Technical Reference #3269

3269.

Proprotein Convertase PC7 Enhances the Activation of the EGF Receptor Pathway through Processing of the EGF Precursor Estelle Rousselet; Suzanne Benjannet; Edwidge Marcinkiewicz; Marie-Claude Asselin; Claude Lazure; and Nabil G. Seidah, Clinical Research Institute of Montreal, Journal of Biological Chemistry, 286(3269), (2011)
Link To Paper

Abstract:
Although the processing profile of the membrane-bound epidermal growth factor precursor (pro-EGF) is tissue-specific; it has not been investigated at the cellular level nor have the cognate proteinases been defined. Among the proprotein convertases (PCs); only the membrane-bound PC7; the most ancient and conserved basic amino acid-specific PC family member; induces the processing of pro-EGF into an 115-kDa transmembrane form (EGF-115) at an unusual VHPR2902A motif. Because sitedirected mutagenesis revealed that Arg290 is not critical; the generation of EGF-115 by PC7 is likely indirect. This was confirmed by testing a wide range of protease inhibitors; which revealed that the production of EGF-115 is most probably achieved via the activation by PC7 of a latent serine and/or cysteine protease(s). EGF-115 is more abundant at the cell surface than pro-EGF and is associated with a stronger EGF receptor (EGFR) activation; as evidenced by higher levels of phosphorylated ERK1/2. This suggests that the generation of EGF-115 represents a regulatory mechanism of juxtacrine EGFR activation. Thus; PC7 is distinct from the other PCs in its ability to enhance the activation of the cell surface EGFR.

Materials & Methods:
Immunofluorescence—Cells were plated on collagen-coated culture dishes (MatTek) and then transfected the following day. Twenty four hours post-transfection; the cells were fixed for 15 min with 4% paraformaldehyde 1% methanol for PC7 and pro-EGF cell surface labeling to slightly permeabilized cell to have access to the V5 tag or 5 min at 20 °C with 100% methanol for intracellular staining. Cell fixed with paraformaldehyde were incubated 5 min in 150 mM glycine to quench aldehyde groups. Cells were then incubated overnight with primary antibodies. PC7 was detected with rabbit anti-PC7 (1:1;000 (35)) and pro-EGF with mAb V5 (1:200; Invitrogen). Antigen-antibody complexes were revealed by 1 h of incubation with the corresponding species-specific Alexa Fluor (488; 555; or 647)- tagged antibodies (Molecular Probes); and the nuclei were stained by Hoechst 33258 for 1 min (100 g/ml; Sigma). Cells were covered with 1;4-diazabicyclo[2.2.2]octane (Sigma) in PBS; 90% glycerol; and immunofluorescence analyses were performed with a confocal microscope (Zeiss LSM-710).

Microscopic Technique
immunofluorescence analyses on a confocal microscope

Cell Type(s)
HEK293 cells