Interaction with mLin-7 alters the targeting of endocytosed transmembrane proteins in mammalian epithelial cells.
To investigate the targeting mechanism for proteins bound to the mammalian Lin-7 (mLin-7) PDZ domain, we created receptor protein chimeras composed of the carboxyl-terminal amino acids of LET-23 fused to truncated nerve growth factor receptor/P75. mLin-7 bound to the chimera with a wild-type LET-23 carboxyl-terminal tail (P75t-Let23WT), but not a mutant tail (P75t-Let23MUT). In Madin-Darby canine kidney (MDCK) cells, P75t-Let23WT localized to the basolateral plasma membrane domain, whereas P75t-Let23MUT remained apical. Furthermore, mutant mLin-7 constructs acted as dominant interfering proteins and inhibited the basolateral localization of P75t-Let23WT. The mechanisms for this differential localization were examined further, and, initially, we found that P75t-Let23WT and P75t-Let23MUT were delivered equally to the apical and basolateral plasma membrane domains. Although basolateral retention of P75t-Let23WT, but not P75t-Let23MUT, was observed, the greatest difference in receptor localization was seen in the rapid trafficking of P75t-Let23WT to the basolateral plasma membrane domain after endocytosis, whereas P75t-Let23MUT was degraded in lysosomes, indicating that mLin-7 binding can alter the fate of endocytosed proteins. Altogether, these data support a model for basolateral protein targeting in mammalian epithelial cells dependent on protein-protein interactions with mLin-7, and also suggest a dynamic role for mLin-7 in endosomal sorting.
Straight
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SW
Howard Hughes Medical Institute, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.
Chen
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L
Karnak
D
D
Margolis
B
B
D000818:Animals; D019921:Biotinylation; D029742:Caenorhabditis elegans Proteins; D002460:Cell Line; D016764:Cell Polarity; D004285:Dogs; D004705:Endocytosis / Q000502:physiology*; D004847:Epithelial Cells / Q000378:metabolism*; D015801:Helminth Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D014162:Transfection
D029742:Caenorhabditis elegans Proteins; D015801:Helminth Proteins; C099173:LIN-7 protein, C elegans; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins