Proteomic identification of desmoglein 2 and activated leukocyte cell adhesion molecule as substrates of ADAM17 and ADAM10 by difference gel electrophoresis

Abstract

In contrast with the early view of metalloproteases as simple extracellular matrix-degrading entities, recent findings show that they are highly specific modulators of different signaling pathways involved, positively or negatively, in tumor development. Thus, before considering a given metalloprotease a therapeutic target, it seems advisable to characterize its function by identifying its repertoire of substrates. Here, we present a proteomic approach to identify ADAM17 substrates by difference gel electrophoresis. We found that the shedding of the extracellular domain of the transferrin receptor and those of two cell-cell adhesion molecules, activated leukocyte cell adhesion molecule (ALCAM) and desmoglein 2 (Dsg-2), is increased in cells overexpressing ADAM17. Genetic evidence shows that while ADAM17 is responsible for the shedding of ALCAM, both ADAM17 and ADAM10 can act on Dsg-2. Activation of the epidermal growth factor receptor leads to the upregulation of the shedding of Dsg-2 and to the concomitant upregulation of ADAM17, but not ADAM10, supporting the ability of overexpressed ADAM17 to shed Dsg-2. These results unveil a role of ADAM10 and ADAM17 in the shedding of cell-cell adhesion molecules. Since loss of cell adhesion is an early event in tumor development, these results suggest that ADAM17 is a useful target in anticancer therapy.

FIG. 1.

Proteomic identification of shedding substrates. A. Expression of ADAM17 in parental and stably transfected A431 cells. Lysates from the indicated cells were analyzed by Western blotting with polyclonal antibodies against the cytoplasmic domain of ADAM17. As a control, the same lysates were analyzed with antiactin antibodies. B. Schematic showing the protocol used. The conditioned media (CM) of A431 cells transfected with TACE and treated with or without BB-94 were subjected to WGA-agarose chromatography. The purified glycoproteins were then labeled with the cyanin dyes Cy3 and Cy5 and analyzed by 2D electrophoresis. C. The 2D gel was scanned with different wavelengths to visualize protein patterns corresponding to proteins labeled with Cy3 and Cy5. D. Experimental (red curve) and normalized model (blue curve) frequency distribution of volume ratios for the spots detected in the fluorescence images of the DIGE experiment. The volume of each individual protein spot, represented as a single data point, is plotted in the right axis. Spots in red represent proteins with a greater-than-1.5-fold decrease in the conditioned medium of cells treated with BB94. Spots in blue represent proteins with a greater-than-1.5-fold increase.

FIG. 2.

Analysis of the shedding of TfnR, ALCAM, and Dsg-2 in parental A431 cells and in the same cells transfected with ADAM17. A. Schematics showing the N terminus (N), signal peptide (SP), transmembrane region (TM), and C terminus (C) of the TfnR, ALCAM, and Dsg-2. The prodomain of Dsg-2, processed by furin-like proprotein convertases, is shown as a shaded box following the SP. The thick lines represent the peptides observed by mass spectrometry analysis and the numbers their location in each protein. B. Parental A431 cells or the same cells permanently transfected with ADAM17 were incubated with or without BB-94. Cells were washed, lysed, and cell lysates (L) analyzed by Western blotting with antibodies against the extracellular domains of TfnR, ALCAM, and Dsg-2 as indicated. Conditioned media (M) were concentrated by WGA-agarose chromatography and analyzed similarly. The protein loading corresponding to cell lysates was normalized with antiactin. The protein loading corresponding to medium samples was normalized by Ponceau red staining of the blots. These controls are not shown to simplify the figure. The averages from three independent experiments ± standard deviations are shown. **, Student's t test values (P < 0.01) comparing soluble material produced by A431 to that produced by parental ADAM17 cells.

FIG. 3.

Analysis of the shedding of ALCAM and Dsg-2 in cells genetically deficient for ADAM 17. A. Wild-type CHO cells (WT) or M2 mutant cells were lysed and cell lysates analyzed by Western blotting with antibodies against ADAM17 or ADAM10 as indicated. B. Lysates (L) from wild-type CHO cells or M2 cell mutants and medium samples (M) obtained after 48 h of serum starvation were concentrated by WGA-agarose chromatography and analyzed by Western blotting with antibodies against the extracellular domain of ALCAM. C. Cell lysates and medium samples obtained as for panel B were treated without or with N-glycosidase F as indicated and analyzed by Western blotting as for panel B. D. WT CHO or M2 cells were transiently transfected with the cDNA encoding human Dsg-2. Cell lysates or conditioned media from transfected cells were analyzed by Western blotting with antibodies against the extracellular domain of Dsg-2.

FIG. 4.

Analysis of the shedding of Dsg-2 in cells genetically deficient for ADAM10. A. MEFs from control mice (+/+) or ADAM10 knockout mice (−/−) were lysed and analyzed by Western blotting with polyclonal antibodies against ADAM10 or ADAM17. B. +/+ or −/− MEFs were transiently cotransfected with the cDNA encoding Dsg-2 and a control DNA (−) or the cDNA encoding ADAM10. Cell lysates (L) or conditioned media (M) obtained as for Fig. 3 from transfected cells were analyzed by Western blotting with antibodies against Dsg-2. C. −/− MEFs were transiently cotransfected with the cDNA encoding Dsg-2 and a control DNA (−/−) or ADAM10 (−/− ADAM10), treated with or without BB-94, and lysed. Cell lysates and medium samples were obtained and analyzed as for panel B. D. Microsomal fractions from ADAM10^−/− MEFs permanently transfected with Dsg-2 were extracted with PBS, 1% Triton X-100, 1 M NaCl, or 0.1 M Na₂CO₃ (pH 12.0). Similar proportions of each fraction were analyzed by Western blotting with antibodies against Dsg-2. The efficiency of the extraction was verified by Ponceau red staining of total proteins.

FIG. 5.

Analysis of the shedding of Dsg-2 in cells treated with EGF. A. Parental A431 cells were treated with or without EGF and BB-94 as indicated and lysed. Cell lysates (L) and medium samples (M) were analyzed by Western blotting with antibodies against Dsg-2, ADAM17, or ADAM10 as indicated. Note that when a lysis buffer without BB-94 is used, only pro-ADAM10 is detected in cell lysates from A431 cells (bottom left). Addition of BB-94 allows the detection of mature ADAM10 (bottom left), and thus a buffer containing this metalloprotease inhibitor was used in the right bottom panel. Western blots were quantified, and the averages from three independent experiments ± standard deviations or the averages from two experiments are shown. B. A431 cells were treated with or without EGF and C225 (a monoclonal antibody that blocks the activation of the EGFR) for 48 h. Cell lysates and medium samples were analyzed by Western blotting with antibodies against Dsg-2, ADAM17, phospho-EGFR, or EGFR as indicated.

FIG. 6.

Hypothetical models for the generation of different Dsg-2 species. A. Schematic showing the shedding of Dsg-2 by ADAM10 and ADAM17. Activation of the EGFR leads to the upregulation of ADAM17 and the increase of the production of sDsg-2. Inhibition of ADAM10 and ADAM17 leads to the increase in the availability of Dsg-2 and the cleavage by an unidentified proteolytic activity that generates cell-associated P100 Dsg-2. B. Both full-length Dsg-2 and P100 Dsg-2 could be substrates of ADAM10 and ADAM17.