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First published online 11 March 2003
doi: 10.1242/jcs.00394

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Furin interacts with proMT1-MMP and integrin {alpha}V at specialized domains of renal cell plasma membrane

Gaétan Mayer1, Guy Boileau2 and Moïse Bendayan1,*

1 Department of Pathology and Cell Biology, Université de Montréal, Montreal, Quebec, H3C 3J7, Canada
2 Department of Biochemistry, Université de Montréal, Montreal, Quebec, H3C 3J7, Canada



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  		Fig. 1. Expression of endogenous furin in normal rat glomerulus. (A) Cryosection labeled with the rabbit anti-furin-N-terminus and FITC-labeled secondary antibodies. The fluorescence revealing furin antigenic sites is found in perinuclear regions (asterisks) of glomerular cells and along basement membranes (arrows). Bar, 10 µm. (B-D) Immunogold electron microscopy. (B) Golgi area within the cell body of a podocyte. Gold particles are present over the Golgi cisternae (G) and the rough endoplasmic reticulum (RER). The nucleus (N) and mitochondria (M) display very few gold particles. (C) Glomerular wall. The plasma membranes of the endothelium (End) and podocytes (P) are decorated by gold particles. Slit diaphragms (arrowheads) of podocytes are preferentially labeled. (D) Over the endothelial cell, labeling for furin is particularly focalized around the fenestrations (arrows). (E) Control conditions. The anti-furin antibody was preadsorbed with an excess of its antigen. The glomerular wall is exempt of gold particles. (B-E) Bars, 200 nm. Abbreviations: Cap, capillary lumen; GBM, glomerular basement membrane; US, urinary space. (F) Glomeruli homogenates were resolved by 10% SDS-PAGE and proteins were electrotransferred onto nitrocellulose membranes. The rabbit anti-furin-N-terminus and anti-proMT1-MMP antibodies were used for immunoblotting and were revealed by chemiluminescence. Single bands were obtained for furin (~98 kDa) and proMT1-MMP (~63 kDa).

 


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  		Fig. 2. In vivo overexpressed furin is localized to the Golgi and the cell surface. 24 hours after the injection of plasmids pCDNA3/RSV/Furin (Fur) (A,C) and pCDNA3/RSV/Furin-HA (Fur-HA) (B,D,E) into the tail vein of mice, kidneys were prepared for immunocytochemistry. (A) Furin immunofluorescence on cryosection is found over most of the glomerular cells. (B) Cryosection of kidney transduced with the HA-tagged furin and labeled with the anti-HA antibody; the immunofluorescence is restricted to those cells expressing the exogenous furin-HA. (A,B) The labeling is found in the perinuclear region (asterisks), in vesicular structures and on plasma membranes (arrows). (C-E) Immunogold on furin- and furin-HA-transduced cells. The many gold particles revealing furin (C) and furin-HA (D) are located at the cell surface of podocytes (P) and endothelial cells (End). Labeling is preferentially associated with the slit diaphragms (arrowheads) and endothelial fenestrations (arrows). (E) Golgi apparatus within the cell body of a podocyte transduced with furin-HA. The gold particles are found concentrated on one side of the Golgi, most probably the TGN. (A,B) Bars, 5 µm. (C-E) Bars, 200 nm.

 


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  		Fig. 3. Subcellular localization of proMT1-MMP to the slit diaphragm. Immunogold on normal rat kidney using the pro-domain-specific anti-proMT1-MMP IgGs with protein-A/10-nm-gold. ProMT1-MMP antigenic sites are mainly located at the base of the podocyte foot processes (P), along the GBM at points of insertion of the slit diaphragms (arrows). The gold particles revealing the precursor are also found over the ablumenal side of the endothelium (End), facing the GBM (not depicted on this micrograph). Abbreviations: Cap, capillary lumen; GBM, glomerular basement membrane; US, urinary space. Bar, 200 nm.

 


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  		Fig. 4. Furin colocalizes with proMT1-MMP in intracellular compartments as well as at the plasma membrane of glomerular cells. (A-D) Double labeling of furin (10 nm gold particles) and proMT1-MMP (5 nm gold particles). (A) Both labels are present over the Golgi apparatus (G). Several close colocalizations of the large and small gold particles are visible (arrows). (B) Furin and proMT1-MMP labelings are found in close proximity (encircled) over podocytic vacuoles (V) and the basal surface of podocytes. (C) Higher magnification depicting colocalization of furin and proMT1-MMP (encircled) over the slit diaphragm area and the endothelial fenestrations (D). Bars, 200 nm. (E,F) Immunoprecipitation of total membrane fractions of glomeruli (Memb), treated with DSP (lane 1) or untreated (lane 2), with the anti-proMT1-MMP. The material recovered was separated by 10% SDS-PAGE under reducing conditions and transferred onto nitrocellulose. (E) Immunoblotting with the rabbit anti-furin-N-terminus antibody revealed one band of ~98 kDa (arrowhead; lane 1). (F) The same blot shown in E, stripped and reprobed for proMT1-MMP. One band at ~63 kDa (arrowhead) is detected independently of chemical crosslinking (lanes 1,2). Notice that, because the immunoprecipitation and the western blotting were conducted with rabbit polyclonal antibodies, there is an immunoglobulin heavy chain band at ~55 kDa in each lane. Lane 3, control. Untreated isolated membrane immunoprecipitated with normal rabbit Ig (ctl Ig).

 


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  		Fig. 5. Cleavage of MT1-MMP pro-domain by furin in isolated glomerular membranes. (A,B) Total membrane fractions of isolated rat glomeruli, untreated (lane 1) or digested with recombinant soluble furin (lane 2), were resolved by 7.5% SDS-PAGE and analysed by western blotting. (A) The 63 kDa band revealing proMT1-MMP in native membranes (lane 1) disappears upon addition of soluble recombinant furin (lane 2), indicating that cleavage of the pro-domain has occurred. (B) In order to assess the integrity of other glomerular membrane proteins, the same blot as in A was stripped and reprobed for the ß3 integrin subunit. The appearance of a ~110 kDa band in both lanes indicates the specificity of the limited proteolysis of proMT1-MMP by furin.

 


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  		Fig. 6. {alpha}V and furin colocalize to the slit diaphragm. (A) Immunogold with the rabbit anti-integrin-{alpha}V antibody and protein-A/10-nm-gold. The gold particles are located over the slit diaphragms (arrowheads) and endothelial fenestrations (arrows). (B) Double immunogold labeling. The {alpha}V (5 nm gold particles) and furin (10 nm gold particles) colocalize to the base of podocytes and over the slit diaphragms (encircled). Bars, 200 nm. (C,D) Total membrane fractions of isolated glomeruli (Memb), treated with DSP (lane 1) or untreated (lane 2), were immunoprecipitated with the anti-{alpha}V-integrin antibody. The resulting material was separated by 10% SDS-PAGE under reducing conditions and analysed by western blotting. (C) The detection of anti-furin antibodies revealed one band of ~98 kDa (arrowhead; lane 1). (D) After stripping the blot shown in C and reprobing it for {alpha}V, one band at ~25 kDa (arrowhead), the molecular mass of the reduced {alpha}V C-terminus, is detected independently of chemical crosslinking (lanes 1,2). This result supports the specificity of the immunoprecipitations. Lane 3, control. Untreated isolated membrane immunoprecipitated with normal rabbit Ig (ctl Ig).

 

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© The Company of Biologists Ltd 2003