A regulated interaction between alpha5beta1 integrin and osteopontin.
The extracellular matrix protein osteopontin (OPN) interacts with a number of integrins, namely alphavbeta1, alphavbeta3, alphavbeta5, alpha9beta1, alpha8beta1, and alpha4beta1. We have investigated the interaction of alpha5beta1 integrin with OPN using K562 cells, which only express alpha5beta1. alpha5beta1 is in a low activation state in this cell line, but can be stimulated to a higher activation state by the phorbol ester TPA. Treating K562 wild-type cells (K562-WT) with TPA stimulated an interaction between alpha5beta1 and OPN. No interaction was seen in the absence of TPA. alpha5beta1 selectively interacted with a GST fusion protein of the N-terminal fragment of OPN (aa17-168), which is generated in vivo by thrombin cleavage of OPN. Expression of the alpha4 integrin in K562 cells (K562-alpha4beta1) stimulated alpha5beta1-dependent binding to aa17-168 in the absence of TPA, suggesting that alpha4beta1 activates alpha5beta1 in K562 cells. Adhesion via alpha5beta1 is mediated by the Arg-Gly-Asp (RGD) motif of OPN, as mutating this sequence to Arg-Ala-Asp (RAD) blocked binding of both cell types. These data demonstrate that thrombin cleavage regulates the adhesive properties of OPN and that alpha5beta1 integrin can interact with thrombin-cleaved osteopontin when in a high activation state.
Barry
S T
ST
Molecular Pharmacology, Glaxo Wellcome Medicines Research Centre, Gunnels Wood Road, Stevenage, SG1 2NY, United Kingdom. stb38557@glaxowellcome.co.uk
Ludbrook
S B
SB
Murrison
E
E
Horgan
C M
CM
D000595:Amino Acid Sequence; D002448:Cell Adhesion / Q000187:drug effects; D016207:Cytokines / Q000502:physiology; D006801:Humans; D020014:K562 Cells; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides; D053495:Osteopontin; D010446:Peptide Fragments / Q000378:metabolism; D017469:Receptors, Fibronectin / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012795:Sialoglycoproteins / Q000502:physiology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013917:Thrombin / Q000378:metabolism; D014162:Transfection
D016207:Cytokines; D009842:Oligopeptides; D010446:Peptide Fragments; D017469:Receptors, Fibronectin; D011993:Recombinant Fusion Proteins; C506895:SPP1 protein, human; D012795:Sialoglycoproteins; D053495:Osteopontin; C047981:arginyl-glycyl-aspartic acid; D013917:Thrombin; D013755:Tetradecanoylphorbol Acetate