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-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.
Longhurst
Celia M
CM
Vascular Biology Center of Excellence and the Department of Pharmacology, University of Tennessee Health Science Center and the Veterans Administration Medical Center, Memphis, Tennessee 38163, USA.
Jacobs
Jonathan D
JD
White
Melanie M
MM
Crossno
Joseph T
JT
Fitzgerald
Deborah A
DA
Bao
Jianxong
J
Fitzgerald
Thomas J
TJ
Raghow
Rajendra
R
Jennings
Lisa K
LK
D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D001665:Binding Sites; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D002448:Cell Adhesion; D002465:Cell Movement; D002478:Cells, Cultured; D006224:Cricetinae; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D005353:Fibronectins / Q000494:pharmacology*; D017353:Gene Deletion; D008562:Membrane Glycoproteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry; D010641:Phenotype; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D060245:Tetraspanin 29; D014162:Transfection
D015703:Antigens, CD; D018076:DNA, Complementary; D005353:Fibronectins; D008562:Membrane Glycoproteins; D010455:Peptides; D011994:Recombinant Proteins; D060245:Tetraspanin 29; D002118:Calcium