Whitaker Hayley C, Stanbury David P B, Brinham Claire, Girling Joanne, Hanrahan Sarah, Totty Nick, Neal David E
Uro-Oncology Research Group, CRUK Cambridge Research Institute, Li Ka Shing Centre, Robinson Way, Cambridge, UK.
Prostate. 2007 Jun 15;67(9):943-54. doi: 10.1002/pros.20580.
Membrane proteins provide the interface between the cell and its environment and are responsible for cell adhesion, mobility, and intracellular signaling. Previous studies have focused on the LNCaP whole cell proteome and transcriptome but little is known about proteins at the prostate cell membrane and how they change in response to androgens.
Following treatment with R1881 or vehicle, membrane proteins of the prostate cancer LNCaP cell line were tagged with biotin using EZ-link sulfo-NHS-LC-biotin. Using the tag membrane proteins were purified and separated using two-dimensional gel electrophoresis and identified using mass spectrometry. E-cadherin and low density lipoprotein receptor (LDLR) were used as positive controls and also investigated following bicalutamide treatment. Membrane localization and androgen-regulation of proteins was confirmed using sub-cellular fractionation, Western blotting and microscopy.
We have demonstrated efficient and specific protein biotinylation and purification of LNCaP plasma membrane proteins using Western analysis. E-cadherin and LDLR were regulated at the cell surface in response to R1881 and bicalutamide. Mass spectrometry identified several androgen-regulated membrane associated proteins including Prx-3 and GRP78 which are known to localize to other cellular compartments as well as the plasma membrane. We confirmed the localization of the identified proteins in LNCaP cells by co-localization with E-cadherin and immunohistochemistry of prostate tissue.
Cell surface biotinylation is an effective technique for identifying membrane proteins in the LNCaP prostate cancer cell line. We have demonstrated the identification of androgen-regulated membrane proteins and their validation in tissue samples.
膜蛋白提供了细胞与其环境之间的界面，负责细胞黏附、迁移和细胞内信号传导。先前的研究聚焦于LNCaP全细胞蛋白质组和转录组，但对于前列腺细胞膜上的蛋白质以及它们如何响应雄激素变化却知之甚少。
用R1881或溶剂处理后，使用EZ-link磺基-NHS-LC-生物素对前列腺癌LNCaP细胞系的膜蛋白进行生物素标记。利用该标记，通过二维凝胶电泳对膜蛋白进行纯化和分离，并用质谱进行鉴定。E-钙黏蛋白和低密度脂蛋白受体（LDLR）用作阳性对照，并在比卡鲁胺处理后进行研究。通过亚细胞分级分离、蛋白质印迹法和显微镜检查来确认蛋白质的膜定位和雄激素调节作用。
我们通过蛋白质印迹分析证明了LNCaP质膜蛋白的高效和特异性蛋白质生物素化及纯化。E-钙黏蛋白和LDLR在细胞表面受R1881和比卡鲁胺的调节。质谱鉴定出几种雄激素调节的膜相关蛋白，包括已知定位于其他细胞区室以及质膜的Prx-3和GRP78。我们通过与E-钙黏蛋白共定位和前列腺组织免疫组化，证实了LNCaP细胞中已鉴定蛋白质的定位。
细胞表面生物素化是鉴定LNCaP前列腺癌细胞系中膜蛋白的有效技术。我们已证明了雄激素调节的膜蛋白的鉴定及其在组织样本中的验证。
