| Co-Investigator(Kenkyū-buntansha) |
SHIRAGA Masamichi Osaka University, Graduate School of Medicine, Medical Staff, 医学部附属病院, 医員(臨床研究)
TOMIYAMA Yoshiaki Osaka University, Graduate School of Medicine, Assistant Professor, 医学系研究科, 助手 (80252667)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2003: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 2002: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Research Abstract |
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
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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
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