Induction of lateral lumens through disruption of a monoleucine-based basolateral-sorting motif in betacellulin.
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 cancer-associated 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.
Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232, USA Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA Epithelial Biology Center, Vanderbilt University School of Medicine, Nashville, TN 37232, USA bhuminder.singh@vanderbilt.edu.
Bhuminder
B
Singh
Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232, USA Epithelial Biology Center, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.
Galina
G
Bogatcheva
Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.
Alina
A
Starchenko
Interdisciplinary Graduate Program, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.
Justine
J
Sinnaeve
Epithelial Biology Center, Vanderbilt University School of Medicine, Nashville, TN 37232, USA Department of Surgery, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.
Lynne A
LA
Lapierre
Vanderbilt Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, TN 37232, USA Cell Imaging Shared Resource, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.
Janice A
JA
Williams
Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA Epithelial Biology Center, Vanderbilt University School of Medicine, Nashville, TN 37232, USA Department of Surgery, Vanderbilt University School of Medicine, Nashville, TN 37232, USA Department of Veteran Affairs Medical Center, Nashville, TN 37232, USA.
James R
JR
Goldenring
Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232, USA Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA Epithelial Biology Center, Vanderbilt University School of Medicine, Nashville, TN 37232, USA Department of Veteran Affairs Medical Center, Nashville, TN 37232, USA.
Robert J
RJ
Coffey
D000595:Amino Acid Sequence; D000818:Animals; D066259:Betacellulin / Q000235:genetics / Q000378:metabolism; D016764:Cell Polarity; D004285:Dogs; D066255:EGF Family of Proteins; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D061985:Madin Darby Canine Kidney Cells; D009154:Mutation; D021382:Protein Sorting Signals / Q000235:genetics; D017434:Protein Structure, Tertiary
D066259:Betacellulin; D066255:EGF Family of Proteins; D021382:Protein Sorting Signals; D066246:ErbB Receptors
Betacellulin; EGFR signaling; Epithelial polarity; Lateral lumens; MDCK cells; Polarized trafficking