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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 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.
© 2015. Published by The Company of Biologists Ltd.
Epidermal growth factor (EGF) family ligands are derived by proteolytic cleavage of the ectodomains of integral membrane precursors. Previously, we established that tumor necrosis factor alpha-converting enzyme (TACE/ADAM17) is a physiologic transforming growth factor-alpha (TGF-alpha) sheddase, and we also demonstrated enhanced shedding of amphiregulin (AR) and heparin-binding (HB)-EGF upon restoration of TACE activity in TACE-deficient EC-2 fibroblasts. Here we extended these results by showing that purified soluble TACE cleaved single sites in the juxtamembrane stalks of mouse pro-HB-EGF and pro-AR ectodomains in vitro. For pro-HB-EGF, this site matched the C terminus of the purified human growth factor, and we speculate that the AR cleavage site is also physiologically relevant. In contrast, ADAM9 and -10, both implicated in HB-EGF shedding, failed to cleave the ectodomain or cleaved at a nonphysiologic site, respectively. Cotransfection of TACE in EC-2 cells enhanced phorbol myristate acetate-induced but not constitutive shedding of epiregulin and had no effect on betacellulin (BTC) processing. Additionally, soluble TACE did not cleave the juxtamembrane stalks of either pro-BTC or pro-epiregulin ectodomains in vitro. Substitution of the shorter pro-BTC juxtamembrane stalk or truncation of the pro-TGF-alpha stalk to match the pro-BTC length reduced TGF-alpha shedding from transfected cells to background levels, whereas substitution of the pro-BTC P2-P2' sequence reduced TGF-alpha shedding less dramatically. Conversely, substitution of the pro-TGF-alpha stalk or lengthening of the pro-BTC stalk, especially when combined with substitution of the pro-TGF-alpha P2-P2' sequence, markedly increased BTC shedding. These results indicate that efficient TACE cleavage is determined by a combination of stalk length and scissile bond sequence.
Several polypeptide growth factors related to epidermal growth factor (EGF) have been identified recently, including transforming growth factor-alpha (TGF-alpha), amphiregulin (AR), heparin-binding EGF-like growth factor (HB-EGF), and betacellulin (BTC). These peptides all bind to the EGF receptor (EGFr). In an effort to understand redundancy within this peptide family and interactions among these related peptides, we compared the biological activities of EGF, TGF-alpha, AR, and HB-EGF in an EGF-responsive, nontransformed intestinal epithelial line (RIE-1) and also determined the effect of individual EGF-related peptides on the expression of related family members in these cells. TGF-alpha, AR, HB-EGF, and EGF were equipotent in stimulating [3H]thymidine incorporation by RIE-1 cells and bound the EGFr with equivalent affinity. Each EGF-related peptide induced the mRNA expression of the remaining family members, including BTC. HB-EGF and AR mRNAs were induced rapidly (within 30 min) and to a greater extent than TGF-alpha and BTC mRNAs, suggesting heterogeneity in the molecular mechanisms for induction. This same pattern was observed for all EGF-related peptides tested. A similar pattern of mRNA induction was observed in secondary cultures of human keratinocytes and in LIM1215 colon adenocarcinoma cells. Nuclear run-on analysis showed that induction of AR and HB-EGF is, at least in part, regulated at the level of gene transcription. Concurrent treatment with HB-EGF and cycloheximide resulted in superinduction of HB-EGF and AR, suggesting that these peptides are immediate early genes in RIE-1 cells. Our results demonstrate an equivalent biological response to EGF-related peptides in RIE-1 cells and further indicate that extensive auto-induction and cross-induction occur within the EGF-related peptide family in several EGF-responsive epithelial cell types.