Cell and Heparin Binding in the Distal Long Arm of Laminin: Identification of Active and Cryptic Sites with Recombinant and Hybrid Glycoprotein

The long arm of laminin, which binds heparin and cells, consists of three polypeptides (A, B1, and B2) joined in a coiled-coil rod attached to a terminal A chain globule (G). Previously, we found that recombinant globular domain (rG) supported heparin and myoblast binding (Yurchenco, P. D., U. Sung, M. D. Ward, Y. Yamada, and J. J. O'Rear. 1993. J. Biol. Chem. 268:8356-8365). To further analyze long arm functions, we expressed the distal moiety of the mouse laminin A chain extending from the middle of the rod to the carboxyl terminus (rAiG). This larger glycoprotein, secreted by Sf9 insect ceils infected with recombinant baculovirus, was intercalated in vitro into the corresponding disulfide-linked B chain segments of laminin fragment E8 (distal long arm rod and proximal globule). The hybrid molecule (B-rAiG) possessed a structure similar to laminin long arm as judged by electron microscopy and limited proteolysis. By joining rAiG with E8-B chains, the affinity of G domain for heparin decreased from that observed with rAiG and rG to one similar to native protein. HT1080 cells adhered to E8, rAiG, and B-rAiG, less well to rG, and not to denatured E8/ B-rAiG, the A and B chain moieties of E8, or to a mixture of rG and E8-B chains. Cell adhesion to E8 and B-rAiG, in contrast to rAiG, was inhibited with antibodies specific for or6 and #1 integrin chains. Since intercalation (a) restored a conformationally dependent a6B1 integrin recognition site present in native protein, (b) inactivated a cryptic cell binding activity in the A chain, and (c) inhibited a heparin binding site present in proximal G domain, we conclude that biological activities of laminin are different from that of its isolated subunits. AMININ is a major glycoprotein of basement membranes which plays both architectural (Yurchenco et al., 1985, 1990, 1992, 1993) and cell-interactive roles, The latter functions, mediated by a variety of cell surface receptors, include regulation of cell adhesion, spreading, migration, growth, and differentiation (reviewed in Beck et al., 1990 and Mecham, 1991). The three subunit chains of laminin (A, B1 and B2) are bound together through disulfide and noncovalent linkages forming an asymmetrical, multidomain molecule with one long and three short arms. The long arm is composed of a rod-like coiled-coil (subdivided into domains I and II by an a-helical interruption of the B1 chain) derived from all three chains and representing the region of chain union, and a large distal globule (G domain) formed only by the COOH-terminal moiety of the A chain (Beck et al., 1990; Hunter et al., 1990, 1992; Paulsson et al., 1985). This terminal globule is further subdivided into five homologous subdomains, G1-G5, which have been recognized in negatively stained preparations of the long arm (Beck et al., 1990). The middle of the c~-helical rod and the distal region of subdomain G3 (the latter in what appears to be a hinge region) contain protease-sensitive sites which can be cleaved by elastase (Beck et al., 1990) to generate fragment E8 (containing the distal half of the rod and the proximal portion of the G domain with a single disulfide link between the COOH-termini of the B1 and B2 chain segments) and fragment E3 (the distal end of the G domain starting at residue 2666). The long arm has been found to play major cell-interactive roles, stimulating neurite outgrowth, promoting cell migration, and providing a crucial ligand for tubular differentiation in kidney development (Goodman et al., 1992; Klein et al., 1988; Sorokin, 1990). A variety of integrins (tx3B1, o~6B1, tx7B1), cell surface galactosyl-transferase, and other receptors have been reported to recognize this region of laminin (Aumailley et al., 1990; Begovic and Shur, 1990; Gehlsen et al., 1992; Kleinman et al., 1991; Kramer et al., 1991; Sonnenberg et al., 1990; vonder Mark et al., 1991). The a6/~l integrin has been shown to bind to fragment E8 (AumaiUey et al., 1990; Sormenberg et al., 1990) and antibodies to this fragment block adhesion of many cells to laminin. Antibodies specific for fragment E8 (Klein et al., 1988) and for the or6 integrin subunit (Sorokin et al., 1990) were observed to separately prevent the conversion of mesenchyme © The Rockefeller University Press, 0021-9525/93/12/1255/14 $2.00 The Journal of Cell Biology, Volume 123, Number 5, December 1993 1255-1268 1255 on M ay 4, 2017 D ow nladed fom Published December 1, 1993