The Requirement of the Glutamic Acid Residue at the Third Position from the Carboxyl Termini of the Laminin γ Chains in Integrin Binding by Laminins

Hiroyuki Ido,Aya Nakamura,Reiko Kobayashi,Shunsuke Ito,Shaoliang Li,Sugiko Futaki,Kiyotoshi Sekiguchi

doi:10.1074/jbc.m609402200

Abstract

Laminins are the major cell-adhesive proteins in the basement membrane, consisting of three subunits termed alpha, beta, and gamma. The putative binding site for integrins has been mapped to the G domain of the alpha chain, although trimerization with beta and gamma chains is necessary for the G domain to exert its integrin binding activity. The mechanism underlying the requirement of beta and gamma chains in integrin binding by laminins remains poorly understood. Here, we show that the C-terminal region of the gamma chain is involved in modulation of the integrin binding activity of laminins. We found that deletion of the C-terminal three but not two amino acids within the gamma1 chain completely abrogated the integrin binding activity of laminin-511. Furthermore, substitution of Gln for Glu-1607, the amino acid residue at the third position from the C terminus of the gamma1 chain, also abolished the integrin binding activity, underscoring the role of Glu-1607 in integrin binding by the laminin. We also found that the conserved Glu residue of the gamma2 chain is necessary for integrin binding by laminin-332, suggesting that the same mechanism operates in the modulation of the integrin binding activity of laminins containing either gamma1 or gamma2 chains. However, the peptide segment modeled after the C-terminal region of gamma1 chain was incapable of either binding to integrin or inhibiting integrin binding by laminin-511, making it unlikely that the Glu residue is directly recognized by integrin. These results, together, indicate a novel mechanism operating in ligand recognition by laminin binding integrins.

Highlights

Ment membrane architecture and influence cell adhesion, spreading, and migration through binding to their cell surface receptors, the integrin family of cell adhesion receptors [5,6,7,8,9]
Solid-phase binding assays with the ␣6␤1 integrin showed that the control laminin-511 (LN511-␥1) was fully active in binding to the ␣6␤1 integrin, whereas the mutant laminin-511 lacking the C-terminal eight amino acids of the ␥1 chain (LN511-␥1⌬8AA) exhibited only marginal activity comparable with that of another mutant laminin-511 lacking the LG3–5 modules of the ␣5 chain, which have been shown to be devoid of integrin binding activity [19]
Two E8 mutants with either deletion of the C-terminal three amino acids (E8-␥1⌬3AA) or substitution of Gln for Glu-1607 (E8␥1EQ) were almost devoid of integrin binding activity, further confirming the importance of Glu-1607 in integrin binding by laminin-511. These results demonstrated that the loss of integrin binding activity by deletion of the C-terminal three amino acids or E1607Q substitution was not due to a defect in disulfide bond formation between the ␤1 and ␥1 chains at their C termini, but rather, it was due to the critical role of the region containing Glu-1607 in sustaining the active conformation of the G domain or to its involvement in integrin binding by comprising a part of the integrin recognition surface

Summary

EXPERIMENTAL PROCEDURES

Antibodies, Proteins, and Peptides—mAbs against the human laminin ␣5 chain (5D6; Ref. 27), the human laminin ␥1 chain (C12SW), and the human laminin ␣3 chain (2B10) were produced in our laboratory. Expression vectors for GST fusion proteins containing FNTPSIEKP (GST-␥1(8AA)), QVTRGDVFT (sequence derived from vitronectin; GST-RGD), or QVTRGEVFT (GSTRGE) were constructed by inserting cDNA fragments encoding the individual peptide sequences into the EcoRI/NotI restriction sites of pGEX4T-1 (Amersham Biosciences). Recombinant ␣6␤1, ␣3␤1, and ␣V␤3 integrins were produced using the same expression system and purified from the conditioned media using anti-FLAG columns (Sigma) described previously [20]. After dialysis against TBS, the purity of these recombinant proteins was verified by SDS-PAGE followed by Coomassie Brilliant Blue staining or immunoblotting with anti-His tag (for ␣5E8), anti-hemagglutinin tag (for ␤1E8), or anti-FLAG tag (for ␥1E8) mAbs. GST fusion proteins (GST-␥1(8AA), GST-RGD, and GSTRGE) were induced in Escherichia coli with 0.1 mM isopropyl␤-D-thiogalactopyranoside and purified on glutathione-Sepharose 4B columns (Amersham Biosciences) after lysis of the cells by sonication. After fixation with formaldehyde and subsequent staining with Diff-Quik (International Reagents Corp., Kobe, Japan), attached cells were counted in three independent wells using Scion Image software (Scion Corp., Frederick, MD)

RESULTS

DISCUSSION

Futaki and Kiyotoshi Sekiguchi