Interaction with mLin-7 alters the targeting of endocytosed transmembrane proteins in mammalian epithelial cells

• Published in: Molecular biology of the cell 2001-05, Vol.12 (5), p.1329-1340
• Main Authors: Straight, S W, Chen, L, Karnak, D, Margolis, B
• Format: Article
• Language: English
• Subjects: Animals; Biotinylation; Caenorhabditis elegans Proteins; Cell Line; Cell Polarity; Dogs; Endocytosis - physiology; Epithelial Cells - metabolism; Helminth Proteins - chemistry; Helminth Proteins - genetics; Helminth Proteins - metabolism; Humans; Immunoblotting; Membrane Proteins - chemistry; Membrane Proteins - genetics; Membrane Proteins - metabolism; Mutation - genetics; Protein Binding; Protein Structure, Tertiary; Protein Transport; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Signal Transduction - physiology; Transfection
• ISSN: 1059-1524; 1939-4586
• DOI: 10.1091/mbc.12.5.1329

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.