Polarity and lipid raft association of the components of the ciliary neurotrophic factor receptor complex in Madin-Darby canine kidney cells
Polarity and lipid raft association of the components of the ciliary neurotrophic factor receptor complex in Madin-Darby canine kidney cells
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DOI：
10.1242/jcs.01049
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发表时间：
2004-04-15
影响因子：
4
通讯作者：
Graeve, L
中科院分区：
文献类型：
--
作者：
Buk, DM;Waibel, M;Graeve, L
睫状神经营养因子（CNTF）通过由糖基磷脂酰肌醇（GPI）锚定的CNTF受体（CNTF- r）、白血病抑制因子受体（liff - r）和白细胞介素-6 （IL-6）信号传感器gp130组成的三方受体复合物传递信号。我们最近报道了gp130在极化上皮模型细胞系Madin-Darby犬肾（MDCK）中内源性表达，并且我们已经证明了该蛋白的优先基底外侧定位。在目前的研究中，我们发现MDCK细胞也表达LIF- r，并通过激活信号传感器和转录激活因子3 （STAT3）的酪氨酸磷酸化来响应人LIF的刺激，但两者都以非极化的方式。这表明MDCK细胞可能是LIE的靶细胞。我们进一步在MDCK细胞中稳定地表达了人类CNTF-R，并通过两种不同的实验发现了一个根尖定位。与这些发现一致的是，当CNTF被添加到细胞顶端时，对CNTF- r阳性细胞的刺激只导致STAT3的激活。这些数据表明，CNTF受体复合物的每个亚基在极化细胞中具有不同的分布，这可能反映了各自细胞因子在体内发挥的不同作用。由于目前认为脂筏参与信号转导和蛋白质分选，我们使用不同的方案研究了三种受体复合物组分与膜筏的关联。尽管CNTF-R与脂筏的定量分离与使用的方法无关，但当使用洗涤剂进行分离时，发现gp130和LIF-R仅与脂筏部分相关。这些发现可能表明，这三种受体复合物亚基要么定位于同一种筏体，但对液体有序环境的亲和力不同，要么它们定位于不同类型的筏体。CNTF-、LIF-和il -6依赖性STAT3激活对降胆固醇药物甲基- β -环糊精（MCD）敏感，这表明脂质筏的完整性对il -6型细胞因子诱导的STAT3激活很重要。
Ciliary neurotrophic factor (CNTF) signals via a tripartite receptor complex consisting of the glycosylphosphatidylinositol (GPI)-anchored CNTF receptor (CNTF-R), the leukaemia inhibitory factor receptor (LIF-R) and the interleukin-6 (IL-6) signal transducer gp130. We have recently reported that gp130 is endogenously expressed in the polarised epithelial model cell line Madin-Darby canine kidney (MDCK) and we have demonstrated a preferential basolateral localisation of this protein. In the present study we show that MDCK cells also express the LIF-R and respond to stimulation with human LIF by activation of tyrosine phosphorylation of signal transducer and activator of transcription-3 (STAT3), both however in an unpolarised fashion. This suggests that MDCK cells may be target cells for LIE We have furthermore stably expressed the human CNTF-R in MDCK cells and by two different assays we found an apical localisation. Consistent with these findings, stimulation of CNTF-R-positive cells resulted only in an activation of STAT3 when CNTF was added apically. These data demonstrate that each subunit of the CNTF receptor complex has a distinct distribution in polarised cells which may reflect the different roles the respective cytokines play in vivo.Since it is currently believed that lipid rafts are involved in signal transduction as well as protein sorting we studied the association of the three receptor complex components with membrane rafts using different protocols. Whereas the CNTF-R cofractionated quantitatively with lipid rafts independently of the method used, gp130 and the LIF-R were found to associate with lipid rafts only partially when detergents were used for isolation. These findings could indicate that either the three receptor complex subunits are localised to the same kind of raft but with different affinities to the liquid-ordered environment, or that they are localised to different types of rafts. CNTF-, LIF-, and IL-6-dependent STAT3 activation was sensitive to the cholesterol-depleting drug methyl-beta-cyclodextrin (MCD) suggesting that the integrity of lipid rafts is important for IL-6-type cytokine-induced STAT activation.