Abstract
The NH(2)-terminal portion (putative ligand-binding domain) of alpha 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 beta-propeller structure. To map ligand recognition sites on the alpha(5) subunit we have taken the approach of constructing and expressing alpha(V)/alpha(5) chimeras. Although the NH(2)-terminal repeats of alpha(5) and alpha(V) are >50% identical at the amino acid level, alpha(5)beta(1) and alpha(V)beta(1) show marked differences in their ligand binding specificities. Thus: (i) although both integrins recognize the Arg-Gly-Asp (RGD) sequence in fibronectin, the interaction of alpha(5)beta(1) but not of alpha(V)beta(1) with fibronectin is strongly dependent on the "synergy" sequence Pro-His-Ser-Arg-Asn; (ii) alpha(5)beta(1) binds preferentially to RGD peptides in which RGD is followed by Gly-Trp (GW) whereas alpha(V)beta(1) has a broader specificity; (iii) only alpha(5)beta(1) recognizes peptides containing the sequence Arg-Arg-Glu-Thr-Ala-Trp-Ala (RRETAWA). Therefore, amino acid residues involved in ligand recognition by alpha(5)beta(1) can potentially be identified in gain-of-function experiments by their ability to switch the ligand binding properties of alpha(V)beta(1) to those of alpha(5)beta(1). By introducing appropriate restriction enzyme sites, or using site-directed mutagenesis, parts of the NH(2)-terminal repeats of alpha(V) were replaced with the corresponding regions of the alpha(5) subunit. Chimeric subunits were expressed on the surface of Chinese hamster ovary-B2 cells (which lack endogenous alpha(5)) as heterodimers with hamster beta(1). Stable cell lines were generated and tested for their ability to attach to alpha(5)beta(1)-selective ligands. Our results demonstrate that: (a) the first three NH(2)-terminal repeats contain the amino acid sequences that determine ligand binding specificity and the same repeats include the epitopes of function blocking anti-alpha subunit mAbs; (b) the divalent cation-binding sites (in repeats 4-7) do not confer alpha(5)beta(1)- or alpha(V)beta(1)-specific ligand recognition; (c) amino acid residues Ala(107)-Tyr(226) of alpha(5) (corresponding approximately to repeats 2 and 3) are sufficient to change all the ligand binding properties of alpha(V)beta(1) to those of alpha(5)beta(1); (d) swapping a small part of a predicted loop region of alpha(V) with the corresponding region of alpha(5) (Asp(154)-Ala(159)) is sufficient to confer selectivity for RGDGW and the ability to recognize RRETAWA.
Highlights
The NH2-terminal portion of ␣ subunits contains 7 homologous repeats, the last 3 or 4 of which possess divalent cation binding sequences
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-␣ subunit mAbs; (b) the divalent cation-binding sites do not confer ␣51- or ␣V1-specific ligand recognition; (c) amino acid residues Ala107–Tyr226 of ␣5 are sufficient to change all the ligand binding properties of ␣V1 to those of ␣51; (d) swapping a small part of a predicted loop region of ␣V with the corresponding region of ␣5 (Asp154-Ala159) is sufficient to confer selectivity for RG
In this report we have sought to identify the regions of the integrin ␣ subunit that are involved in ligand recognition using ␣V/␣5 chimeras
Summary
Monoclonal Antibodies and Peptides—MAbs 16 and 11 recognizing the human ␣5 subunit, and mAb 13 recognizing the human 1 subunit were gifts from Dr K. The cell population was incubated with mAb P3G8 or mAb11, and with anti-mouse or anti-rat IgG-coated magnetic beads (Dako) to select for cells expressing wild-type or chimeric integrins. Cell Attachment Assay—Chinese hamster ovary-B2 cells, or cells transfected with chimeric or wild-type integrins were detached using 0.05% (w/v) trypsin, 0.02% (w/v) EDTA in PBS, washed with 150 mM NaCl, 25 mM HEPES, pH 7.4, 1 mg/ml BSA, resuspended in the same buffer with 1 mM MgCl2 and 1 mM CaCl2 (buffer A) to a concentration of 5 ϫ 105/ml, and incubated at 37 °C for 20 min. To estimate the reference value for 100% attachment, cells in quadruplicate wells coated with poly-L-lysine (Sigma) (500 g/ml) were fixed immediately by direct addition of 100 l of 5% (w/v) glutaraldehyde for 30 min at room temperature. Each experiment shown is representative of at least three separate experiments
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