Trafficking of the EGFR ligand Spitz regulates its signaling activity in polarized tissues

Abstract

SummaryEpidermal growth factor receptor (EGFR) ligands undergo a complex series of processing events during their maturation to active signaling proteins. Like its mammalian homologs, the predominant Drosophila EGFR ligand Spitz is produced as a transmembrane pro-protein. In the secretory pathway, Spitz is cleaved within its transmembrane domain to release the extracellular signaling domain. This domain is modified with an N-terminal palmitate group that tethers it to the plasma membrane. We found that the pro-protein can reach the cell surface in the absence of proteolysis, but that it fails to activate the EGFR. To address why the transmembrane pro-protein is inactive, whereas membrane association through the palmitate group promotes activity, we generated a panel of chimeric constructs containing the Spitz extracellular region fused to exogenous transmembrane proteins. Although the orientation of the EGF domain and its distance from the plasma membrane varies in these chimeras, they are all active in vivo. Thus, tethering Spitz to the membrane via a transmembrane domain at either terminus does not prevent activity. Conversely, removing the N-terminal palmitate group from the C-terminally tethered pro-protein does not render it active. Furthermore, we show that the Spitz transmembrane pro-protein can activate the EGFR in a tissue culture assay, indicating that its failure to signal in vivo is not due to structural features. In polarized imaginal disc cells, unprocessed Spitz pro-protein localizes to apical puncta, whereas the active chimeric Spitz constructs are basolaterally localized. Taken together, our data support the model that localized trafficking of the pro-protein restricts its ability to activate the receptor in polarized tissues.

ESCRT-0 complex modulates Rbf-mutant cell survival by regulating Rhomboid endosomal trafficking and EGFR signaling

“…A recent report has shown that unprocessed Spi can be observed at the apical surface of the polarized epithelia in the presence of Star, but such Spi is inactive owing to the localization of the receptor in the basal lateral region (Steinhauer et al, 2013). In that report, it is suggested that Spi is endocytosed, cleaved by Rhomboid in endosomes and released to the basal lateral region to activate EGFR signaling (Steinhauer et al, 2013).…”

“…In that report, it is suggested that Spi is endocytosed, cleaved by Rhomboid in endosomes and released to the basal lateral region to activate EGFR signaling (Steinhauer et al, 2013). Alternatively, Spi can potentially enter the Rhomboid-containing endosomes from Golgi and be released to the appropriate location after processing by Rhomboid (Yogev et al, 2010).…”

“…For example, although Spi is strictly dependent on Rhomboid and/or Star to activate EGFR, Krn can undergo low-level cleavage and activate EGFR independent of Rhomboid and/or Star . Another possibility is that in some tissues that are not polarized, EGFR signaling can potentially be activated by the unprocessed ligands exported by Star (Steinhauer et al, 2013). In this case, Stam and Hrs mainly regulate EGFR downregulation after ligand binding.…”

Drosophila Condensin II subunit Chromosome-associated Protein-D3 regulates cell fate determination through non-cell-autonomous signaling

“…One possibility is that the EGFR ligand Spitz is responsible for the signaling between the cells, and that in CAP-D3-deficient cells there might be a defect in Spitz localization. It was recently shown that the localization of transmembrane Spitz to the apical region of imaginal disc cells renders it incapable of activating EGFR in neighboring cells (Steinhauer et al, 2013). Both overexpression of secreted Spitz and knockdown of Argos, which associates predominantly with Spitz to prevent EGFR signaling (Vinos and Freeman, 2000), rescue the loss-of-ACV phenotype in CAP-D3-deficient cells (Fig.…”

Drosophila Vps4 promotes Epidermal growth factor receptor signaling independently of its role in receptor degradation

“…It is possible that EGFR is trafficked by distinct routes in imaginal discs and in follicle cells. Grk is internalized through the apical domain of follicle cells (Tanentzapf et al, 2000), whereas active forms of Spi are localized basolaterally in discs (Steinhauer et al, 2013). A role for Vps4 in targeting the EGFR to the appropriate membrane domain would, however, not explain the requirement for Vps4 in cultured S2 cells, which lack apical-basal polarity.…”