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Determination of the CD148-Interacting Region in Thrombospondin-1.

Takahashi K, Sumarriva K, Kim R, Jiang R, Brantley-Sieders DM, Chen J, Mernaugh RL, Takahashi T
PLoS One. 2016 11 (5): e0154916

PMID: 27149518 · PMCID: PMC4858292 · DOI:10.1371/journal.pone.0154916

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.

MeSH Terms (15)

Animals Binding Sites Cell Proliferation Cells, Cultured Endothelial Cells Gene Knockdown Techniques Humans Immunoblotting Immunoprecipitation Mice, Inbred C57BL Neovascularization, Physiologic Peptide Fragments Protein Tyrosine Phosphatases Receptor-Like Protein Tyrosine Phosphatases, Class 3 Thrombospondin 1

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