The integrin alpha(9)beta(1) binds to a novel recognition sequence (SVVYGLR) in the thrombin-cleaved amino-terminal fragment of osteopontin.
The integrin alpha(9)beta(1) mediates cell adhesion to tenascin-C and VCAM-1 by binding to sequences distinct from the common integrin-recognition sequence, arginine-glycine-aspartic acid (RGD). A thrombin-cleaved NH(2)-terminal fragment of osteopontin containing the RGD sequence has recently been shown to also be a ligand for alpha(9)beta(1). In this report, we used site-directed mutagenesis and synthetic peptides to identify the alpha(9)beta(1) recognition sequence in osteopontin. alpha(9)-transfected SW480, Chinese hamster ovary, and L-cells adhered to a recombinant NH(2)-terminal osteopontin fragment in which the RGD site was mutated to RAA (nOPN-RAA). Adhesion was completely inhibited by anti-alpha(9) monoclonal antibody Y9A2, indicating the presence of a non-RGD alpha(9)beta(1) recognition sequence within this fragment. Alanine substitution mutagenesis of 13 additional conserved negatively charged amino acid residues in this fragment had no effect on alpha(9)beta(1)-mediated adhesion, but adhesion was dramatically inhibited by either alanine substitution or deletion of tyrosine 165. A synthetic peptide, SVVYGLR, corresponding to the sequence surrounding Tyr(165), blocked alpha(9)beta(1)-mediated adhesion to nOPN-RAA and exposed a ligand-binding-dependent epitope on the integrin beta(1) subunit on alpha(9)-transfected, but not on mock-transfected cells. These results demonstrate that the linear sequence SVVYGLR directly binds to alpha(9)beta(1) and is responsible for alpha(9)beta(1)-mediated cell adhesion to the NH(2)-terminal fragment of osteopontin.
Yokosaki
Y
Y
Department of Internal Medicine, National Hiroshima Hospital, 513 Jike, Saijoh, Higashi-Hiroshima 739-0041, Japan. yokosaki@hiroshima.hosp.go.jp
Matsuura
N
N
Sasaki
T
T
Murakami
I
I
Schneider
H
H
Higashiyama
S
S
Saitoh
Y
Y
Yamakido
M
M
Taooka
Y
Y
Sheppard
D
D
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D016466:CHO Cells; D006224:Cricetinae; D006801:Humans; D016023:Integrins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D053495:Osteopontin; D011485:Protein Binding; D012795:Sialoglycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013917:Thrombin / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
D016023:Integrins; C506895:SPP1 protein, human; D012795:Sialoglycoproteins; C113256:integrin alpha 9 beta 1; D053495:Osteopontin; D013917:Thrombin