Molecular Basis of Ligand Recognition by Integrin α5β1

By Paul Mould, J. Askari, Martin J. Humphries. Journal of Biological Chemistry, 2000

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The NH2-terminal portion (putative ligand-binding domain) of α subunits contains 7 homologous repeats, the last 3 or 4 of which possess divalent cation binding sequences. These repeats are predicted to form a seven-bladed β-propeller structure. To map ligand recognition sites on the α5 subunit we have taken the approach of constructing and expressing αV/α5 chimeras. Although the NH2-terminal repeats of α5 and αV are >50% identical at the amino acid level, α5β1 and αVβ1show marked differences in their ligand binding specificities. Thus: (i) although both integrins recognize the Arg-Gly-Asp (RGD) sequence in fibronectin, the interaction of α5β1 but not of αVβ1 with fibronectin is strongly dependent on the “synergy” sequence Pro-His-Ser-Arg-Asn; (ii) α5β1 binds preferentially to RGD peptides in which RGD is followed by Gly-Trp (GW) whereas αVβ1 has a broader specificity; (iii) only α5β1 recognizes peptides containing the sequence Arg-Arg-Glu-Thr-Ala-Trp-Ala (RRETAWA). Therefore, amino acid residues involved in ligand recognition by α5β1 can potentially be identified in gain-of-function experiments by their ability to switch the ligand binding properties of αVβ1 to those of α5β1. By introducing appropriate restriction enzyme sites, or using site-directed mutagenesis, parts of the NH2-terminal repeats of αV were replaced with the corresponding regions of the α5 subunit. Chimeric subunits were expressed on the surface of Chinese hamster ovary-B2 cells (which lack endogenous α5) as heterodimers with hamster β1. Stable cell lines were generated and tested for their ability to attach to α5β1-selective ligands. Our results demonstrate that: (a) the first three NH2-terminal repeats contain the amino acid sequences that determine ligand binding specificity and the same repeats include the epitopes of function blocking anti-α subunit mAbs; (b) the divalent cation-binding sites (in repeats 4–7) do not confer α5β1- or αVβ1-specific ligand recognition; (c) amino acid residues Ala107–Tyr226 of α5(corresponding approximately to repeats 2 and 3) are sufficient to change all the ligand binding properties of αVβ1 to those of α5β1; (d) swapping a small part of a predicted loop region of αV with the corresponding region of α5 (Asp154-Ala159) is sufficient to confer selectivity for RGDGW and the ability to recognize RRETAWA.

Subfields: Immunology and Allergy, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/be3c9d5b9e3252df6fd5f456149f736596a5dfa1

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