Molecular basis of ligand recognition by integrin alpha(5)beta(1) - I. Specificity of ligand binding is determined by amino acid sequences in the second and third NH2-terminal repeats of the alpha subunit
Ap. Mould et al., Molecular basis of ligand recognition by integrin alpha(5)beta(1) - I. Specificity of ligand binding is determined by amino acid sequences in the second and third NH2-terminal repeats of the alpha subunit, J BIOL CHEM, 275(27), 2000, pp. 20324-20336
The NH2-terminal portion (putative ligand-binding domain) of a subunits con
tains 7 homologous repeats, the last 3 or 4 of which possess divalent catio
n binding sequences. These repeats are predicted to form a seven-bladed bet
a-propeller structure. To map ligand recognition sites on the alpha(5) subu
nit we have taken the approach of constructing and expressing alpha(V)/alph
a(5) chimeras. Although the NH2-terminal repeats of alpha(5) and alpha(V) a
re >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 fibro
nectin, the interaction of alpha(5)beta(1) but not of alpha(V)beta(1) with
fibronectin is strongly dependent on the "synergy" sequence Pra-His-Ser-Arg
-Asn; (ii) alpha(5)beta(1) binds preferentially to RGD peptides in which RG
D is followed by Gly-TrP (GW) whereas alpha(V)beta(1) has a broader specifi
city; (iii) only alpha(5)beta(1) recognizes peptides containing the sequenc
e Arg-Arg-Glu-Thr-Ala-Trp-Ala (RRETAWA). Therefore, amino acid residues inv
olved in ligand recognition by alpha(5)beta(1) can potentially be identifie
d in gain-of-function experiments by their ability to switch the ligand bin
ding properties of alpha(V)beta(1) to those of alpha(5)beta(1). By introduc
ing appropriate restriction enzyme sites, or using site-directed mutagenesi
s, parts of the NH2-terminal repeats of a, were replaced with the correspon
ding regions of the a, subunit. Chimeric subunits were expressed on the sur
face of Chinese hamster ovary-B2 cells (which lack endogenous alpha(5)) as
heterodimers with hamster beta(1). Stable cell lines were generated and tes
ted for their ability to attach to alpha(5)beta(1)-selective ligands. Our r
esults demonstrate that: (a) the first three NH2-terminal repeats contain t
he amino acid sequences that determine ligand binding specificity and the s
ame repeats include the epitopes of function blocking anti-a 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 r
esidues Ala(107)-Tyr(226) of alpha(5) (corresponding approximately to repea
ts 2 and 3) are sufficient to change all the ligand binding properties of a
lpha(V)beta(1) to these of alpha(5)beta(1); (d) swapping a small part of a
predicted loop region of alpha(5) with the corresponding region of alpha(5)
(Asp(154)-Ala(159)) is sufficient to confer selectivity for RGDGW and the
ability to recognize RRETAWA.