Involvement of a di-leucine motif in targeting of ABCC1 to the basolateral plasma membrane of polarized epithelial cells

Localization of ATP-binding cassette transporter isoform C1 (ABCC1) to the basolateral membrane of polarized cells is crucial for export of a variety of cellular metabolites; however, the mechanism regulating basolateral targeting of the transporter is poorly understood. Here we describe identification of a basolateral targeting signal in the first cytoplasmic loop domain (CLD1) of human ABCC1. Comparison of the CLD1 amino acid sequences from ABCC1 to ABCC2 revealed that ABCC1 possesses a characteristic sequence, E²⁹⁵EVEALI³⁰¹, which is comprised of a cluster of acidic glutamate residues followed by a di-leucine motif. This characteristic sequence is highly conserved among vertebrate ABCC1 orthologs and is positioned at a site that is structurally equivalent to the apical targeting signal previously described in ABCC2. Alanine scanning mutagenesis of this sequence in full-length human ABCC1 showed that both L³⁰⁰ and I³⁰¹ residues were required for basolateral targeting of ABCC1 in polarized HepG2 and MDCK cells. Conversely, E²⁹⁵, E²⁹⁶, and E²⁹⁸ residues were not required for basolateral localization of the transporter. Therefore, a di-leucine motif within the CLD1 is a basolateral targeting determinant of ABCC1.     

PDZ-binding motifs are unable to ensure correct polarized protein distribution in the absence of additional localization signals

The C-terminal PDZ-binding motifs are required for polarized apical/basolateral localization of many membrane proteins. To determine the specificity of the PDZ-binding motifs in establishing cellular distribution, we utilized a 111-amino acid region from the C-terminus of cystic fibrosis transmembrane conductance regulator (CFTR) that is able to direct apical localization of fused reporter proteins. Substitution of the C-terminal PDZ-binding motif of CFTR with corresponding motifs necessary for basolateral localization of other membrane proteins did not lead to the redistribution of the fusion protein to the basolateral membrane. Instead, some fusion proteins remained localized to the apical membrane, whereas others showed no specific distribution. The specificity of the PDZ-based interactions was substantially increased when specific amino acids located upstream of the classical PDZ-binding motifs were included. However, even the presence of a longer C-terminal motif from a basolateral protein could not ensure basolateral distribution of the fusion protein. Our results indicate that the C-terminal PDZ-binding motifs are not the primary signals for polarized protein distribution, although they are required for targeting and/or stabilization of protein at the given location.     

Rab10 is involved in basolateral transport in polarized Madin-Darby canine kidney cells

The sorting of newly synthesized membrane proteins to the cell surface is an important mechanism of cell polarity. To identify more of the molecular machinery involved, we investigated the function of the small GTPase Rab10 in polarized epithelial Madin-Darby canine kidney cells. We find that GFP-tagged Rab10 localizes primarily to the Golgi during early cell polarization. Expression of an activated Rab10 mutant inhibits biosynthetic transport from the Golgi and missorts basolateral cargo to the apical membrane. Depletion of Rab10 by RNA interference has only mild effects on biosynthetic transport and epithelial polarization, but simultaneous inhibition of Rab10 and Rab8a more strongly impairs basolateral sorting. These results indicate that Rab10 functions in trafficking from the Golgi at early stages of epithelial polarization, is involved in biosynthetic transport to the basolateral membrane and may co-operate with Rab8.     

Rab14 regulates apical targeting in polarized epithelial cells

Epithelial cells display distinct apical and basolateral membrane domains, and maintenance of this asymmetry is essential to the function of epithelial tissues. Polarized delivery of apical and basolateral membrane proteins from the trans Golgi network (TGN) and/or endosomes to the correct domain requires specific cytoplasmic machinery to control the sorting, budding and fission of vesicles. However, the molecular machinery that regulates polarized delivery of apical proteins remains poorly understood. In this study, we show that the small guanosine triphosphatase Rab14 is involved in the apical targeting pathway. Using yeast two-hybrid analysis and glutathione S-transferase pull down, we show that Rab14 interacts with apical membrane proteins and localizes to the TGN and apical endosomes. Overexpression of the GDP mutant form of Rab14 (S25N) induces an enlargement of the TGN and vesicle accumulation around Golgi membranes. Moreover, expression of Rab14-S25N results in mislocalization of the apical raft-associated protein vasoactive intestinal peptide/MAL to the basolateral domain but does not disrupt basolateral targeting or recycling. These data suggest that Rab14 specifically regulates delivery of cargo from the TGN to the apical domain.     

Activation of ERK signaling upon alternative protease nexin-1 internalization mediated by syndecan-1

Protease nexin-1 (PN-1), an inhibitor of serine proteases, contributes to tissue homeostasis and influences the behavior of some tumor cells. The internalization of PN-1 protease complexes is considered to be mediated by the low-density lipoprotein receptor related protein 1 (LRP1). In this study, both wild-type and LRP1-/- mouse embryonic fibroblasts (MEF) were shown to internalize PN-1. Receptor associated protein (RAP) interfered with PN-1 uptake only in wild-type MEF cells, indicating that another receptor mediates PN-1 uptake in the absence of LRP1. In LRP1-/- MEF cells, inhibitor sensitivity and kinetic values (t(1/2) at 45 min) of PN-1 uptake showed a similarity to syndecan-1-mediated endocytosis. In these cells, PN-1 uptake was increased by overexpression of full-length syndecan-1 and decreased by RNA interference targeting this proteoglycan. Most important, in contrast to PKA activation known to be triggered by LRP1-mediated internalization, our study shows that syndecan-1-mediated internalization of PN-1 stimulated the Ras-ERK signaling pathway.     

Coupling assembly of the E-cadherin/beta-catenin complex to efficient endoplasmic reticulum exit and basal-lateral membrane targeting of E-cadherin in polarized MDCK cells

The E-cadherin/catenin complex regulates Ca++-dependent cell-cell adhesion and is localized to the basal-lateral membrane of polarized epithelial cells. Little is known about mechanisms of complex assembly or intracellular trafficking, or how these processes might ultimately regulate adhesion functions of the complex at the cell surface. The cytoplasmic domain of E-cadherin contains two putative basal-lateral sorting motifs, which are homologous to sorting signals in the low density lipoprotein receptor, but an alanine scan across tyrosine residues in these motifs did not affect the fidelity of newly synthesized E-cadherin delivery to the basal-lateral membrane of MDCK cells. Nevertheless, sorting signals are located in the cytoplasmic domain since a chimeric protein (GP2CAD1), comprising the extracellular domain of GP2 (an apical membrane protein) and the transmembrane and cytoplasmic domains of E-cadherin, was efficiently and specifically delivered to the basal-lateral membrane. Systematic deletion and recombination of specific regions of the cytoplasmic domain of GP2CAD1 resulted in delivery of <10% of these newly synthesized proteins to both apical and basal-lateral membrane domains. Significantly, >90% of each mutant protein was retained in the ER. None of these mutants formed a strong interaction with beta-catenin, which normally occurs shortly after E-cadherin synthesis. In addition, a simple deletion mutation of E-cadherin that lacks beta-catenin binding is also localized intracellularly. Thus, beta-catenin binding to the whole cytoplasmic domain of E-cadherin correlates with efficient and targeted delivery of E-cadherin to the lateral plasma membrane. In this capacity, we suggest that beta-catenin acts as a chauffeur, to facilitate transport of E-cadherin out of the ER and the plasma membrane.     

Identification of a novel mono-leucine basolateral sorting motif within the cytoplasmic domain of amphiregulin

Epithelial cells establish apical and basolateral (BL) membranes with distinct protein and lipid compositions. To achieve this spatial asymmetry, the cell utilizes a variety of mechanisms for differential sorting, delivery and retention of cell surface proteins. The EGF receptor (EGFR) and its ligand, amphiregulin (AREG), are transmembrane proteins delivered to the BL membrane in polarized epithelial cells. Herein, we show that the cytoplasmic domain of AREG (ACD) contains dominant BL sorting information; replacement of the cytoplasmic domain of apically targeted nerve growth factor receptor with the ACD redirects the chimera to the BL surface. Using sequential truncations and site-directed mutagenesis of the ACD, we identify a novel BL sorting motif consisting of a single leucine C-terminal to an acidic cluster (EEXXXL). In adaptor protein (AP)-1B-deficient cells, newly synthesized AREG is initially delivered to the BL surface as in AP-1B-expressing cells. However, in these AP-1B-deficient cells, recycling of AREG back to the BL surface is compromised, leading to its appearance at the apical surface. These results show that recycling, but not delivery, of AREG to the BL surface is AP-1B dependent.     

The Rab8 GTPase selectively regulates AP-1B-dependent basolateral transport in polarized Madin-Darby canine kidney cells

The AP-1B clathrin adaptor complex plays a key role in the recognition and intracellular transport of many membrane proteins destined for the basolateral surface of epithelial cells. However, little is known about other components that act in conjunction with AP-1B. We found that the Rab8 GTPase is one such component. Expression of a constitutively activated GTP hydrolysis mutant selectively inhibited basolateral (but not apical) transport of newly synthesized membrane proteins. Moreover, the effects were limited to AP-1B-dependent basolateral cargo; basolateral transport of proteins containing dileucine targeting motifs that do not interact with AP-1B were targeted normally despite overexpression of mutant Rab8. Similar results were obtained for a dominant-negative allele of the Rho GTPase Cdc42, previously implicated in basolateral transport but now shown to be selective for the AP-1B pathway. Rab8-GFP was localized to membranes in the TGN-recycling endosome, together with AP-1B complexes and the closely related but ubiquitously expressed AP-1A complex. However, expression of active Rab8 caused a selective dissociation of AP-1B complexes, reflecting the specificity of Rab8 for AP-1B-dependent transport.     

The PDZ-binding motif of MCC is phosphorylated at position -1 and controls lamellipodia formation in colon epithelial cells

In this study, we describe a new post-translational modification at position -1 of the PDZ-binding motif in the mutated in colorectal cancer (MCC) protein and its role in lamellipodia formation. Serine 828 at position -1 of this motif is phosphorylated, which is predicted to increase MCC binding affinity with the polarity protein Scrib. We show that endogenous MCC localizes at the active migratory edge of cells, where it interacts with Scrib and the non-muscle motor protein Myosin-IIB. Expression of MCC harboring a phosphomimetic mutation MCC-S828D strongly impaired lamellipodia formation and resulted in accumulation of Myosin-IIB in the membrane cortex fraction. We propose that MCC regulates lamellipodia formation by binding to Scrib and its downstream partner Myosin-IIB in a multiprotein complex. Importantly, we propose that the function of this complex is under the regulation of a newly described phosphorylation of the PDZ-binding motif at position -1.     

Identification of a basolateral membrane targeting signal within the cytoplasmic domain of myelin/oligodendrocyte glycoprotein

Oligodendrocytes possess two distinct membrane compartments--uncompacted plasma membrane (cell body, processes) and compact myelin. Specific targeting mechanisms must exist to establish and maintain these membrane domains. Polarized epithelial cells have the best characterized system for targeting components to apical and basolateral compartments. Since oligodendrocytes arise from neuroepithelial cells, we investigated whether they might utilize targeting paradigms similar to polarized epithelial cells. Myelin/oligodendrocyte glycoprotein (MOG) is a transmembrane Ig-like molecule restricted to uncompacted oligodendroglial plasma membrane. We stably expressed MOG in Madin-Darby canine kidney (MDCK) Type II epithelial cells, which have been extensively used in protein-targeting studies. Data from surface biotinylation assays and confocal microscopy revealed that MOG sorts exclusively to the basolateral membrane of MDCK cells. Expression vectors containing progressive truncations of MOG from the cytoplasmic C-terminus were expressed in MDCK cells to localize basolateral sorting signals. A loss of only four C-terminal residues results in some MOG expression at the apical surface. More strikingly, removal of the C-terminal membrane associated hydrophobic domain from MOG results in complete loss of basolateral sorting and specific targeting to the apical membrane. These data suggest that myelinating oligodendrocytes may utilize a sorting mechanism similar to that of polarized epithelia.     

N-Glycosylation of the MUC1 mucin in epithelial cells and secretions

The MUC1 mucin is an important tumor-associated antigen that shows extensive glycosylation in vivo. The O-glycosylation of this molecule, which has been well characterized in many cell types and tissues, is important in conferring the unusual biochemical and biophysical properties on a mucin. N-Glycosylation is crucial to the folding, sorting, membrane trafficking, and secretion of many proteins. Here, we evaluated the N-glycosylation of MUC1 derived from two sources: endogenous MUC1 isolated from human milk and a recombinant epitope-tagged MUC1F overexpressed in Caco2 colon carcinoma cells. N-Glycans on purified MUC1F/MUC1 were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), gas chromatography-mass spectrometry (GC-MS), and CAD-ESI-MS/MS. The spectra indicate that MUC1F N-glycans have compositions consistent with high-mannose structures (Hex(5-9)HexNAc(2)) and complex/hybrid-type glycans (NeuAc(0-3)Fuc(0-3)Hex(3-8)HexNAc(3-7)). Many of the N-glycan structures are identical on MUC1F and native MUC1; however, a marked difference is seen between the N-glycans on membrane-bound and secreted forms of the native molecule.     

Distribution and function of AP-1 clathrin adaptor complexes in polarized epithelial cells

Expression of the epithelial cell-specific heterotetrameric adaptor complex AP-1B is required for the polarized distribution of many membrane proteins to the basolateral surface of LLC-PK1 kidney cells. AP-1B is distinguished from the ubiquitously expressed AP-1A by exchange of its single 50-kD mu subunit, mu1A, being replaced by the closely related mu1B. Here we show that this substitution is sufficient to couple basolateral plasma membrane proteins, such as a low-density lipoprotein receptor (LDLR), to the AP-1B complex and to clathrin. The interaction between LDLR and AP-1B is likely to occur in the trans-Golgi network (TGN), as was suggested by the localization of functional, epitope-tagged mu1 by immunofluorescence and immunoelectron microscopy. Tagged AP-1A and AP-1B complexes were found in the perinuclear region close to the Golgi complex and recycling endosomes, often in clathrin-coated buds and vesicles. Yet, AP-1A and AP-1B localized to different subdomains of the TGN, with only AP-1A colocalizing with furin, a membrane protein that uses AP-1 to recycle between the TGN and endosomes. We conclude that AP-1B functions by interacting with its cargo molecules and clathrin in the TGN, where it acts to sort basolateral proteins from proteins destined for the apical surface and from those selected by AP-1A for transport to endosomes and lysosomes.     

Current-voltage relations of the basolateral membrane in tight amphibian epithelia: Use of nystatin to depolarize the apical membrane

Summary Exposure of the mucosal side of toad(Bufo bufo) urinary bladder and frog(Rana ridibunda) skin to the polyene ionophore nystatin, resulted in stable preparations in which the apical resistance was negligible compared to the basolateral resistance. The preparations support passive K currents in both directions and an amiloride-insensitive Na current in the apicalserosal direction which is blocked by ouabain. The nystatintreated toad bladder was used to study the electrical properties of the basolateral membrane by means of current-voltage curves recorded transepithelially. The K current showed strong rectification at cellular potentials negative with respect to the interstitial space. The ouabain-sensitive current increased with membrane voltage at negative voltages but saturated above+20 mV.     

Vps34p differentially regulates endocytosis from the apical and basolateral domains in polarized hepatic cells

Using a microinjection approach to study apical plasma membrane protein trafficking in hepatic cells, we found that specific inhibition of Vps34p, a class III phosphoinositide 3 (PI-3) kinase, nearly perfectly recapitulated the defects we reported for wortmannin-treated cells (Tuma, P.L., C.M. Finnegan, J.-H Yi, and A.L. Hubbard. 1999. J. Cell Biol. 145:1089-1102). Both wortmannin and injection of inhibitory Vps34p antibodies led to the accumulation of resident apical proteins in enlarged prelysosomes, whereas transcytosing apical proteins and recycling basolateral receptors transiently accumulated in basolateral early endosomes. To understand how the Vps34p catalytic product, PI3P, was differentially regulating endocytosis from the two domains, we examined the PI3P binding protein early endosomal antigen 1 (EEA1). We determined that EEA1 distributed to two biochemically distinct endosomal populations: basolateral early endosomes and subapical endosomes. Both contained rab5, although the latter also contained late endosomal markers but was distinct from the transcytotic intermediate, the subapical compartment. When PI3P was depleted, EEA1 dissociated from basolateral endosomes, whereas it remained on subapical endosomes. From these results, we conclude that PI3P, via EEA1, regulates early steps in endocytosis from the basolateral surface in polarized WIF-B cells. However, PI3P must use different machinery in its regulation of the apical endocytic pathway, since later steps are affected by Vps34p inhibition.     

The intracellular carboxyl terminal domain of Vangl proteins contains plasma membrane targeting signals

Vangl1 and Vangl2 are integral membrane proteins that play a critical role in establishing planar cell polarity (PCP) in epithelial cells and are required for convergent extension (CE) movements during embryogenesis. Their proper targeting to the plasma membrane (PM) is required for function. We created discrete deletions at the amino and carboxy termini of Vangl1 and monitored the effect of the mutations on PM targeting in Madin–Darby canine kidney cells. Our results show that the Vangl1 amino terminus lacks PM targeting determinants, and these are restricted to the carboxy terminus, including the predicted PDZBM motif at the C‐terminus.     

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.     

The glycan-binding protein galectin-1 controls survival of epithelial cells along the crypt-villus axis of small intestine

Intestinal epithelial cells serve as mechanical barriers and active components of the mucosal immune system. These cells migrate from the crypt to the tip of the villus, where different stimuli can differentially affect their survival. Here we investigated, using in vitro and in vivo strategies, the role of galectin-1 (Gal-1), an evolutionarily conserved glycan-binding protein, in modulating the survival of human and mouse enterocytes. Both Gal-1 and its specific glyco-receptors were broadly expressed in small bowel enterocytes. Exogenous Gal-1 reduced the viability of enterocytes through apoptotic mechanisms involving activation of both caspase and mitochondrial pathways. Consistent with these findings, apoptotic cells were mainly detected at the tip of the villi, following administration of Gal-1. Moreover, Gal-1-deficient (Lgals1^−/−) mice showed longer villi compared with their wild-type counterparts in vivo. In an experimental model of starvation, fasted wild-type mice displayed reduced villi and lower intestinal weight compared with Lgals1^−/− mutant mice, an effect reflected by changes in the frequency of enterocyte apoptosis. Of note, human small bowel enterocytes were also prone to this pro-apoptotic effect. Thus, Gal-1 is broadly expressed in mucosal tissue and influences the viability of human and mouse enterocytes, an effect which might influence the migration of these cells from the crypt, the integrity of the villus and the epithelial barrier function.     

Early response to ErbB2 over‐expression in polarized Caco‐2 cells involves partial segregation from ErbB3 by relocalization to the apical surface and initiation of survival signaling

In several human cancers, ErbB2 over‐expression facilitates the formation of constitutively active homodimers resistant to internalization which results in progressive signal amplification from the receptor, conducive to cell survival, proliferation, or metastasis. Here we report on studies of the influence of ErbB2 over‐expression on localization and signaling in polarized Caco‐2 and MDCK cells, two established models to study molecular trafficking. In these cells, ErbB2 is not over‐expressed and shares basolateral localization with ErbB3. Over‐expression of ErbB2 by transient transfection resulted in partial separation of the receptors by relocalization of ErbB2, but not ErbB3, to the apical surface, as shown by biotinylation of the apical or basolateral surfaces. These results were confirmed by immunofluorescence and confocal microscopy. Polarity controls indicated that the relocalization of ErbB2 is not the result of depolarization of the cells. Biotinylation and confocal microscopy also showed that apical, but not basolateral ErbB2 is activated at tyrosine 1139. This phosphotyrosine binds adaptor protein Grb2, as confirmed by immunoprecipitation. However, we found that it does not initiate the canonical Grb2‐Ras‐Raf‐Erk pathway. Instead, our data supports the activation of a survival pathway via Bcl‐2. The effects of ErbB2 over‐expression were abrogated by the humanized anti‐ErbB2 monoclonal antibody Herceptin added only from the apical side. The ability of apical ErbB2 to initiate an altered downstream cascade suggests that subcellular localization of the receptor plays an important role in regulating ErbB2 signaling in polarized epithelia. J. Cell. Biochem. 111: 643–652, 2010. © 2010 Wiley‐Liss, Inc.     

Protein trafficking in the exocytic pathway of polarized epithelial cells

Ten years ago, we knew much about the function of polarized epithelia from the work of physiologists, but, as cell biologists, our understanding of how these cells were constructed was poor. We knew proteins were sorted and targeted to different plasma membrane domains and that, in some cells, the Golgi was the site of sorting, but we did not know the mechanisms involved. Between 1991 and the present, significant advances were made in defining sorting motifs for apical and basal-lateral proteins, describing the sorting machinery in the trans-Golgi network (TGN) and plasma membrane, and in understanding how cells specify delivery of transport vesicles to different membrane domains. The challenge now is to extend this knowledge to defining molecular mechanisms in detail in vitro and comprehending the development of complex epithelial structures in vivo.     

Collagen receptors mediate early events in the attachment of epithelial (MDCK) cells

Summary Madin-Darby canine kidney (MDCK) cells kept in suspension culture for 12–15 hr displayed high-affinity binding sites for¹²⁵I-lathyritic (soluble) collagen (120,000/cell,K _D =30nm) and preferred collagens types I and IV over laminin or fibronectin as substrates during the first hour of attachment. On the other hand, after 4 hr, attachment to all four substrates was equally efficient. Upon challenge with a collagen substrate, the high-affinity sites were rapidly recruited on it (T1/2=6 min). Their occupancy by soluble collagen triggered the exocytosis of a second large population of low-affinity collagen binding sites that included laminin and seems to be involved in a second cell-attachment mechanism. These results are compatible with a twostep model of MDCK cell attachment to the substrate: first, via high-affinity collagen binding sites, and second, via laminin of cellular origin.