A dileucine motif targets E-cadherin to the basolateral cell surface in Madin-Darby canine kidney and LLC-PK1 epithelial cells.
E-cadherin is a major adherens junction protein of epithelial cells, with a central role in cell-cell adhesion and cell polarity. Newly synthesized E-cadherin is targeted to the basolateral cell surface. We analyzed targeting information in the cytoplasmic tail of E-cadherin by utilizing chimeras of E-cadherin fused to the ectodomain of the interleukin-2alpha (IL-2alpha) receptor expressed in Madin-Darby canine kidney and LLC-PK(1) epithelial cells. Chimeras containing the full-length or membrane-proximal half of the E-cadherin cytoplasmic tail were correctly targeted to the basolateral domain. Sequence analysis of the membrane-proximal tail region revealed the presence of a highly conserved dileucine motif, which was analyzed as a putative targeting signal by mutagenesis. Elimination of this motif resulted in the loss of Tac/E-cadherin basolateral localization, pinpointing this dileucine signal as being both necessary and sufficient for basolateral targeting of E-cadherin. Truncation mutants unable to bind beta-catenin were correctly targeted, showing, contrary to current understanding, that beta-catenin is not required for basolateral trafficking. Our results also provide evidence that dileucine-mediated targeting is maintained in LLC-PK(1) cells despite the altered polarity of basolateral proteins with tyrosine-based signals in this cell line. These results provide the first direct insights into how E-cadherin is targeted to the basolateral membrane.
Miranda
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KC
Institute for Molecular Bioscience, the Department of Biochemistry, and the Department of Physiology & Pharmacology, University of Queensland, Brisbane, Queensland 4072, Australia.
Khromykh
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Christy
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Gottardi
C J
CJ
Yap
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Stow
J L
JL
Teasdale
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D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001485:Basement Membrane / Q000378:metabolism; D015820:Cadherins / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D017931:DNA Primers; D004285:Dogs; D004847:Epithelial Cells / Q000378:metabolism; D018374:LLC-PK1 Cells; D007930:Leucine / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D013552:Swine
D015820:Cadherins; D017931:DNA Primers; D007930:Leucine