Identification of the ligand binding site for the integrin alpha9 beta1 in the third fibronectin type III repeat of tenascin-C.
The integrin alpha9 subunit forms a single heterodimer, alpha9 beta1 that mediates cell adhesion to a site within the third fibronectin type III repeat of tenascin-C (TNfn3). In contrast to at least 3 other integrins that bind to this region of tenascin-C, alpha9 beta1 does not recognize the common integrin recognition motif, Arg-Gly-Asp (RGD). In this report, we have used substitution mutagenesis to identify a unique ligand recognition sequence in TNfn3. We introduced mutations substituting alanine for each of the acidic residues in or adjacent to each of the exposed loops predicted from the solved crystal structure. Most of these mutations had little or no effect on adhesion of alpha9-transfected SW480 colon carcinoma cells, but mutations of either of two acidic residues in the B-C loop region markedly reduced attachment of these cells. In contrast, cells expressing the integrin alphav beta3, previously reported to bind to the RGD sequence in the adjacent F-G loop, attached to all mutant fragments except one in which the RGD site was mutated to RAA. The peptide, AEIDGIEL, based on the sequence of human tenascin-C in this region blocked the binding of alpha9-transfected cells, but not beta3-transfected cells to wild type TNfn3. This sequence contains a tripeptide, IDG, homologous to the sequences LDV, IDA, and LDA in fibronectin and IDS in VCAM-1 recognized by the closely related integrin alpha4 beta1. These findings support the idea that this tripeptide motif serves as a ligand binding site for the alpha4/alpha9 subfamily of integrins.
Yokosaki
Y
Y
Department of Internal Medicine, National Hiroshima Hospital, 513 Jike, Saijoh Higashi-Hiroshima 739-0041, Japan. yokosaki@hirosima.hosp.go.jp
Matsuura
N
N
Higashiyama
S
S
Murakami
I
I
Obara
M
M
Yamakido
M
M
Shigeto
N
N
Chen
J
J
Sheppard
D
D
D000595:Amino Acid Sequence; D001665:Binding Sites; D015815:Cell Adhesion Molecules / Q000378:metabolism; D019281:Dimerization; D000939:Epitopes; D006801:Humans; D039001:Integrin alpha Chains; D019012:Integrin beta1 / Q000276:immunology / Q000378:metabolism*; D016023:Integrins / Q000378:metabolism*; D008024:Ligands; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012091:Repetitive Sequences, Nucleic Acid; D016415:Sequence Alignment; D019063:Tenascin / Q000378:metabolism*
D015815:Cell Adhesion Molecules; D000939:Epitopes; D039001:Integrin alpha Chains; D019012:Integrin beta1; D016023:Integrins; D008024:Ligands; D009842:Oligopeptides; D019063:Tenascin; C084613:integrin alpha9