title: Determination of the CD148-Interacting Region in Thrombospondin-1.
abstract: CD148 is a transmembrane protein tyrosine phosphatase that is expressed in multiple cell types, including vascular endothelial cells and duct epithelial cells. Previous studies have shown a prominent role of CD148 to reduce growth factor signals and suppress cell proliferation and transformation. Further, we have recently shown that thrombospondin-1 (TSP1) serves as a functionally important ligand for CD148. TSP1 has multiple structural elements and interacts with various cell surface receptors that exhibit differing effects. In order to create the CD148-specific TSP1 fragment, here we investigated the CD148-interacting region in TSP1 using a series of TSP1 fragments and biochemical and biological assays. Our results demonstrate that: 1) CD148 binds to the 1st type 1 repeat in TSP1; 2) Trimeric TSP1 fragments that contain the 1st type repeat inhibit cell proliferation in A431D cells that stably express wild-type CD148 (A431D/CD148wt cells), while they show no effects in A431D cells that lack CD148 or express a catalytically inactive form of CD148. The anti-proliferative effect of the TSP1 fragment in A431D/CD148wt cells was largely abolished by CD148 knockdown and antagonized by the 1st, but not the 2nd and 3rd, type 1 repeat fragment. Furthermore, the trimeric TSP1 fragments containing the 1st type repeat increased the catalytic activity of CD148 and reduced phospho-tyrosine contents of EGFR and ERK1/2, defined CD148 substrates. These effects were not observed in the TSP1 fragments that lack the 1st type 1 repeat. Last, we demonstrate that the trimeric TSP1 fragment containing the 1st type 1 repeat inhibits endothelial cell proliferation in culture and angiogenesis in vivo. These effects were largely abolished by CD148 knockdown or deficiency. Collectively, these findings indicate that the 1st type 1 repeat interacts with CD148, reducing growth factor signals and inhibiting epithelial or endothelial cell proliferation and angiogenesis.
authors.affiliation: Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America.
authors.forename: Keiko
authors.initials: K
authors.lastname: Takahashi
authors.affiliation: Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America.
authors.forename: Katherine
authors.initials: K
authors.lastname: Sumarriva
authors.affiliation: Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America.
authors.forename: Rachel
authors.initials: R
authors.lastname: Kim
authors.affiliation: Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America.
authors.forename: Rosie
authors.initials: R
authors.lastname: Jiang
authors.affiliation: Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America.|Department of Cancer Biology, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America.
authors.forename: Dana M
authors.initials: DM
authors.lastname: Brantley-Sieders
authors.affiliation: Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America.|Department of Cancer Biology, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America.
authors.forename: Jin
authors.initials: J
authors.lastname: Chen
authors.affiliation: Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America.
authors.forename: Raymond L
authors.initials: RL
authors.lastname: Mernaugh
authors.affiliation: Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America.|Department of Cancer Biology, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America.
authors.forename: Takamune
authors.initials: T
authors.lastname: Takahashi
mesh_terms: D000818:Animals; D001665:Binding Sites; D049109:Cell Proliferation; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000378:metabolism; D055785:Gene Knockdown Techniques; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D008810:Mice, Inbred C57BL; D018919:Neovascularization, Physiologic / Q000502:physiology; D010446:Peptide Fragments / Q000378:metabolism; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism; D054631:Receptor-Like Protein Tyrosine Phosphatases, Class 3 / Q000378:metabolism; D019700:Thrombospondin 1 / Q000378:metabolism*
chemical_list: D010446:Peptide Fragments; D019700:Thrombospondin 1; C585483:Thbs1 protein, mouse; D017027:Protein Tyrosine Phosphatases; C098066:Ptprj protein, mouse; D054631:Receptor-Like Protein Tyrosine Phosphatases, Class 3