| Budget Amount *help |
¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 2001: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2000: ¥2,100,000 (Direct Cost: ¥2,100,000)
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| Research Abstract |
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
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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
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