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J. Biol. Chem., Vol. 281, Issue 43, 32929-32940, October 27, 2006

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Laminin 1 Chain LG4 Module Promotes Cell Attachment through Syndecans and Cell Spreading through Integrin 21^*

Kentaro Hozumi¹, Nobuharu Suzuki, Peter K. Nielsen², Motoyoshi Nomizu, and Yoshihiko Yamada³

From the Molecular Biology Section, Craniofacial Developmental Biology and Regeneration Branch, NIDCR, National Institutes of Health, Bethesda, Maryland 20892-4370 and School of Pharmacy, Tokyo University of Pharmacy and Life Science, Hachioji, Tokyo 192-0392, Japan

The laminin 1 chain is a subunit of laminin-1, a heterotrimeric basement membrane protein. The LG4-5 module at the C terminus of laminin 1 contains major binding sites for heparin, sulfatide, and -dystroglycan and plays a critical role in early embryonic development. We previously identified active synthetic peptides AG73 and EF-1 from the sequence of laminin1 LG4 for binding to syndecan and integrin 21, respectively. However, their activity and functional relationship within the laminin-1 and LG4 as well as the functional relation between these sites and -dystroglycan binding sites in LG4 are not clear. To address these questions, we created mutant recombinant LG4 proteins containing alanine substitutions within the AG73 (M1), EF-1 (M2, M3), and -dystroglycan binding sites (M4, M5) and analyzed their activities. We found that recombinant proteins rec-M1 and rec-M5, containing mutations within M1 and M5, respectively, did not bind heparin or lymphoid cell lines expressing syndecans. These results suggest that LG4 binds to heparin and syndecans through M1 and M5. Rec-M1 and rec-M5 reduced fibroblast attachment, whereas mutant rec-M2 and rec-M3 retained cell attachment activity but did not promote cell spreading. Fibroblast attachment to rec-LG4 was inhibited by heparin but not by integrin antibodies. Spreading of fibroblasts on rec-LG4 was inhibited by anti-integrin 2 and 1 but not by anti-integrin 1 and 6. These results suggest that the M1 and M5 sites are necessary for cell attachment on LG4 through syndecans and that the EF-1 site is for cell spreading activity through integrin 21. In contrast, laminin-1-mediated fibroblast attachment and spreading were not inhibited by heparin or antiintegrin 2. Our findings indicate that LG4 has a unique function distinct from laminin-1 and suggest that laminin 1 LG4-5 may also be produced by a proteolytic cleavage in certain tissues where it exerts its activity.

Received for publication, June 14, 2006 , and in revised form, August 29, 2006.

^* This work was supported in part by the Intramural Research Program of the National Institutes of Health (NIDCR) (to Y. Y.) and the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to M. N.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by Japan Society for the Promotion of Science (JSPS) Research Fellowship for Japanese Biomedical and Behavioral Researchers at the National Institutes of Health.

² Present address: Dept. of Protein Chemistry, Diabetes Research Unit, Novo Nordisk A/S, Novo Allé 6BS.58.5, DK-2880 Bagsvaerd, Denmark.

³ To whom correspondence should be addressed: Bldg. 30, Rm. 407, NIDCR, NIH, 30 Convent Dr. MSC 4370, Bethesda, MD 20892-4370. Tel.: 301-496-2111; Fax: 301-402-0897; E-mail: yoshi.yamada{at}nih.gov.

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