Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.
Proc Natl Acad Sci U S A. 2012 Feb 7;109(6):1985-90. doi: 10.1073/pnas.1106171109. Epub 2012 Jan 23.
CD148 is a receptor-type protein tyrosine phosphatase that is expressed in several cell types, including vascular endothelial cells and duct epithelial cells. Growing evidence demonstrates a prominent role for CD148 in negative regulation of growth factor signals, suppressing cell proliferation and transformation. However, its extracellular ligand(s) remain unknown. To identify the ligand(s) of CD148, we introduced HA-tagged CD148 into cultured endothelial cells and then isolated its interacting extracellular protein(s) by biotin surface labeling and subsequent affinity purifications. The binding proteins were identified by mass spectrometry. Here we report that soluble thrombospondin-1 (TSP1) binds to the extracellular part of CD148 with high affinity and specificity, and its binding increases CD148 catalytic activity, leading to dephosphorylation of the substrate proteins. Consistent with these findings, introduction of CD148 conferred TSP1-mediated inhibition of cell growth to cells which lack CD148 and TSP1 inhibition of growth. Further, we demonstrate that TSP1-mediated inhibition of endothelial cell growth is antagonized by soluble CD148 ectodomain as well as by CD148 gene silencing. These findings provide evidence that CD148 functions as a receptor for TSP1 and mediates its inhibition of cell growth.
CD148 是一种受体型蛋白酪氨酸磷酸酶，在多种细胞类型中表达，包括血管内皮细胞和胆管上皮细胞。越来越多的证据表明 CD148 在负向调控生长因子信号方面发挥着重要作用，抑制细胞增殖和转化。然而，其细胞外配体（s）仍然未知。为了鉴定 CD148 的配体（s），我们将 HA 标记的 CD148 导入培养的内皮细胞中，然后通过生物素表面标记和随后的亲和纯化来分离其相互作用的细胞外蛋白（s）。通过质谱鉴定结合蛋白。在这里，我们报告可溶性血小板反应蛋白-1（TSP1）以高亲和力和特异性与 CD148 的细胞外部分结合，其结合增加了 CD148 的催化活性，导致底物蛋白去磷酸化。与这些发现一致的是，CD148 的引入赋予了缺乏 CD148 和 TSP1 的细胞 TSP1 介导的生长抑制作用，以及 TSP1 对生长的抑制作用。此外，我们证明 TSP1 介导的内皮细胞生长抑制作用可以被可溶性 CD148 胞外结构域以及 CD148 基因沉默拮抗。这些发现为 CD148 作为 TSP1 的受体并介导其抑制细胞生长提供了证据。
