Labeling and identification of LNCaP cell surface proteins: a pilot study.
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.


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.


CONCLUSIONS
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.
Whitaker
Hayley C
HC
Uro-Oncology Research Group, CRUK Cambridge Research Institute, Li Ka Shing Centre, Robinson Way, Cambridge, UK. hayley.whitaker@cancer.org.uk
Stanbury
David P B
DP
Brinham
Claire
C
Girling
Joanne
J
Hanrahan
Sarah
S
Totty
Nick
N
Neal
David E
DE
D000728:Androgens / Q000494:pharmacology; D019921:Biotinylation; D045744:Cell Line, Tumor; D015180:Electrophoresis, Gel, Two-Dimensional; D000091342:Endoplasmic Reticulum Chaperone BiP; D006801:Humans; D008297:Male; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D015741:Metribolone / Q000494:pharmacology; D009363:Neoplasm Proteins / Q000302:isolation & purification / Q000378:metabolism; D010865:Pilot Projects; D011471:Prostatic Neoplasms / Q000473:pathology*
D000728:Androgens; D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D015741:Metribolone