Chinese hamster ovary cell motility to fibronectin is modulated by the second extracellular loop of CD9. Identification of a putative fibronectin binding site

CD9, a member of the tetraspanin family of proteins, is characterized by four transmembrane domains and two extracellular loops. Surface expression of CD9 on Chinese hamster ovary (CHO) cells dramatically enhances spreading and motility on fibronectin. To elucidate the mechanistic basis of CD9-fibro...

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Bibliographic Details
Published in:The Journal of biological chemistry 2002-09, Vol.277 (36), p.32445
Main Authors: Longhurst, Celia M, Jacobs, Jonathan D, White, Melanie M, Crossno, Jr, Joseph T, Fitzgerald, Deborah A, Bao, Jianxong, Fitzgerald, Thomas J, Raghow, Rajendra, Jennings, Lisa K
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
Antigens, CD - chemistry
Antigens, CD - metabolism
Antigens, CD - physiology
Binding Sites
Calcium - metabolism
Cell Adhesion
Cell Movement
Cells, Cultured
CHO Cells
Cricetinae
DNA, Complementary - metabolism
Dose-Response Relationship, Drug
Enzyme-Linked Immunosorbent Assay
Fibronectins - pharmacology
Gene Deletion
Membrane Glycoproteins
Molecular Sequence Data
Mutagenesis, Site-Directed
Peptides - chemistry
Phenotype
Protein Structure, Tertiary
Recombinant Proteins - metabolism
Sequence Homology, Amino Acid
Tetraspanin 29
Transfection
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Summary:CD9, a member of the tetraspanin family of proteins, is characterized by four transmembrane domains and two extracellular loops. Surface expression of CD9 on Chinese hamster ovary (CHO) cells dramatically enhances spreading and motility on fibronectin. To elucidate the mechanistic basis of CD9-fibronectin interaction, binding to fibronectin was investigated using purified and recombinant forms of CD9. The affinity of fibronectin for CD9 in enzyme-linked immunosorbent assay was 81 +/- 25 nm. The binding of fibronectin to immobilized CD9 was enhanced by Ca(2+) ions. Protein binding and peptide competition studies demonstrated that peptide 6 derived from CD9 extracellular loop 2 (amino acids 168-192) contained part of the fibronectin-binding domain. Additionally, enhanced adhesion of CD9-CHO-B2 cells to fibronectin was significantly reduced by peptide 6. CD9-CHO cells had a 5-fold increase in motility to fibronectin as compared with mock-transfected controls, an effect that correlated with CD9 cell surface density. Truncation of CD9 extracellular loop 2 and peptide 6 caused inhibition of CD9-CHO cell motility to fibronectin. Deletion of CD9 extracellular loop 1 had no significant effect on CHO cell motility. These findings demonstrate a critical role for CD9 extracellular loop 2 in cell motility to fibronectin and clarify the mechanism by which CD9-fibronectin interaction modulates cell adhesion and motility.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M204420200