Osteopontin N-terminal domain contains a cryptic adhesive sequence recognized by alpha9beta1 integrin.
Osteopontin is an adhesive glycoprotein implicated in numerous diseases associated with inflammation and remodeling. There are several structural domains in osteopontin that are of particular interest. The RGD motif is a cell attachment sequence shown to be critical for cell adhesion through alphav-containing integrins. In close proximity to the RGD domain is the thrombin cleavage site. Previous observations suggest that thrombin cleavage of osteopontin occurs in vivo and may be physiologically important. To study the functional significance of osteopontin cleavage by thrombin, we made glutathione S-transferase-osteopontin fusion proteins. These proteins contain either the N- or C-terminal domains expected to be formed following thrombin cleavage at the Arg169-Ser170 peptide bond. We compared these osteopontin fragments with native osteopontin in their ability to support adhesion of several different cell lines and identified the receptors mediating these interactions. Our data show that the N-terminal osteopontin fragment, which contains the RGD domain, supports adhesion of a melanoma cell line that is unable to bind native osteopontin. This suggests that osteopontin adhesive interactions may be regulated by thrombin cleavage. We also demonstrate that osteopontin contains a cryptic binding activity, which can be recognized by a novel osteopontin receptor. This receptor has been identified as the alpha9beta1 integrin.
Department of Pathology, University of Washington, Seattle, Washington 98195, USA. ceci@u.washington.edu
L L
LL
Smith
H K
HK
Cheung
L E
LE
Ling
J
J
Chen
D
D
Sheppard
R
R
Pytela
C M
CM
Giachelli
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002448:Cell Adhesion; D002460:Cell Line; D016207:Cytokines / Q000737:chemistry* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005434:Flow Cytometry; D006801:Humans; D039001:Integrin alpha Chains; D019012:Integrin beta1 / Q000378:metabolism*; D016023:Integrins / Q000378:metabolism*; D009842:Oligopeptides / Q000378:metabolism; D053495:Osteopontin; D011994:Recombinant Proteins / Q000737:chemistry; D012795:Sialoglycoproteins / Q000737:chemistry* / Q000378:metabolism; D014407:Tumor Cells, Cultured
D016207:Cytokines; D039001:Integrin alpha Chains; D019012:Integrin beta1; D016023:Integrins; D009842:Oligopeptides; D011994:Recombinant Proteins; C506895:SPP1 protein, human; D012795:Sialoglycoproteins; C084613:integrin alpha9; D053495:Osteopontin; C047981:arginyl-glycyl-aspartic acid