Involvement of the cytoplasmic juxtamembrane region of matriptase in its exclusive localization to the basolateral membrane domain of Madin–Darby canine kidney epithelial cells

Abstract

Matriptase is a type II transmembrane serine protease. This protease is strongly expressed in simple epithelial cells such as enterocytes and kidney tubular cells in which the plasma membranes are separated into apical and basolateral domains. Although matriptase was found previously to occur exclusively on the basolateral membrane of enterocytes, the underlying mechanism of localization is unclear. In the present study, a full-length rat matriptase and a chimera consisting of the cytoplasmic and transmembrane regions of the protease and green fluorescent protein (designated as 1-86GFP) were found to localize exclusively to the basolateral membrane domain when expressed in Madin-Darby canine kidney epithelial cells. Mutagenesis analysis of 1-86GFP revealed that the matriptase cytoplasmic juxtamembrane amino acid residues (Lys45, Val47, and Arg50) play a role in mediating the localization in the cells. This study provides the first evidence that matriptase carries information for its localization in simple epithelia.

The Occurrence of Matriptase C-Terminal Fragments on the Apical and Basolateral Sides of Madin–Darby Canine Kidney Epithelial Cells

“…4) We and others found recently by immunofluorescence microscopy that the matriptase CTF occurs on basolateral membranes but not on apical membranes when a cDNA encoding full-length matriptase was transfected into Madin-Darby canine kidney (MDCK) epithelial cells (this cell line has been widely used as a model of simple epithelial cells). 5,6) These findings suggest that when matriptase is expressed in simple epithelium, it can interact with potential substrates occurring on the basolateral side (e.g., pro-HGF) but not with those occurring on the apical side (e.g., prostasin zymogen), but there remains the possibility that matriptase CTF is also delivered to the apical side of the epithelium. In fact, a soluble matriptase (matriptase CTF) has been found in biological fluids, including milk and urine.…”

“…6) In addition, the NTF was co-purified with CTF from Triton extract of MDCK cells, suggesting that there exist CTF molecules associated with NTF even after cleavage. 6) In the present study, confluent MDCK cells expressing matriptase-Myc/(His) 6 cultured on TranswellÔ were incubated with serum-free medium, which can be condensed by ultrafiltration. Domainselective biotinylation and Western blotting with Tmc172 showed exclusive localization of matriptase NTF (indicated by a 24-kDa band) on the basolateral side under the present experimental conditions (apical, less than 5%, N ¼ 4) (Fig.…”

“…4,6) We have found that matriptase-Myc/(His) 6 is processed post-translationally via cleavage after Gly149, and that the NTF part occurs on the basolateral side but poorly on the apical side when expressed in MDCK cells. 6) In addition, the NTF was co-purified with CTF from Triton extract of MDCK cells, suggesting that there exist CTF molecules associated with NTF even after cleavage. 6) In the present study, confluent MDCK cells expressing matriptase-Myc/(His) 6 cultured on TranswellÔ were incubated with serum-free medium, which can be condensed by ultrafiltration.…”

“…Note that the 24-kDa was not produced when the blot was incubated with Tmc172 that had been preadsorbed with the antigenic peptide. 4,6) Domain-selective biotinylation and Western blotting using anti-Myc antibody did not permit evaluation of distribution of matriptase CTF on the cell-surface membranes (data not shown). Strikingly, however, a 93-kDa protein, which indicates proteolytically-inactive, single-chain matriptase CTF (Fig.…”

“…Strikingly, however, a 93-kDa protein, which indicates proteolytically-inactive, single-chain matriptase CTF (Fig. 1), 6) was found to occur in both the apical and basolateral media (Fig. 2, middle panel, indicated by solid arrowhead).…”