Center for Vascular and Inflammatory Diseases, University of Maryland School of Medicine, Baltimore, Maryland, USA.
PLoS One. 2013 Jul 26;8(7):e70432. doi: 10.1371/journal.pone.0070432. Print 2013.
The PDGF signaling pathway plays a major role in several biological systems, including vascular remodeling that occurs following percutaneous transluminal coronary angioplasty. Recent studies have shown that the LDL receptor-related protein 1 (LRP1) is a physiological regulator of the PDGF signaling pathway. The underlying mechanistic details of how this regulation occurs have yet to be resolved. Activation of the PDGF receptor β (PDGFRβ) leads to tyrosine phosphorylation of the LRP1 cytoplasmic domain within endosomes and generates an LRP1 molecule with increased affinity for adaptor proteins such as SHP-2 that are involved in signaling pathways. SHP-2 is a protein tyrosine phosphatase that positively regulates the PDGFRβ pathway, and is required for PDGF-mediated chemotaxis. We investigated the possibility that LRP1 may regulate the PDGFRβ signaling pathway by binding SHP-2 and competing with the PDGFRβ for this molecule.
METHODOLOGY/PRINCIPAL FINDINGS: To quantify the interaction between SHP-2 and phosphorylated forms of the LRP1 intracellular domain, we utilized an ELISA with purified recombinant proteins. These studies revealed high affinity binding of SHP-2 to phosphorylated forms of both LRP1 intracellular domain and the PDGFRβ kinase domain. By employing the well characterized dynamin inhibitor, dynasore, we established that PDGF-induced SHP-2 phosphorylation primarily occurs within endosomal compartments, the same compartments in which LRP1 is tyrosine phosphorylated by activated PDGFRβ. Immunofluorescence studies revealed colocalization of LRP1 and phospho-SHP-2 following PDGF stimulation of fibroblasts. To define the contribution of LRP1 to SHP-2-mediated PDGF chemotaxis, we employed fibroblasts expressing LRP1 and deficient in LRP1 and a specific SHP-2 inhibitor, NSC-87877. Our results reveal that LRP1 modulates SHP-2-mediated PDGF-mediated chemotaxis.
CONCLUSIONS/SIGNIFICANCE: Our data demonstrate that phosphorylated forms of LRP1 and PDGFRβ compete for SHP-2 binding, and that expression of LRP1 attenuates SHP-2-mediated PDGF signaling events.
血小板衍生生长因子（PDGF）信号通路在多个生物学系统中发挥重要作用，包括经皮腔内冠状动脉成形术后的血管重塑。最近的研究表明，低密度脂蛋白受体相关蛋白 1（LRP1）是 PDGF 信号通路的生理调节剂。但这种调节的具体机制仍有待解决。PDGF 受体β（PDGFRβ）的激活导致内体中 LRP1 胞质结构域的酪氨酸磷酸化，并产生一种与参与信号通路的衔接蛋白（如 SHP-2）亲和力增加的 LRP1 分子。SHP-2 是一种蛋白酪氨酸磷酸酶，正向调节 PDGFRβ 通路，是 PDGF 介导的趋化作用所必需的。我们研究了 LRP1 通过与 SHP-2 结合并与 PDGFRβ 竞争该分子，从而调节 PDGFRβ 信号通路的可能性。
方法/主要发现：为了量化 SHP-2 与 LRP1 细胞内结构域磷酸化形式之间的相互作用，我们利用纯化的重组蛋白进行 ELISA 分析。这些研究表明 SHP-2 与 LRP1 细胞内结构域和 PDGFRβ 激酶结构域的磷酸化形式具有高亲和力结合。通过使用经过充分表征的动力蛋白抑制剂 dynasore，我们确定 PDGF 诱导的 SHP-2 磷酸化主要发生在内体区室中，这是 LRP1 被激活的 PDGFRβ 酪氨酸磷酸化的相同区室。免疫荧光研究显示，PDGF 刺激成纤维细胞后，LRP1 和磷酸化 SHP-2 发生共定位。为了确定 LRP1 对 SHP-2 介导的 PDGF 趋化作用的贡献，我们使用表达 LRP1 并缺乏 LRP1 和特定 SHP-2 抑制剂 NSC-87877 的成纤维细胞进行实验。我们的结果表明，LRP1 调节 SHP-2 介导的 PDGF 趋化作用。
结论/意义：我们的数据表明，磷酸化的 LRP1 和 PDGFRβ 竞争 SHP-2 结合，LRP1 的表达减弱了 SHP-2 介导的 PDGF 信号事件。
