Proteomic identification of differentially expressed plasma membrane proteins in renal cell carcinoma by stable isotope labelling of a von Hippel-Lindau transfectant cell line model.
The von Hippel-Lindau (VHL) tumour suppressor gene plays a central role in development of clear cell renal cell carcinoma (RCC). Using a cell line pair generated from the VHL-defective RCC cell line UMRC2 by transfection with vector control or VHL (-/+VHL) and stable isotope labelling with amino acids in cell culture (SILAC) followed by enrichment of plasma membrane proteins by cell surface biotinylation/avidin-affinity chromatography and analysis by GeLC-MS/MS, VHL-associated changes in plasma membrane proteins were analysed. Comparative analysis of -/+VHL cells identified 19 differentially expressed proteins which were confirmed by reciprocal SILAC labelling. These included several proteins previously reported to be VHL targets, such as transferrin receptor 1 and the alpha 3 and beta1 integrin subunits and novel findings including upregulation of CD166 and CD147 in VHL-defective cells. Western blotting confirmed these changes and also revealed VHL-dependent alterations in protein form for CD147 and CD166, which in the case of CD166 was shown to be due to differential glycosylation. Analysis of patient-matched normal and malignant renal tissues confirmed these differences were also present in vivo in a subset of clear cell RCCs. These results illustrate the potential of this approach for identifying VHL-dependent proteins that may be important in tumorigenesis.
Aggelis
Vassilis
V
Cancer Research UK Clinical Centre, St. James's University Hospital, Leeds, UK.
Craven
Rachel A
RA
Peng
Jianhe
J
Harnden
Patricia
P
Cairns
David A
DA
Maher
Eamonn R
ER
Tonge
Robert
R
Selby
Peter J
PJ
Banks
Rosamonde E
RE