Newton Christopher S, Loukinova Elena, Mikhailenko Irina, Ranganathan Sripriya, Gao Yamei, Haudenschild Christian, Strickland Dudley K
Department of Surgery and Physiology, University of Maryland School of Medicine, 15601 Crabbs Branch Way, Rockville, Maryland 20855, USA.
J Biol Chem. 2005 Jul 29;280(30):27872-8. doi: 10.1074/jbc.M505410200. Epub 2005 Jun 8.
Activation of the platelet-derived growth factor receptor-beta (PDGFR-beta) leads to tyrosine phosphorylation of the cytoplasmic domain of LRP and alters its association with adaptor and signaling proteins, such as Shc. The mechanism of the PDGF-induced LRP tyrosine phosphorylation is not well understood, especially since PDGF not only activates PDGF receptor but also binds directly to LRP. To gain insight into this mechanism, we used a chimeric receptor in which the ligand binding domain of the PDGFR-beta was replaced with that from the macrophage colony-stimulating factor (M-CSF) receptor, a highly related receptor tyrosine kinase of the same subfamily, but with different ligand specificity. Activation of the chimeric receptor upon the addition of M-CSF readily mediated the tyrosine phosphorylation of LRP. Since M-CSF is not recognized by LRP, these results indicated that growth factor binding to LRP is not necessary for this phosphorylation event. Using a panel of cytoplasmic domain mutants of the chimeric M-CSF/PDGFR-beta, we confirmed that the kinase domain of PDGFR-beta is absolutely required for LRP tyrosine phosphorylation but that PDGFR-beta-mediated activation of phosphatidylinositol 3-kinase, RasGAP, SHP-2, phospholipase C-gamma, and Src are not necessary for LRP tyrosine phosphorylation. To identify the cellular compartment where LRP and the PDGFR-beta may interact, we employed immunofluorescence and immunogold electron microscopy. In WI-38 fibroblasts, these two receptors co-localized in coated pits and endosomal compartments following PDGF stimulation. Further, phosphorylated forms of the PDGFR-beta co-immunoprecipitated with LRP following PDGF treatment. Together, these studies revealed close association between activated PDGFR-beta and LRP, suggesting that LRP functions as a co-receptor capable of modulating the signal transduction pathways initiated by the PDGF receptor from endosomes.
血小板衍生生长因子受体-β（PDGFR-β）的激活会导致低密度脂蛋白受体相关蛋白（LRP）胞质结构域的酪氨酸磷酸化，并改变其与衔接蛋白和信号蛋白（如Shc）的结合。血小板衍生生长因子（PDGF）诱导的LRP酪氨酸磷酸化机制尚不清楚，尤其是因为PDGF不仅能激活PDGF受体，还能直接与LRP结合。为了深入了解这一机制，我们使用了一种嵌合受体，其中PDGFR-β的配体结合结构域被巨噬细胞集落刺激因子（M-CSF）受体的相应结构域所取代，M-CSF受体是同一亚家族中高度相关的受体酪氨酸激酶，但具有不同的配体特异性。添加M-CSF后，嵌合受体的激活很容易介导LRP的酪氨酸磷酸化。由于LRP不识别M-CSF，这些结果表明，生长因子与LRP的结合对于这一磷酸化事件并非必需。使用一组嵌合M-CSF/PDGFR-β的胞质结构域突变体，我们证实PDGFR-β的激酶结构域对于LRP酪氨酸磷酸化是绝对必需的，但PDGFR-β介导的磷脂酰肌醇3激酶、RasGAP、SHP-2、磷脂酶C-γ和Src的激活对于LRP酪氨酸磷酸化并非必需。为了确定LRP和PDGFR-β可能相互作用的细胞区室，我们采用了免疫荧光和免疫金电子显微镜技术。在WI-38成纤维细胞中，PDGF刺激后，这两种受体共定位于有被小窝和内体区室。此外，PDGF处理后，磷酸化形式的PDGFR-β与LRP共免疫沉淀。总之，这些研究揭示了活化的PDGFR-β与LRP之间的密切关联，表明LRP作为一种共受体，能够在内体中调节由PDGF受体启动的信号转导通路。
