title: Molecular basis of ligand recognition by integrin alpha 5beta 1. I. Specificity of ligand binding is determined by amino acid sequences in the second and third NH2-terminal repeats of the alpha subunit.
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.
authors.affiliation: Wellcome Trust Centre for Cell-Matrix Research, School of Biological Sciences, University of Manchester, Manchester M13 9PT, United Kingdom. paul.mold@man.ac.uk
authors.forename: A P
authors.initials: AP
authors.lastname: Mould
authors.forename: J A
authors.initials: JA
authors.lastname: Askari
authors.forename: M J
authors.initials: MJ
authors.lastname: Humphries
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001665:Binding Sites; D016466:CHO Cells; D002413:Cations, Divalent / Q000378:metabolism; D006224:Cricetinae; D006801:Humans; D016023:Integrins / Q000235:genetics / Q000378:metabolism*; D008024:Ligands; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides / Q000378:metabolism; D017469:Receptors, Fibronectin / Q000235:genetics / Q000378:metabolism*; D019030:Receptors, Vitronectin; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D020449:Repetitive Sequences, Amino Acid; D016415:Sequence Alignment; D013379:Substrate Specificity; D014162:Transfection
chemical_list: D000911:Antibodies, Monoclonal; D002413:Cations, Divalent; D016023:Integrins; D008024:Ligands; D009842:Oligopeptides; D017469:Receptors, Fibronectin; D019030:Receptors, Vitronectin; D011993:Recombinant Fusion Proteins; C094340:integrin alphavbeta1; C047981:arginyl-glycyl-aspartic acid