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BACKGROUND: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Original publication

DOI

10.1002/pros.20580

Type

Journal article

Journal

Prostate

Publication Date

15/06/2007

Volume

67

Pages

943 - 954

Keywords

Androgens, Biotinylation, Cell Line, Tumor, Electrophoresis, Gel, Two-Dimensional, Humans, Male, Membrane Proteins, Metribolone, Neoplasm Proteins, Pilot Projects, Prostatic Neoplasms