Structural Requirement of Carboxyl-terminal Globular Domains of Laminin α3 Chain for Promotion of Rapid Cell Adhesion and Migration by Laminin-5

doi:10.1074/jbc.M001326200

Structural Requirement of Carboxyl-terminal Globular Domains of Laminin α3 Chain for Promotion of Rapid Cell Adhesion and Migration by Laminin-5*

From the ^‡Division of Cell Biology, Kihara Institute for Biological Research and ^§Graduate School of Integrated Sciences, Yokohama City University, 641-12 Maioka-cho, Totsuka-ku, Yokohama 244-0813, Japan

Abstract

The basement membrane protein laminin-5, a heterotrimer of laminin α3, β3, and γ2 chains, potently promotes cellular adhesion and motility. It has been supposed that the carboxyl-terminal globular region of the α3 chain consisting of five distinct domains (G1 to G5) is important for its interaction with integrins. To clarify the function of each G domain, we transfected cDNAs for the full-length (wild type (WT)) and five deletion derivatives (ΔGs) of the α3 chain into human fibrosarcoma cell line HT1080, which expressed and secreted the laminin β3 and γ2 chains but not the α3 chain. The transfectants with the α3 chain cDNAs lacking G5 (ΔG₅), G4–5 (ΔG_4–5), G3–5 (ΔG_3–5), and G2–5 (ΔG_2–5) secreted laminin-5 variants at levels comparable to that with WT cDNA. However, the transfectant with the cDNA without any G domains (ΔG_1–5) secreted little laminin-5, suggesting that the G domains are essential for the efficient assembly and secretion of the heterotrimer α3β3γ2. The transfectants with WT, ΔG₅, and ΔG_4–5 cDNAs survived in serum-free medium longer than those with ΔG_3–5, ΔG_2–5, and ΔG_1–5 cDNAs. The transfectants with WT, ΔG₅, and ΔG_4–5cDNAs secreted apparently the same size of laminin-5, which lacked G4 and G5 due to proteolytic cleavage between G3 and G4, and these laminin-5 forms potently promoted integrin α₃β₁-dependent cell adhesion and migration. However, the laminin-5 forms of ΔG_3–5 and ΔG_2–5 hardly promoted the cell adhesion and motility. These findings demonstrate that the G3 domain, but not the G4 and G5 domains, of the α3 chain is essential for the potent promotion of cell adhesion and motility by laminin-5.

Footnotes

↵* This work was supported by a Grant-in-aid from the Ministry of Education, Science, Sports and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¶ Both authors contributed equally to this work.
↵‖ To whom correspondence should be addressed: Division of Cell Biology, Kihara Institute for Biological Research, Yokohama City University, 642-12 Maioka-cho, Totsuka-ku, Yokohama 244-0813. Tel.: 81-45-820-1905; Fax: 81-45-820-1901; E-mail: miyazaki@yokohama-cu.ac.jp.
Published, JBC Papers in Press, May 2, 2000, DOI 10.1074.jbc.M001326200
↵2 Y. Kikkawa and K. Miyazaki, unpublished data.
↵3 Y. Tsubota, H. Mizushima, T. Hirosaki, S. Higashi, H. Yasumitsu, and K. Miyazaki, unpublished data.
↵4 T. Hirosaki, H. Mizushima, K. Moriyama, and K. Miyazaki, unpublished data.
Abbreviations:

DME/F12

Dulbecco's modified Eagle's medium/Ham's F12 medium

FCS

fetal calf serum

G domains

carboxyl-terminal globular domains of laminin α3 chain

PCR

polymerase chain reaction

ΔG_x

laminin α3 chain lacking Gx domain

WT

wild type of laminin α3 chain

PAGE

polyacrylamide gel electrophoresis

CHAPS

3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid
- Received February 16, 2000.
- Revision received February 16, 2000.
The American Society for Biochemistry and Molecular Biology, Inc.