|Budget Amount *help
¥3,300,000 (Direct Cost : ¥3,300,000)
Fiscal Year 1999 : ¥1,400,000 (Direct Cost : ¥1,400,000)
Fiscal Year 1998 : ¥1,900,000 (Direct Cost : ¥1,900,000)
Integrins are the major receptors for the extracellular matrix adhesive glycoproteins such as fibronectins and laminins. Binding of extracellular ligands to integrins transduces signals through tyrosine phosphorylation of adaptor proteins (such as focal adhesion Kinase, paxillin, tensin, pl30Cas) followed by activation of the rho family of small GTP-binding proteins (i.e. rac, rho, cdc42) and ras/MAP kinase signaling cascades. In this investigation, we focused our efforts on the interaction of integrins with laminins (LNs), the major basement membrane adhesive proteins. The major findings of this study are as follows.
(1) Puification and Characterization of LN-8 : We screened the expression of five different LN α chains in more than 35 human cell lines by RT-PCR. The T98G glioma cells were found to express only α4 chain among the five LNα chains. We also produced a monoclonal antibody recognizing α4 chain on immunoblots and found that the antibody identified 600 kDa band in the culture
supernatants of T98G cells under nonreducing conditions. We grew T98G cells in a large scale and harvested the spent medium (approximately 4 liter) which was concentrated by ammonium sulfate precipitation, followed by gel filtration and immunoaffinity chromatography using the anti-LN β1 monoclonal antibody 4F5. The resulting protein was apparently homogeneous on SDS-PAGE under nonreducing conditions and identified to be LN-8 since it consisted of α4/β1/γi chains. The puified LN-8 was less potent in mediating adhesion of T98G cells than LN-5 and LN-10/11 and comparable to LN-1 in its cell-adhesive activity. Cell adhesion onto LN-8 was mediated through integrin α6β1and α3β1, two major LN-binding integrins.
(2) Cytoskeletal Modulation on LN-10/11 : Recently, we have succeeded in purifying LN-10/11 containing the α5 chain in its intact form (Kikkawa et al. J. Biol. Chem. 247, 15854-15859, 1998). We examined whether adhesion onto LN-10/11 could induce formation of stress fibers and focal adhesions, the hallmark of phenotypes of cells adhered onto fibronectin-coated substrates. Despite its very potent cell-adhesive activity, LN-10/11 failed to induce stress fibers or focal adhesions in cells adhered to LN-10/11. We also found that activation of rho was not detected on LN10/11, consistent with its failure in inducing stress fibers and focal adhesions.
(3) Identification of the 30 kDa Protein That Tightly Associates with Integrin α3β1 As CD151 : We produced polyclonal antibodies against the cytoplasmic domain of the integrin α3 subunit and purified integrin α3β1 from human placenta. Integrin α3β1 thus purified gave three major bands on SDS-PAGE, i.e. 150 kda/120 kDa bands corresponding to the α3 and β1 chains and an unexpected 30 kDa band. The 30 kDa band was identified as CD 151, one of the transmembrane 4 superfamily proteins, by immunoprecipitation using the anti-CD 151 antibody Provided by Dr. Hitoshi Hasegawa (Ehime University Medical School). We also produced two monoclonal antibodies both recognizing the 30 kDa Protein and the antibodies were confirmed to recognize CD 151 based on their reactivity with the NIH3T3 cells transfected with the CD 151 cDNA. These monoclonal antilbodies should prove to be useful in the study of physiological funcions of CD 151. Less