Identification of dual alpha 4beta1 integrin binding sites within a 38 amino acid domain in the N-terminal thrombin fragment of human osteopontin.
Previous work from our laboratory demonstrates that the alpha(4)beta(1) integrin is an adhesion receptor for OPN and that alpha(4)beta(1) binding site(s) are present in the N-terminal thrombin fragment of osteopontin (OPN) (Bayless, K. J., Meininger, G. A., Scholtz, J. M., and Davis, G. E. (1998) J. Cell Sci. 111, 1165-1174). The work presented here identifies two alpha(4)beta(1) binding sites within a recombinantly produced N-terminal thrombin fragment of human OPN. Initial experiments, using wild-type OPN containing an RGD sequence or an OPN-RGE mutant, showed identical alpha(4)beta(1)-dependent cell adhesive activity. A strategy to localize alpha(4)beta(1) binding sites within the thrombin fragment of osteopontin involved performing a series of truncation analyses. Removal of the last 39 amino acids (130) completely eliminated adhesion, indicating all binding activity was present within that portion of the molecule. Combined mutation and deletion analyses of this region revealed the involvement of dual alpha(4)beta(1) binding sites. Synthetic peptides for both regions in OPN, ELVTDFPTDLPAT (131) and SVVYGLR (162), were found to block alpha(4)beta(1)-dependent adhesion. The first peptide when coupled to Sepharose bound the alpha(4)beta(1) integrin directly whereas a mutated ELVTEFPTELPAT peptide showed a dramatically reduced ability to bind. These data collectively demonstrate that dual alpha(4)beta(1) integrin binding sites are present in a 38 amino acid domain within the N-terminal thrombin fragment of OPN.
Bayless
K J
KJ
Department of Pathology and Laboratory Medicine, Texas A&M University Health Science Center, College Station, Texas 77843, USA.
Davis
G E
GE
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002846:Chromatography, Affinity; D006801:Humans; D039041:Integrin alpha4beta1; D016023:Integrins / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D053495:Osteopontin; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D016201:Receptors, Lymphocyte Homing / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012795:Sialoglycoproteins / Q000737:chemistry* / Q000378:metabolism*; D013552:Swine; D013917:Thrombin / Q000737:chemistry*
D039041:Integrin alpha4beta1; D016023:Integrins; D010446:Peptide Fragments; D010750:Phosphoproteins; D016201:Receptors, Lymphocyte Homing; D011994:Recombinant Proteins; C506895:SPP1 protein, human; D012795:Sialoglycoproteins; C506897:Spp1 protein, rat; D053495:Osteopontin; D013917:Thrombin