Abstract
In this study, the role of the amphiregulin precursor (pro-AR) cytoplasmic domain in the basolateral sorting and cell-surface processing of pro-AR in polarized epithelial cells has been investigated using Madin-Darby canine kidney cells stably expressing various human pro-AR forms. Our results demonstrate that newly synthesized wild-type pro-AR (50 kDa) is delivered directly to the basolateral membrane domain with >95% efficiency, where it is sequentially cleaved within the ectodomain to release several soluble amphiregulin (AR) forms. Analyses of a pro-AR cytoplasmic domain truncation mutant (ARTL27) and two pro-AR secretory mutants (ARsec184 and ARsec190) indicated that the pro-AR cytoplasmic domain is not required for efficient delivery to the plasma membrane, but does contain essential basolateral sorting information. We show that the pro-AR cytoplasmic domain truncation mutant (ARTL27) is not sorted in polarized Madin-Darby canine kidney cells, with approximately 65% of the newly synthesized protein delivered to the apical cell surface. Under base-line conditions, ARTL27 was preferentially cleaved from the basolateral surface with 4-fold greater efficiency compared with cleavage from the apical membrane domain. However, ARTL27 ectodomain cleavage could be stimulated equivalently from either membrane domain by a variety of different stimuli. The metalloprotease inhibitor BB-94 could inhibit both base-line and stimulus-induced ectodomain cleavage of wild-type pro-AR and ARTL27. These results indicate that the pro-AR cytoplasmic domain is required for basolateral sorting, but is not essential for ectodomain processing. Preferential constitutive cleavage of ARTL27 from the basolateral cell surface also suggests that the metalloprotease activity involved in base-line and stimulus-induced ARTL27 ectodomain cleavage may be regulated differently in the apical and basolateral membrane domains of polarized epithelial cells.
Highlights
All six EGF receptor (EGFR) ligands are synthesized as transmembrane precursors that are cleaved to release biologically active soluble growth factors, which act in an autocrine and/or a paracrine manner [2]
We show that ARTL27 is preferentially cleaved from the basolateral membrane domain under constitutive conditions; efficient stimulated cleavage can occur from either membrane domain of polarized epithelial cells
Basolateral distribution of EGFRs has important consequences for defining how functional and productive ligand-mediated receptor signaling can occur in polarized epithelial cells
Summary
In the case of HB-EGF, data from previous reports indicate that membrane-anchored and soluble forms of HB-EGF may have distinct EGFR signaling activities [10, 11] Taken together, these observations suggest an important regulatory role for ectodomain cleavage in EGF-like ligand activities. Cytoplasmic domain sorting information defines spatial compartmentalization of different EGFR ligands in polarized epithelial cells, but can determine ligand accessibility to basolateral EGFRs and modulate constitutive release at the different membrane domains [1, 17, 18]. We demonstrate that newly synthesized pro-AR is directly sorted to the basolateral membrane domain in polarized MDCK cells, where it is sequentially cleaved to release soluble AR forms into the basolateral conditioned medium.
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