# Shedding of GPP130 by PC7 and Furin: Potential Implication in Lung Cancer Progression
## 2 Results
### GPP130 Is Cleaved in Post-ER Acidic Comparments
(**B**) Incuocyte analysis of cellular proliferation of A549 cells overexpressing GPP130-FL, soluble GPP130, the N-terminal fragment (aa 1-67) generated by S1-cleavage and the C-terminal product of the S2-cleavage (aa 278-696) or with PIRES-empty vector (EV) [\(n=3\)]. Additionally, cellular proliferation of naive A549 cells incubated with the conditioned media of HEK293 over expressing sol-GPP130 compared to A549 cells overexpressing soluble GPP130 or the PIRES-empty vector (EV) is also depicted (experimental design shown in the inlet). (**C**) Palmitoylation of GPP130 regulates the proliferation of A549 cells. Incuocyte analysis of cellular proliferation of naive A549 cells overexpressing soluble GPP130 aa(1-67) compared to its non-palmitoylated C6A mutant or the PIRES-empty vector (EV) [\(n=3\)]. The data are normalized to day 0 and proliferation is followed for 96 h. The data are normalized to day 0 and proliferation is followed for 96 h. The statistically significant results (* \(p<0.05\), ** \(p<0.005\)) at the 96 h timepoint are depicted as separate bar graphs. Compared to the EV control, WT GPP130 and sol-GPP130 enhance the growth of A549 cells by \(\sim\)35% and \(-\)85%, respectively (Figure 6B). In contrast, aa 1-67 and aa 278-696 did not significantly affect growth. This suggests that the proliferation-promoting domain of GPP130 may reside in aa 71-696. We next tested whether the secreted sol-GPP130 would have a paracrine growth-enhancing effect. Thus, we either overexpressed a cDNA encoding sol-GPP130 or treated naive A549 cells with media obtained from HEK293 cells overexpressing sol-GPP130 (media swap, Figure 6B). Even though the extent of enhanced cellular growth by media-swapped sol-GPP130 is smaller than that caused by overexpression of this fragment, clearly, sol-GPP130 in the media could enhance cellular growth in a paracrine fashion (Figure 6B). The lesser effect on cellular growth observed could be due to the different media used for maintaining and producing the conditioned media from HEK293 cells (DMEM media) and that used to maintain A549 cells (F12K media), leading us to dilute in a ratio of 1:3 the media of HEK293 cells (and hence the secreted sol-GPP130) by the F12K media. Furthermore, it is possible that expression of sol-GPP130 (artificially) could result in an intracellular activity on growth that is distinct from that seen extracellularly upon the addition of sol-GPP130 to the cells. Indeed, only in the overexpressed sol-GPP130 condition can we detect this form in the cells (Supplementary Figure S1), but not in overexpressed full-length GPP130 + PC7 or Furin (Figures 1 and 4). This suggests a lack of accumulation in cells of the shed form produced by PC7 or Furin cleavage, which may be rapidly secreted. The extracellular form of sol-GPP130 may interact with a receptor(s) at the cell surface and affect growth accordingly, whereas the intracellular GPP130 may have a different mechanism to control cellular growth. We also noticed a Cys6 in the cytosolic tail of GPP130, a potential site of S-palmitoylation, wherein a possible covalent attachment of palmitic acid could take place. Accordingly, we generated a C6A mutant of GPP130 aa(1-67), namely, GPP130 aa(1-67)C6A that could not be palmitoided. Notably, the A549 cellular proliferation data show that upon blocking the potential Cys-palmitoylation of the N-terminal membrane-bound domain, there is a very significant \(\sim\)2.4-fold increase in proliferation (Figure 6C).