Laminin Isoforms Containing the γ3 Chain Are Unable to Bind to Integrins due to the Absence of the Glutamic Acid Residue Conserved in the C-terminal Regions of the γ1 and γ2 Chains

Abstract

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 ␣3␤1, ␣6␤1, ␣6␤4, and ␣7␤1, 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 (␣5␤1␥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...

Laminins containing the β2 and γ3 chains regulate astrocyte migration and angiogenesis in the retina

“…This suggests that the laminin γ3 chain may regulate astrocyte-ILM interactions during migration. This is consistent with the observation that the presence of the γ3 chain in a laminin inhibits integrin binding (Ido et al, 2008) and may alter the balance between adhesion and migration.…”

Basement Membranes: Cell Scaffoldings and Signaling Platforms

“…Binding of these integrins depends on contributions arising from g1 and b1 subunit coiled-coil domain sequences located in the vicinity of the LG domains (Ido et al 2007;Taniguchi et al 2009). A g1-subunit glutamic acid residue (E1607) important for integrin-binding is absent in the g3 laminin subunit (Ido et al 2008). Although little is known about this subunit that is otherwise quite homologous to g1, this new finding may Figure 1.…”

The C-terminal Region of Laminin β Chains Modulates the Integrin Binding Affinities of Laminins

“…Construction of Expression Vectors-Expression vectors for the human laminin ␣1 (pcDNA3.1-␣1), ␣2 (pcDNA3.1-␣2), ␣5 (pcDNA3.1-␣5), ␤1 (pCEP4-␤1), and ␤2 (pcDNA3.1-␤2) chains were prepared as described previously (16,21,34). An expression vector for a FLAG-tagged laminin ␤2 chain was constructed by inserting the following nucleic acid sequence encoding a FLAG tag between positions 96 and 97 from the starting codon of the ␤2 mRNA: 5Ј-CAA GCA CCA ATC GAT GAT TAC AAG GAC GAC GAT GAC AAG-3Ј (the FLAG tag sequence is shown with underlines).…”

“…Expression vectors for individual E8 fragments of the human laminin ␣1, ␣2, ␣5, ␤1, and ␥1 chains (designated ␣1E8, ␣2E8, ␣5E8, ␤1E8, and ␥1E8, respectively) were prepared as described (20,21). His 6 , HA, and FLAG tags were added to the recombinant ␣E8 (␣1E8, ␣2E8, and ␣5E8), ␤1E8, and ␥1E8 chains, respectively.…”

“…Recently, we found that the C-terminal region of laminin ␥ chains, particularly the glutamic acid residue at the third position from the C terminus, is critically involved in laminin recognition by integrins (20). Deletion or substitution of this glutamic acid residue, which is conserved between the ␥1 and ␥2 chains, abrogates the integrin binding activities of laminin isoforms containing these ␥ chains, whereas isoforms containing the ␥3 chain, whose C-terminal tail is shorter than those of the ␥1 and ␥2 chains and lacks the glutamic acid residue, are unable to bind to integrins (20,21). Despite the emerging evidence for roles of the ␣ and ␥ chains in integrin binding by laminins, it remains unknown whether the ␤ chains contribute to laminin recognition by integrins.…”