人：“慢性免疫性血小板减少性紫癜中的抗 αvβ3 抗体”Thromb Haemost 85(1)。

H.Kashiwagi, Y.Tomiyama, et al.: "Analyses of genetic abnormalities in type I CD36 deficiency in Japan : identification and cell biological characterization of two novel mutations that cause CD36 deficiency in man"Hum Genet. 108(6). 459-466 (2001)

H.Kashiwagi、Y.Tomiyama 等人：“日本 I 型 CD36 缺乏症的遗传异常分析：导致人类 CD36 缺乏症的两种新突变的鉴定和细胞生物学特征”Hum Genet。