Apical Enrichment of Human EGF Precursor in Madin-Darby Canine Kidney Cells Involves Preferential Basolateral Ectodomain Cleavage Sensitive to a Metalloprotease Inhibitor

Dempsey, Peter J.; Meise, Katherine S.; Yoshitake, Yoshino; Nishikawa, Katsuzo; Coffey, Robert J.

doi:10.1083/jcb.138.4.747

Cited by 87 publications

(100 citation statements)

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“…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)(4)(5). These ligands have distinct motifs that govern their basolateral sorting (6)(7)(8).…”

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confidence: 99%

Transformation of polarized epithelial cells by apical mistrafficking of epiregulin

Singh

Bogatcheva²,

Washington

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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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...

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confidence: 99%

Transformation of polarized epithelial cells by apical mistrafficking of epiregulin

Singh

Bogatcheva²,

Washington

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…EGF is delivered equally to both the apical and basolateral surface, but is selectively cleaved from the basolateral surface (Dempsey et al, 1997). In this study, we have shown that BTC is preferentially localized to the basolateral surface under steady-state conditions in polarized MDCK cells.…”

Section: Preferential Sorting Of Btc In Polarized Epithelial Cellsmentioning

confidence: 68%

“…These studies have established that spatially controlled ligand trafficking regulates EGFR activity. For example, EGF is delivered to both the apical and basolateral surfaces, but is preferentially cleaved from the basolateral surface (Dempsey et al, 1997). Basolateral EGF delivery is dependent on a PxxP motif (where x denotes any amino acid) within its cytoplasmic domain (Groenestege et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

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

Singh

Bogatcheva

Starchenko

et al. 2015

Journal of Cell Science

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Directed delivery of EGF receptor (EGFR) ligands to the apical or basolateral surface is a crucial regulatory step in the initiation of EGFR signaling in polarized epithelial cells. Herein, we show that the EGFR ligand betacellulin (BTC) is preferentially sorted to the basolateral surface of polarized MDCK cells. By using sequential truncations and site-directed mutagenesis within the BTC cytoplasmic domain, combined with selective cell-surface biotinylation and immunofluorescence, we have uncovered a monoleucine-based basolateral-sorting motif (EExxxL, specifically 156 EEMETL 161 ). Disruption of this sorting motif led to equivalent apical and basolateral localization of BTC. Unlike other EGFR ligands, BTC mistrafficking induced formation of lateral lumens in polarized MDCK cells, and this process was significantly attenuated by inhibition of EGFR. Additionally, expression of a cancerassociated somatic BTC mutation (E156K) led to BTC mistrafficking and induced lateral lumens in MDCK cells. Overexpression of BTC, especially mistrafficking forms, increased the growth of MDCK cells. These results uncover a unique role for BTC mistrafficking in promoting epithelial reorganization.

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“…There are suggestions of other ligand-shedding activities, including serine proteases (Pandiella et al 1992;Le Gall et al 2004). Tissue-specific heterogeneity in the molecular weight forms of cleaved ligands also suggests that different sheddases exist (Dempsey et al 1997). Furthermore, given that many signaling components are conserved between flies and mammals, a role for rhomboids in mammalian EGFR ligand cleavage is also possible.…”

Section: Active Mammalian Rhomboidsmentioning

confidence: 99%

Regulation of Receptor Tyrosine Kinase Ligand Processing

Adrain

Freeman

2014

Cold Spring Harbor Perspectives in Biology

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A primary mode of regulating receptor tyrosine kinase (RTK) signaling is to control access of ligand to its receptor. Many RTK ligands are synthesized as transmembrane proteins. Frequently, the active ligand must be released from the membrane by proteolysis before signaling can occur. Here, we discuss RTK ligand shedding and describe the proteases that catalyze it in flies and mammals. We focus principally on the control of EGF receptor ligand shedding, but also refer to ligands of other RTKs. Two prominent themes emerge. First, control by regulated trafficking and cellular compartmentalization of the proteases and their ligand substrates plays a key role in shedding. Second, many external signals converge on the shedding proteases and their control machinery. Proteases therefore act as regulatory hubs that integrate information that the cell receives and translate it into precise outgoing signals. The activation of signaling by proteases is therefore an essential element of the cellular communication machinery.C ells must talk to one another. This principle applies throughout the tree of life: from unicellular bacteria, to the trillions of cells that coordinate to make a mammal. Communication between cells requires dedicated machinery, capable of relaying information across membranes. Transmembrane proteins are therefore essential for signaling. Understanding how this is regulated is paramount. In mammals, receptor tyrosine kinases (RTKs) and their ligands are important examples of such machinery (Schlessinger 2000), controlling many biological processes including development, immunity, tissue repair, and metabolic homeostasis (Ullrich and Schlessinger 1990). They are transmembrane proteins with an extracellular ligand-binding motif and an intracellular kinase domain. As discussed in other chapters, a common mode of RTK activation involves receptor dimerization induced by ligand binding (Lemmon and Schlessinger 2010).Regulated access of ligand to receptor, over distance and time, is key to controlling signaling. Ligands are frequently synthesized as transmembrane forms; when they remain membrane-tethered and cannot diffuse, the range over which they can operate is limited to adjacent cells (Massague and Pandiella 1993; Singh and 1 Present address: Instituto

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Apical Enrichment of Human EGF Precursor in Madin-Darby Canine Kidney Cells Involves Preferential Basolateral Ectodomain Cleavage Sensitive to a Metalloprotease Inhibitor

Cited by 87 publications

References 59 publications

Transformation of polarized epithelial cells by apical mistrafficking of epiregulin

Transformation of polarized epithelial cells by apical mistrafficking of epiregulin

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

Regulation of Receptor Tyrosine Kinase Ligand Processing

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