# Shedding of GPP130 by PC7 and Furin: Potential Implication in Lung Cancer Progression
## 2 Results
### GPP130 Is Cleaved in Post-ER Acidic Comparments  
We took advantage of different inhibitors and mutant proteins to determine which cellular compartments GPP130 is cleaved by PC7 and Furin. In HEK293 cells, WB analysis showed that the shedding of GPP130 by PC7 and Furin was abrogated when treated with Brefeldin A (BFA) (Figure 4A), an inhibitor that blocks the transport of proteins from the ER to the cis-Golgi [31]. Shedding was also inhibited when we overexpressed PC7 or Furin in combination with a dominant negative Sar1p (H79G) mutant protein (Figure 4B), a GTP-restricted mutant that blocks COPII vesicle formation [32]. We conclude that the processing of GPP130 occurs in post-ER compartments. It is interesting to note that GPP130 intracellular levels are increased upon both BFA treatment and the co-expression of the Sar1p (H79G) mutant (Figure 4A,B). This suggests that these conditions are, in part, blocking the 'normal' degradative route taken by GPP130 when overexpressed in cells. We next used the alkalizing agent ammonium chloride (NH4 Cl), which blocks the acidification of  
Figure 3: GPP130 cleavage by PC7 and Furin occurs at HRSR70\(\downarrow\)LE and HRSRLEK73\(\downarrow\), respectively, and solely at KPTR27\(\downarrow\)EV for Furin. **(A)** Schematic representation of GPP130 cleavage motifs and released fragments. **(B)** Western blot analysis of cell lysates and media from HEK293 cells overexpressing GPP130-V5 WT and different point mutations (H67A, R68A, R70A, K73A, R148A, K274A, or R277A), with human PC7, human Furin, or pIRES-empty vector (EV) [\(n=3\)]. **(C)** Western blot analysis and quantifications of cell lysates and media from HEK293 cells overexpressing GPP130-V5 WT, GPP130 4 REL (an optimally cleaved mutant), or GPP130 R70A (a PC non-cleavable mutant) co-expressed with human PC7, human Furin, or pIRES-empty vector [\(n=3\)]. The images shown are representative of three independent experiments. intracellular compartments, to test if the shedding occurs in acidic pH compartments, as previously described [30]. Accordingly, treatment of HEK293 cells with NH\({}_{4}\)Cl prevented GPP130 shedding at both S1 and S2 by PC7 and Furin (Figure 4C), suggesting that cleavage at both sites occurs in the acidic environment of the TGN and/or endosomes. Figure 4: GPP130 is cleaved in a post-ER acidic compartment by PC7 and Furin. (**A,B**) Western blot analysis of cell lysates and media from HEK293 cells overexpressing GPP130-V5 WT and human PC7, human Furin, or with pIRES-empty vector (vector) in the presence or absence of BFA (**A**) or (**B**) Sar1p(H79G) variant [\(n=3\)]. Notice the secretion of the s1-fragment by PC7 and Furin and the S2 fragment by Furin. (**C–E**) Western blot analysis and quantifications of cell lysates and media from HEK293 cells overexpressing GPP130-V5 WT, and human PC7, human Furin or with pIRES-empty vector (vector) in the presence or absence of NH\({}_{4}\)Cl (**C**), D6R (**D**) or dec-RVKR-cmk (RVKR) (**E**) [\(n=3\)]. The images shown are representative of three independent experiments. We next compared the effects of the cell surface (non-cell-permeable) inhibitor hexapeptide D-Arg6 (D6R) and the cell-permeable pan-PC general inhibitor decanoyl-RVKR-cmk (RVKR) [33]. The data demonstrated that D6R did not affect either of the two cleavages, supporting the NH\({}_{4}\)Cl data that suggested that neither cleavages occur at the neutral pH of the cell surface (Figure 4D). In contrast, treatment with the irreversible inhibitor dec-RVKR-cmk, which alkalinizes the active site His* in Furin and PC7, abolished almost completely S1 shedding by both PC7 and Furin (Figure 4E), emphasizing that the S1-processing is PC-specific. Unexpectedly, the S2-cleavage by overexpressed Furin is not affected by de-RVKR-cmk, suggesting that it may occur in an acidic environment not readily accessible to dec-RVKR-cmk, possibly late endosomes and/or multivesicular bodies, where GPP130 is known to traffic [21].