An internal deletion in the cytoplasmic tail reverses the apical localization of human NGF receptor in transfected MDCK cells.

Abstract

Abstract. A cDNA encoding the full-length 75-kD human nerve growth factor receptor was transfected into MDCK cells and its product was found to be expressed predominantly (80%) on the apical membrane, as a result of vectorial targeting from an intracellular site . Apical hNGFR bound NGF with low affinity and internalized it inefficiently (6% of surface bound NGF per hour) . Several mutant hNGFRs were analyzed, after transfection in MDCK cells, for polarized surface expression, ligand binding, and endocytosis . Deletion of juxtamembrane attachment sites for a cluster of O-linked sugars did not alter apical localization . A mutant receptor lacking the entire cytoplasmic tail (except for the five proximal amino acids) was also expressed on the apical membrane, suggesting that information for apical sorting was contained in the ectoplasmic or transmembrane domains. However, a 58 T HE plasma membrane of epithelial cells displays distinct apical and basolateral domains with different sets of proteins and lipids (9,50,58) . To establish and maintain these polarized surface domains, epithelial cells use diverse strategies including sorting and packaging of proteins and lipids into different post-Golgi transport vesicles, targeting of these vesicles to the correct domain, and stabilization and/or accurate recycling of the proteins specific for a given domain . Using different epithelial models such as liver, intestine, and kidney cells, progress was recently made in defining the pathways followed by apical and basolateral proteins from the Golgi complex to their respective domains (2, 25, 37) . Two major sorting sites were established by these studies, the trans-Golgi network (TGN) ' (17, 27, 28, 31, 36, 41) and the basolateral membrane (2,25,35,37) . To date, all basolateral proteins studied appear to be targeted directly from an intracellular sorting site (presumably 1. Abbreviations used in this paper: GPI, glycosyl-phosphatidylinositol ; hNGFR, human nerve growth factor ; PIgR, poly-IG receptor ; PLAP, placental alkaline phosphatase ; S-NHS-biotin, sulfo-N-hydroxyl-succinimidobiotin ; TGN, trans-Golgi network; WT, wild type .C The Rockefeller University Press, 0021-9525/91/11/607/12 $2 .00

Mutational and secondary structural analysis of the basolateral sorting signal of the polymeric immunoglobulin receptor.

“…The importance of polar and charged residues has also been suggested in previous studies on internalization and basolateral targeting signals (Ktistakis et al, 1990;Trowbridge, 1991;Le Bivic et al, 1991;Matter et al, 1992). Interestingly, this positively charged cluster is located only two residues away from the transmembrane region of the molecule.…”

“…Most of the signals appear to be colinear with Tyr-containing signals for endocytosis via clathrin-coated pits (Hunziker et al, 1991;Brewer and Roth, 1991;Le Bivic et al, 1991;reviewed in Mostov et al, 1992). In several other cases (e.g., the transferrin receptor and vesicular stomatitis virus G protein) basolateral sorting signals appear not to overlap with endocytosis signals (Hunziker et al, 1991;Dargemont et al, 1993;Thomas et ai., 1993).…”

The epithelial polarity program: machineries involved and their hijacking by cancer

“…Basolateral sorting is guided by basolateral sorting signals (Keller and Simons, 1997;Rodriguez-Boulan et al, 2005), and they include: short tyrosine motifs such as Yxxf or NPXY (Brewer and Roth, 1991;Le Bivic et al, 1991;Matter et al, 1992), dileucine (Hunziker and Fumey, 1994) or monoleucine motifs with nearby acidic patches (Deora et al, 2004), PxxP, found in EGFR and pleomorphic motifs that do not yet fall into established classes, found in NCAM (Le Gall et al, 1994), transferrin receptor (Odorizzi and Trowbridge, 1997) and polymeric IgA receptor (Aroeti and Mostov, 1994) ( Figure 2). Yeast 2 and 3 hybrid assays (Ohno et al, 1995;Janvier et al, 2003) demonstrated that Yxxf motifs interact with the m subunit of AP adaptors (AP1, AP2, AP3, AP4), whereas dileucine motifs of the type [DE]XXXL [LI] bind to g and s1 subunits of AP1 and to d and s3 subunits of AP3 (Kirchhausen, 2000;Bonifacino and Traub, 2003).…”

Basolateral Localization and Transcytosis of Gonadotropin and Thyrotropin Receptors Expressed in Madin-Darby Canine Kidney Cells

“…Such data can be obtained by metabolic labeling of the receptor followed by either domain-selective biotinylation of the receptor (45) or surface immunoprecipitation (36). Both techniques can be applied separately to the basolateral and apical compartments yielding quantitative measurements of receptors present on each surface.…”

“…Surface immunoprecipitation was performed as described (36) using monoclonal antibodies FSHR323 or LHR38 (30 g/ml) or polyclonal antibody TSHR 19-389 (dilution 1/300) added in the apical or in the basolateral compartment during the chase period. Extraction and purification of receptor-antibody complex were performed as described (30,36). All experiments were performed at least twice with triplicate samples.…”