Molecular dissection of the human α2-macroglobulin subunit reveals domains with antagonistic activities in cell signaling
Molecular dissection of the human α2-macroglobulin subunit reveals domains with antagonistic activities in cell signaling
复制标题
DOI：
10.1074/jbc.m801762200
复制
发表时间：
2008-07-18
影响因子：
4.8
通讯作者：
Gonias, Steven L.
中科院分区：
文献类型：
--
作者：
Mantuano, Elisabetta;Mukandala, Gatambwa;Gonias, Steven L.
α(2)-巨球蛋白(αM-2)是一种血浆蛋白水解酶抑制物，它可逆地结合生长因子，并以其激活的形式与低密度脂蛋白受体相关蛋白(LRP-1)结合，LRP-1是一种具有细胞信号活性的内源性受体。因为αM-2中不同的结构域负责其各种功能，我们假设αM-2对细胞生理的总体影响反映了多个结构域的整合活动，其中一些可能是拮抗的。为了验证这一假设，我们将生长因子载体位点和LRP-1识别结构域(RBD)表达为单独的GST融合蛋白(分别为FP3和FP6)。FP6在雪旺细胞和PC12细胞中迅速而有力地激活Akt和ERK/MAP激酶。这种反应被LRP-1基因沉默或与LRP-1拮抗剂受体相关蛋白共同孵育所阻断。突变的Lys(1370)和Lys(1374)也阻断了Fp6的活性，从而阻止了LRP-1的结合。在对神经生长因子-β(NGF-β)的反应中，FP3阻断Akt和ERK/MAP激酶的激活，但不能阻断FP6。在PC12细胞中，FP6促进轴突生长和生长相关蛋白-43的表达，而当加入NGF-β时，FP3拮抗同样的反应。通过蛋白分解和层析从血浆纯化的αM-2中分离得到18 kDa RBD，复制了FP6在PC12细胞中触发LRP-1依赖的细胞信号的能力。我们认为，完整的αM-2对细胞生理的影响反映了与不同结构域相关的活动的渗透程度，如FP3和FP6，它们可能受到细胞微环境中构象变化和其他调控蛋白的异步调节。
alpha(2)-Macroglobulin (alpha M-2) is a plasma protease inhibitor, which reversibly binds growth factors and, in its activated form, binds to low density lipoprotein receptor-related protein (LRP-1), an endocytic receptor with cell signaling activity. Because distinct domains in alpha M-2 are responsible for its various functions, we hypothesized that the overall effects of alpha M-2 on cell physiology reflect the integrated activities of multiple domains, some of which may be antagonistic. To test this hypothesis, we expressed the growth factor carrier site and the LRP-1 recognition domain (RBD) as separate GST fusion proteins (FP3 and FP6, respectively). FP6 rapidly and robustly activated Akt and ERK/MAP kinase in Schwann cells and PC12 cells. This response was blocked by LRP-1 gene silencing or by co-incubation with the LRP-1 antagonist, receptor-associated protein. The activity of FP6 also was blocked by mutating Lys(1370) and Lys(1374), which precludes LRP-1 binding. FP3 blocked activation of Akt and ERK/MAP kinase in response to nerve growth factor-beta (NGF-beta) but not FP6. In PC12 cells, FP6 promoted neurite outgrowth and expression of growth-associated protein-43, whereas FP3 antagonized the same responses when NGF-beta was added. The ability of FP6 to trigger LRP-1-dependent cell signaling in PC12 cells was reproduced by the 18-kDa RBD, isolated from plasma-purified alpha M-2 by proteolysis and chromatography. We propose that the effects of intact alpha M-2 on cell physiology reflect the degree of penetration of activities associated with different domains, such as FP3 and FP6, which may be regulated asynchronously by conformational change and by other regulatory proteins in the cellular microenvironment.