Transformation of polarized epithelial cells by apical mistrafficking of epiregulin

Abstract

Establishment and maintenance of apico-basolateral trafficking pathways are critical to epithelial homeostasis. Loss of polarity and trafficking fidelity are thought to occur as a consequence of transformation; however, here we report that selective mistrafficking of the epidermal growth factor receptor (EGFR) ligand epiregulin (EREG) from the basolateral to the apical cell surface drives transformation. Normally, EREG is preferentially delivered to the basolateral surface of polarized Madin-Darby canine kidney cells. EREG basolateral trafficking is regulated by a conserved tyrosine-based basolateral sorting motif in its cytoplasmic domain (YXXΦ: Y 156 ERV). Both Y156 and V159 are required for basolateral sorting of EREG, because Y156A and V159G substitutions redirect EREG to the apical cell surface. We also show that basolateral sorting of EREG is adaptor protein 1B-independent. Apical mistrafficking of EREG has a distinctive phenotype. In contrast to transient EGFR tyrosine phosphorylation after basolateral EREG stimulation, apical EREG leads to prolonged EGFR tyrosine phosphorylation, which may be related, at least in part, to a lack of negative regulatory Y1045 phosphorylation and subsequent ubiquitylation. Notably, Madin-Darby canine kidney cells stably expressing apically mistrafficked EREG form significantly larger, hyperproliferative, poorly differentiated, and locally invasive tumors in nude mice compared with WT EREG-expressing cells.epithelial transformation | growth factor trafficking | receptor tyrosine kinase | protein sorting U nder normal physiological conditions, binding of endogenous ligand to EGF receptor (EGFR) is required for initiation of signal transduction. In polarized epithelial cells, EGFR is thought to be restricted to the basolateral surfaces (1). All seven EGFR ligands are produced as type 1 transmembrane proteins (2). Newly synthesized ligands are inserted into the plasma membrane, whereupon they undergo ectodomain cleavage to release soluble growth factor. We previously studied the biosynthesis and trafficking of EGF, TGF-α (TGFA), and amphiregulin (AREG) in polarized Madin-Darby canine kidney (MDCK) cells (3-5). These ligands have distinct motifs that govern their basolateral sorting (6-8). Of clinical relevance is a germline mutation in the basolateral sorting motif of EGF in individuals with isolated renal hypomagnesemia (9). In this autosomal recessive disorder, loss of EGF delivery to the basolateral surface impairs EGFR activity in the distal convoluted tubules, which is required for efficient magnesium absorption.Here we undertook the study of another EGFR ligand, epiregulin (EREG). EREG is proteolytically cleaved by a disintegrin and a metalloprotease 17 (ADAM17) to release soluble ligand that binds to and activates ERBB1/EGFR and ERBB4/Her4 (2, 10, 11). Expression of EREG in adults is restricted (12-14), but EREG is overexpressed in a number of human breast and colorectal cancer cell lines and tumors (15)(16)(17)(18)(19). EREG also has been implicated as a "metastasis dri...

Induction of lateral lumens by disruption of a monoleucine-based basolateral sorting motif in betacellulin

“…Previous work from our lab has shown that the basolateral-sorting information of TGFA, AREG and EREG is contained within their respective cytoplasmic domains (Dempsey et al, 2003;Gephart et al, 2011;Singh et al, 2013). The 39-amino-acid cytoplasmic domain of BTC is highly conserved across species (supplementary material Fig.…”

“…S1B) (Nakamura et al, 1995;Singh and Coffey, 2014b). Thus, the EExxxL basolateral-sorting motif is the most common basolateral-sorting motif amongst the family of mammalian EGFR ligands; a PxxP motif in EGF, a dileucine motif in TGFA and a YxxΦ motif in EREG are represented only once (Dempsey et al, 2003;Gephart et al, 2011;Groenestege et al, 2007;Singh et al, 2013).…”

“…EGF receptor (EGFR) signaling plays a vital role in epithelial homeostasis (Vermeer et al, 2003). Although EGFR is largely restricted to the basolateral surface, approximately 10% of EGFRs are present on the apical surface, and EGFR signaling from apical and basolateral surfaces can differ (Singh et al, 2013). EGFR activation is dependent on the binding of one of seven ligands -EGF, transforming growth factor-α (TGFA), amphiregulin (AREG), epiregulin (EREG), heparin-binding EGF-like growth factor (HBEGF), betacellulin (BTC) or epigen (EPGN) (Harris et al, 2003).…”

“…We have previously characterized the trafficking of EGF, TGFA, AREG and EREG in polarized epithelial cells (Dempsey et al, 2003(Dempsey et al, , 1997Gephart et al, 2011;Singh et al, 2013;Singh and Coffey, 2014a,b). These studies have established that spatially controlled ligand trafficking regulates EGFR activity.…”

“…TGFA basolateral sorting depends on a dileucine motif and a cysteine-containing positively-charged juxtamembrane region (HCCQVRKH) (Dempsey et al, 2003). Basolateral sorting of AREG depends on a monoleucine-based motif (EExxxL), whereas a YxxΦ motif directs basolateral-cell-surface delivery of EREG (Brown et al, 2001;Gephart et al, 2011;Singh et al, 2013).…”