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最初被鉴定为玻璃体连接素受体，主要表达于血管或肿瘤组织，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。由于这些原因，开发一种新的治疗方法来明确αvβ3的确切作用并控制它是很重要的。本研究的主要目的是确定整合素αvβ3的配体结合位点，阐明该整合素的功能作用，并开发αvβ3特异性抑制剂。αvβ3 αv亚基配体结合位点的鉴定及功能缺陷αvβ3表达细胞系的建立：通过对293细胞（人胚胎肾细胞）中表达的重组αvβ3进行丙氨酸扫描诱变，成功鉴定了αvβ3亚基配体结合位点。例如，在αv亚基Tyr178被丙氨酸取代的Tyr178Alaαvβ3中，无法结合可溶性配体，包括纤维蛋白原和一种拟配体单克隆抗体（WOW-1 Fab）。接下来，我们将突变体αv引入αv缺陷黑色素瘤细胞，建立表达Tyr178Alaαvβ3的细胞系。表达Tyr178Alaαvβ3的细胞对固定纤维蛋白原和玻璃体连接蛋白的粘附明显减弱。因此，数据证实Tyr178对受体-配体相互作用至关重要。αvβ3突变体的功能分析及αvβ3抑制剂的研究：为了研究Tyr178Alaαvβ3是否抑制内源性整合素的功能，我们检测了在组成型αvβ3野生型的β3-293细胞中瞬时转染Tyr178Alaαvβ3对细胞粘附功能的影响。与模拟转染的β3-293细胞相比，表达Tyr178Alaαvβ3的β3-293细胞对固定纤维蛋白原的粘附明显减弱，而内源性αvβ3的表面表达不受抑制。因此，这些数据表明，过表达Tyr178Alaαvβ3可能会对内源性αvβ3的功能产生显性的负面影响。少

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