Laminins are the major cell adhesive proteins in basement membranes, and consist of three subunits termed ␣, , and ␥. Recently, we found that the Glu residue at the third position from the C termini of the ␥1 and ␥2 chains is critically involved in integrin binding by laminins. However, the ␥3 chain lacks this Glu residue, suggesting that laminin isoforms containing the ␥3 chain may be unable to bind to integrins. To address this possibility, we expressed the E8 fragment of laminin-213 and found that it was incapable of binding to integrins. Similarly, the E8 fragment of laminin-113 was expressed and also found to be inactive in binding to integrins, confirming the distinction between the integrin binding activities of ␥3 chain-containing isoforms and those containing the ␥1 or ␥2 chain. To further address the importance of the Glu residue, we swapped the C-terminal four amino acids of the ␥3 chain with the C-terminal nine amino acids of the ␥1 chain, which contain the Glu residue. The resulting chimeric E8 fragment of laminin-213 became fully active in integrin binding, whereas replacement with the nine amino acids of the ␥1 chain after substitution of Gln for the conserved Glu residue failed to restore the integrin binding activity. These results provide both loss-of-function and gainof-function evidence that laminin isoforms containing the ␥3 chain are unable to bind to integrins due to the absence of the conserved Glu residue, which should play a critical role in integrin binding by laminins.Laminins are heterotrimeric glycoproteins found in basement membranes and consist of three covalently linked chains termed ␣, , and ␥. There are five ␣ chains (␣1-␣5), three  chains (1-3), and three ␥ chains (␥1-␥3) that can give rise to at least 15 different functional laminin isoforms (1-3). These isoforms have been implicated in a wide variety of biological processes involving cell-basement membrane interactions through binding to cell surface receptors including integrins, syndecans, and dystroglycan (1, 4 -9).Integrins are ␣ transmembrane receptors that play critical roles in cell matrix adhesion in multicellular organisms. Several members of the integrin family proteins, including ␣31, ␣61, ␣64, and ␣71, serve as laminin receptors on a variety of cell types (10). The putative binding sites for these integrins have been mapped to the globular (G) 3 domain of the laminin ␣ chains (11-16), although trimerization with  and ␥ chains is necessary for the G domain to exert its integrin binding activity (17)(18)(19). Recently, we found that the C-terminal regions of the ␥ chains are critically involved in integrin binding by laminins (20). Briefly, deletion of the C-terminal three but not two amino acids of the ␥1 chain completely abrogated the integrin binding activity of laminin-511 (␣51␥1), while substitution of Gln for Glu-1607, the amino acid residue at the third position from the C terminus of the ␥1 chain, also abolished the integrin binding activity; thereby underscoring a critical role of Glu-1607 in i...