Polarized Madin-Darby Canine Kidney Cells Research Articles

GPI-anchored proteins are directly targeted to the apical surface in fully polarized MDCK cells

The polarity of epithelial cells is dependent on their ability to target proteins and lipids in a directional fashion. The trans-Golgi network, the endosomal compartment, and the plasma membrane act as sorting stations for proteins and lipids. The site of intracellular sorting and pathways used for the apical delivery of glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are largely unclear. Using biochemical assays and confocal and video microscopy in living cells, we show that newly synthesized GPI-APs are directly delivered to the apical surface of fully polarized Madin–Darby canine kidney cells. Impairment of basolateral membrane fusion by treatment with tannic acid does not affect the direct apical delivery of GPI-APs, but it does affect the organization of tight junctions and the integrity of the monolayer. Our data clearly demonstrate that GPI-APs are directly sorted to the apical surface without passing through the basolateral membrane. They also reinforce the hypothesis that apical sorting of GPI-APs occurs intracellularly before arrival at the plasma membrane.

Polarized Apical Sorting of Guanylyl Cyclase C is Specified by a Cytosolic Signal

Receptor guanylyl cyclases respond to ligand stimulation by increasing intracellular cGMP, thereby initiating a variety of cell-signaling pathways. Furthermore, these proteins are differentially localized at the apical and basolateral membranes of epithelial cells. We have identified a region of 11 amino acids in the cytosolic COOH terminus of guanylyl cyclase C (GCC) required for normal apical localization in Madin-Darby canine kidney (MDCK) cells. These amino acids share no significant sequence homology with previously identified cytosolic apical sorting determinants. However, these amino acids are highly conserved and are sufficient to confer apical polarity to the interleukin-2 receptor alpha-chain (Tac). Additionally, we find two molecular weight species of GCC in lysates prepared from MDCK cells over-expressing GCC but observe only the fully mature species on the cell surface. Using pulse-chase analysis in polarized MDCK cells, we followed the generation of this mature species over time finding it to be detectable only at the apical cell surface. These data support the hypothesis that selective apical sorting can be determined using short, cytosolic amino acid motifs and argue for the existence of apical sorting machinery comparable with the machinery identified for basolateral protein traffic.

Vectorial Secretion of Perivitelline Membrane Glycoprotein ZPC of Japanese Quail (Coturnix japonica) in Polarized Madin-Darby Canine Kidney Cells

The avian perivitelline membrane (PL), which is an investment homologous to the mammalian zona pellucida, is found between the surface of the oocyte and the apical surface of ovarian granulosa cells. Our previous study demonstrated that ZPC, one of the components of PL, is synthesized in ovarian granulosa cells. However, how the secretion of ZPC is regulated in the cells has been insufficiently investigated. We studied the secretion of quail ZPC expressed in polarized Madin-Darby canine kidney (MDCK) cells in a dual-chamber apparatus. Western blot analyses of the conditioned medium demonstrated that the majority of the secreted ZPC were distributed in the apical compartment. When ZPC lacking N-linked oligosaccharides was transfected into the cells, the 31-kDa immunoreactive band was detected in both the apical and the basolateral medium. Interestingly, immunohistochemical observations of the follicular wall demonstrated that the predominant intracellular form of ZPC in the cells localized in the apical side of the perinuclear region apposed to the PL, but not the basolateral side, indicating the possibility that ZPC could be selectively transported toward the apical surface in vivo. Taken together, these results indicated that ZPC expressed in MDCK cells are selectively released to the apical compartment, and that the N-linked carbohydrates might possess information that causes the efficient transport of ZPC to the apical surface of the cells.

Helicobacter pylori CagA induces a transition from polarized to invasive phenotypes in MDCK cells

CagA is a bacterial effector protein of Helicobacter pylori that is translocated via a type IV secretion system into gastric epithelial cells. We previously described that H. pylori require CagA to disrupt the organization and assembly of apical junctions in polarized epithelial cells. In this study, we provide evidence that CagA expression is not only sufficient to disrupt the apical junctions but also perturbs epithelial differentiation. CagA-expressing cells lose apicobasal polarity and cell-cell adhesion, extend migratory pseudopodia, and degrade basement membranes, acquiring an invasive phenotype. Expression of the CagA C-terminal domain, which contains the tyrosine phosphorylated EPIYA motifs, induces pseudopodial activity but is not sufficient to induce cell migration. Conversely, the N terminus targets CagA to the cell-cell junctions. Neither domain is sufficient to disrupt cell adhesion or cell polarity, but coexpressed in trans, the N terminus determines the localization of both polypeptides. We show that CagA induces a morphogenetic program in polarized Madin-Darby canine kidney cells resembling an epithelial-to-mesenchymal transition. We propose that altered cell-cell and cell matrix interactions may serve as an early event in H. pylori-induced carcinogenesis.

FAPP2 is involved in the transport of apical cargo in polarized MDCK cells

Phosphatidylinositol-4-phosphate (PI(4)P) is the main phosphoinositide in the Golgi complex and has been reported to play a pleiotropic role in transport of cargo from the trans-Golgi network to the plasma membrane (PM) in polarized Madin–Darby canine kidney (MDCK) cells. Overexpression of the chimeric fluorescent protein encoding the pleckstrin homology domain, which is specific for PI(4)P, inhibited both apical and basolateral transport pathways. The transport of apical cargo from the Golgi was shown to be specifically decreased by adenovirus-mediated RNA interference directed against PI(4)P adaptor protein (FAPP) 2. FAPP1 depletion had no effect on transport. On the other hand, FAPP2 was not involved in the Golgi-to-PM transport of cargo that was targeted to the basolateral membrane domain. Thus, we conclude that FAPP2 plays a specific role in apical transport in MDCK cells.

Use of influenza C virus glycoprotein HEF for generation of vesicular stomatitis virus pseudotypes

Influenza C virus contains two envelope glycoproteins: CM2, a putative ion channel protein; and HEF, a unique multifunctional protein that performs receptor-binding, receptor-destroying and fusion activities. Here, it is demonstrated that expression of HEF is sufficient to pseudotype replication-incompetent vesicular stomatitis virus (VSV) that lacks the VSV glycoprotein (G) gene. The pseudotyped virus showed characteristic features of influenza C virus with respect to proteolytic activation, receptor usage and cell tropism. Chimeric glycoproteins composed of HEF ectodomain and VSV-G C-terminal domains were efficiently incorporated into VSV particles and showed receptor-binding and receptor-destroying activities but, unlike authentic HEF, did not mediate efficient infection, probably because of impaired fusion activity. HEF-pseudotyped VSV efficiently infected polarized Madin-Darby canine kidney cells via the apical plasma membrane, whereas entry of VSV-G-complemented virus was restricted to the basolateral membrane. These findings suggest that pseudotyping of viral vectors with HEF might be useful for efficient apical gene transfer into polarized epithelial cells and for targeting cells that express 9-O-acetylated sialic acids.

Use of the H,K-ATPase β Subunit to Identify Multiple Sorting Pathways for Plasma Membrane Delivery in Polarized Cells

A dynamic equilibrium between multiple sorting pathways maintains polarized distribution of plasma membrane proteins in epithelia. To identify sorting pathways for plasma membrane delivery of the gastric H,K-ATPase beta subunit in polarized cells, the protein was expressed as a yellow fluorescent protein N-terminal construct in Madin-Darby canine kidney (MDCK) and LLC-PK1 cells. Confocal microscopy and surface-selective biotinylation showed that 80% of the surface amount of the beta subunit was present on the apical membrane in LLC-PK1 cells, but only 40% was present in MDCK cells. Nondenaturing gel electrophoresis of the isolated membranes showed that a significant fraction of the H,K-ATPase beta subunits associate with the endogenous Na,K-ATPase alpha(1) subunits in MDCK but not in LLC-PK cells. Hence, co-sorting of the H,K-ATPase beta subunit with the Na,K-ATPase alpha(1) subunit to the basolateral membrane in MDCK cells may determine the differential distribution of the beta subunit in these two cell types. The major fraction of unassociated monomeric H,K-ATPase beta subunits is detected in the apical membrane. Quantitative analysis showed that half of the apical pool of the beta subunit originates directly from the trans-Golgi network and the other half from transcytosis via the basolateral membrane in MDCK cells. A minor fraction of monomeric beta subunits detected in the basolateral membrane represents a transient pool of the protein that undergoes transcytosis to the apical membrane. Hence, the steady state distribution of the H,K-ATPase beta subunit in polarized cells depends on the balance between (a) direct sorting from the trans-Golgi network, (b) secondary associative sorting with a partner protein, and (c) transcytosis.

Distinct Golgi Populations of Phosphatidylinositol 4-Phosphate Regulated by Phosphatidylinositol 4-Kinases

Phosphatidylinositol 4-phosphate (PI4P) regulates biosynthetic membrane traffic at multiple steps and differentially affects the surface delivery of apically and basolaterally destined proteins in polarized cells. Two phosphatidylinositol 4-kinases (PI4Ks) have been localized to the Golgi complex in mammalian cells, type III PI4Kbeta (PI4KIIIbeta) and type II PI4Kalpha (PI4KIIalpha). Here we report that PI4KIIIbeta and PI4KIIalpha localize to discrete subcompartments of the Golgi complex in Madin-Darby canine kidney (MDCK) cells. PI4KIIIbeta was enriched in early Golgi compartments, whereas PI4KIIalpha colocalized with markers of the trans-Golgi network (TGN). To understand the temporal and spatial control of PI4P generation across the Golgi complex, we quantitated the steady state distribution of a fluorescent PI4P-binding domain relative to cis/medial Golgi and TGN markers in transiently transfected MDCK cells. The density of the signal from this PI4P reporter was roughly 2-fold greater in the early Golgi compartments compared with that of the TGN. Furthermore, this ratio could be modulated in vivo by overexpression of catalytically inactive PI4KIIIbeta and PI4KIIalpha or in vitro by the PI4KIIIbeta inhibitor wortmannin. Our data suggest that both PI4KIIIbeta and PI4KIIalpha contribute to the compartmental regulation of PI4P synthesis within the Golgi complex. We discuss our results with respect to the kinetic effects of modulating PI4K activity on polarized biosynthetic traffic in MDCK cells.

The phosphoinositol-3-kinase-protein kinase B/Akt pathway is critical for Pseudomonas aeruginosa strain PAK internalization.

Several Pseudomonas aeruginosa strains are internalized by epithelial cells in vitro and in vivo, but the host pathways usurped by the bacteria to enter nonphagocytic cells are not clearly understood. Here, we report that internalization of strain PAK into epithelial cells triggers and requires activation of phosphatidylinositol 3-kinase (PI3K) and protein kinase B/Akt (Akt). Incubation of Madin-Darby canine kidney (MDCK) or HeLa cells with the PI3K inhibitors LY294002 (LY) or wortmannin abrogated PAK uptake. Addition of the PI3K product phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] to polarized MDCK cells was sufficient to increase PAK internalization. PtdIns(3,4,5)P3 accumulated at the site of bacterial binding in an LY-dependent manner. Akt phosphorylation correlated with PAK invasion. The specific Akt phosphorylation inhibitor SH-5 inhibited PAK uptake; internalization also was inhibited by small interfering RNA-mediated depletion of Akt phosphorylation. Expression of constitutively active Akt was sufficient to restore invasion when PI3K signaling was inhibited. Together, these results demonstrate that the PI3K signaling pathway is necessary and sufficient for the P. aeruginosa entry and provide the first example of a bacterium that requires Akt for uptake into epithelial cells.

The relationship between cell proliferation, Cl− secretion, and renal cyst growth: A study using CFTR inhibitors

In autosomal-dominant polycystic kidney disease (ADPKD), cAMP-stimulated cell proliferation and Cl- secretion via the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel drive the enlargement of fluid-filled epithelial cysts. To investigate how CFTR blockers inhibit cyst growth, we studied cAMP-dependent Cl- secretion, cell proliferation, and cyst growth using type I Madin Darby canine kidney (MDCK) cells as a model of renal cyst development and growth. We grew MDCK cysts in collagen gels in the presence of the cAMP agonist forskolin, measured Cl- secretion with the Ussing chamber technique, and assayed cell proliferation using nonpolarized and polarized MDCK cells. To inhibit CFTR, we used glibenclamide, 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), genistein, and the specific CFTR inhibitor CFTRinh-172. As controls, we tested the effects of blockers of other types of apical membrane Cl- channels and inhibitors of basolateral membrane ion channels and transporters. In the absence of inhibitors of transepithelial ion transport, forskolin stimulated dramatic cyst growth. CFTR blockers and inhibitors of basolateral membrane ion channels and transporters retarded cyst growth. In contrast, blockers of other types of apical membrane Cl- channels, which were without effect on CFTR, failed to inhibit cyst growth. Inhibition of cyst growth by CFTR blockers was correlated with inhibition of cAMP-stimulated Cl- current (correlation coefficient = 0.81; P < 0.05), but not cell proliferation (correlation coefficient = 0.50; P > 0.05). Our data suggest that CFTR blockers might retard cyst growth predominantly by inhibiting fluid accumulation within the cyst lumen.

Transepithelial pressure pulses induce nucleotide release in polarized MDCK cells.

The release of nucleotides is involved in mechanosensation in various epithelial cells. Intriguingly, kidney epithelial cells are absolutely dependent on the primary cilium to sense changes in apical laminar flow. During fluid passage, the renal epithelial cells are subjected to various mechanical stimuli in addition to changes in the laminar flow rate. In the distal part of the collecting duct, the epithelial cells are exposed to pressure changes and possibly distension during papillary contractions. The aim of the present study was to determine whether nucleotide release contributes to mechanosensation in kidney epithelial cells, thereby establishing whether pressure changes are sufficient to produce nucleotide-mediated responses. Madin-Darby canine kidney (MDCK) cells grown on permeable supports were mounted in a closed double perfusion chamber on an inverted microscope. The intracellular Ca(2+) concentration ([Ca(2+)](i)) was monitored with the Ca(2+)-sensitive fluorescence probe fluo 4. Transepithelial pressure pulses of 30-80 mm Hg produced a transient increase in [Ca(2+)](i) of MDCK cells. This response is independent of the primary cilium, since it is readily observed in immature cells that do not yet express primary cilia. The amplitudes of the pressure-induced Ca(2+) transients varied with the applied chamber pressure in a quantity-dependent manner. The ATPase apyrase and the P2Y antagonist suramin significantly reduced the pressure-induced Ca(2+) transients. Applying apyrase or suramin to both sides of the preparation simultaneously nearly abolished the pressure-induced Ca(2+) response. In conclusion, these observations suggest that rapid pressure changes induce both apical and basolateral nucleotide release that contribute to mechanosensation in kidney epithelial cells.

Signals regulating trafficking of Menkes (MNK; ATP7A) copper-translocating P-type ATPase in polarized MDCK cells.

The Menkes protein (MNK; ATP7A) functions as a transmembrane copper-translocating P-type ATPase and plays a vital role in systemic copper absorption in the gut and copper reabsorption in the kidney. Polarized epithelial cells such as Madin-Darby canine kidney (MDCK) cells are a physiologically relevant model for systemic copper absorption and reabsorption in vivo. In this study, cultured MDCK cells were used to characterize MNK trafficking and enabled the identification of signaling motifs required to target the protein to specific membranes. Using confocal laser scanning microscopy and surface biotinylation we demonstrate that MNK relocalizes from the Golgi to the basolateral (BL) membrane under elevated copper conditions. As previously shown in nonpolarized cells, the metal binding sites in the NH2-terminal domain of MNK were found to be required for copper-regulated trafficking from the Golgi to the plasma membrane. These data provide molecular evidence that is consistent with the presumed role of this protein in systemic copper absorption in the gut and reabsorption in the kidney. Using site-directed mutagenesis, we identified a dileucine motif proximal to the COOH terminus of MNK that was critical for correctly targeting the protein to the BL membrane and a putative PDZ target motif that was required for localization at the BL membrane in elevated copper.

Myristoylated Naked2 escorts transforming growth factor alpha to the basolateral plasma membrane of polarized epithelial cells.

The epidermal growth factor receptor ligands transforming growth factor alpha (TGF alpha) and amphiregulin are delivered to the basolateral surface of polarized epithelial cells where they are cleaved by TACE/ADAM17. Basolateral sorting information resides in their cytoplasmic tail domains, but tail-interacting proteins required for basolateral trafficking have not been identified. Naked (NKD)1 and NKD2 are mammalian homologs of Drosophila Naked Cuticle, which negatively regulates canonical Wnt signaling by binding Dishevelled. We present evidence that NKD2, but not NKD1, binds to basolateral sorting motifs in the cytoplasmic tail of TGF alpha. Processing and cell-surface delivery of TGF alpha are accelerated in NKD2-overexpressing Madin-Darby canine kidney cells. NKD2 is myristoylated on glycine, the second residue. On expression of myristoylation-defective (G2A) NKD2, neither NKD2 nor TGF alpha appears at the basolateral plasma membrane of polarized Madin-Darby canine kidney cells; however, membrane staining for TGF alpha is restored on silencing expression of this mutant NKD2. Amphiregulin does not interact with NKD2 and retains its basolateral localization in G2A-NKD2-expressing cells, as do Na(+), K(+) ATPase alpha 1 and E-cadherin. These data identify an unexpected function for NKD2, i.e., myristoylation-dependent escort of TGF alpha to the basolateral plasma membrane of polarized epithelial cells.

Polarized glycoprotein targeting affects the spread of measles virus in vitro and in vivo.

We have shown previously that basolateral targeting of plasmid-encoded measles virus (MV) F and H protein is dependent on single tyrosine residues in the cytoplasmic tails of the glycoproteins and is essential for fusion activity in polarized epithelial cells. Here, we present data on the functional importance of polarized glycoprotein expression for the cytopathic properties of infectious MV in culture and for pathogenesis in vivo. By the introduction of single point mutations, we generated recombinant viruses in which the basolateral targeting signal of either one or both glycoproteins was destroyed (tyrosine mutants). As a consequence, the mutated glycoproteins were predominantly expressed on the apical membrane of polarized Madin-Darby canine kidney cells. In contrast to parental MV, none of these virus mutants was able to spread by syncytia formation in polarized cells showing that the presence of both MV glycoproteins at the basolateral cell surface is required for cell-to-cell fusion in vitro. Using cotton rats as an animal model that allows MV replication in the respiratory tract, we showed that basolateral glycoprotein targeting is also of importance for the spread of infection in vivo. Whereas parental MV was able to spread laterally within the respiratory epithelium and from there to cells in the underlying tissue, tyrosine mutants infected only single epithelial and very few subepithelial cells. These data strongly suggest that basolateral targeting of MV glycoproteins helps to overcome the epithelial barrier and thereby facilitates the systemic spread of MV infection in vivo.

Hepatocyte growth factor stimulates adenoviral-mediated gene transfer across the apical membrane of epithelial cells.

The apical surface of polarized epithelial cells is relatively resistant to gene delivery by various agents including adenoviral vectors. Hepatocyte growth factor (HGF) dedifferentiates previously well-polarized Madin-Darby canine kidney (MDCK) cell monolayers by altering cell-surface polarity and inhibiting tight junction function. We used an in vitro model of polarized MDCK cells grown on permeable supports to examine the effects of HGF pretreatment on adenoviral (Ad)-mediated gene delivery through the apical surface of epithelial cell monolayers. HGF pretreatment of MDCK cell monolayers for 72 h increased Ad-mediated gene transfer and expression of enhanced green fluorescent protein (EGFP) and luciferase in a dose-dependent fashion. Time-course analysis of HGF-induced stimulation of Ad-mediated gene transfer was seen after 24 h and increased further with pretreatment periods extending to 72 h. HGF pretreatment increased Ad-mediated gene transfer at varying multiplicity of infection (MOI; ranging from 0.2-2000). PCR analysis for adenoviral DNA in control and HGF-pretreated MDCK cells suggested increased entry of viral constructs into HGF-pretreated MDCK cell monolayers. HGF-induced alterations in cell polarity are reversible upon removal of HGF. These data demonstrate that HGF pretreatment of MDCK cells increases the sensitivity of the cells to Ad-mediated gene delivery. The mechanism by which this occurs appears to be through increased entry of adenovirus into epithelial cells. These data provide evidence that biological agents that transiently alter epithelial cell polarity and tight junction function can be used to augment Ad-mediated gene delivery into epithelial cells from the apical surface.

Epithelial cell polarity alters Rho-GTPase responses to Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic human pathogen that preferentially infects damaged epithelial tissues. Previous studies have failed to distinguish whether the increased susceptibility of injured epithelium results from the loss of cell polarity or increased access to the basolateral surface. We have used confluent monolayers of Madin-Darby canine kidney (MDCK) cells cultured on porous filter supports for 1-3 d as a model system to investigate whether the differentiation state of a polarized model epithelium affected the response of epithelial cells to this pathogen. Confluent incompletely polarized MDCK cell monolayers (day 1) efficiently internalized apically applied P. aeruginosa via a pathway that required actin polymerization and activation of Rho-family GTPases and was accompanied by an increase in the amount of activated RhoA. In contrast, P. aeruginosa entry into highly polarized MDCK monolayers (day 3) was 10- to 100-fold less efficient and was insensitive to inhibitors of actin polymerization or of Rho-family GTPase activation. There was no activation of RhoA; instead, Cdc42-GTP levels increased significantly. Basolateral infection of highly polarized MDCK monolayers was less efficient and insensitive to Clostridium difficile Toxin B, whereas basolateral infection of incompletely polarized MDCK monolayers was more efficient and required activation of Rho-family GTPases. Together, our findings suggest that as epithelial barrier differentiates and becomes highly polarized, it becomes resistant to P. aeruginosa infection. Nevertheless, polarized epithelial cells still sense the presence of apically infecting P. aeruginosa, but they may do so through a different group of surface proteins and/or downstream signaling pathways than do incompletely polarized cells.

Hepatocyte growth factor induces MDCK cell morphogenesis without causing loss of tight junction functional integrity.

Hepatocyte growth factor (HGF) induces mitogenesis, motogenesis, and tubulogenesis of cultured Madin-Darby canine kidney (MDCK) epithelial cells. We report that in addition to these effects HGF stimulates morphogenesis of tight, polarized MDCK cell monolayers into pseudostratified layers without loss of tight junction (TJ) functional integrity. We tested TJ functional integrity during formation of pseudostratified layers. In response to HGF, the TJ marker ZO-1 remained in morphologically complete rings and functional barriers to paracellular diffusion of ruthenium red were maintained in pseudostratified layers. Transepithelial resistance (TER) increased transiently two- to threefold during the morphogenetic transition from monolayers to pseudostratified layers and then declined to baseline levels once pseudostratified layers were formed. In MDCK cells expressing the trk/met chimera, both HGF and NGF at concentrations of 2.5 ng/ml induced scattering. However, 2.5 ng/ml HGF did not affect TER. The peak effect of HGF on TER was at a concentration of 100 ng/ml. In contrast, NGF at concentrations as high as 25 microg/ml had no effect on TER or pseudostratified layer morphogenesis of trk/met-expressing cultures. These results suggest that altered presentation of the stimulus, such as through HGF interaction with low-affinity sites, may change the downstream signaling response. In addition, our results demonstrate that HGF stimulates pseudostratified layer morphogenesis while inducing an increase in TER and maintaining the overall tightness of the epithelial layer. Stimulation of epithelial cell movements by HGF without loss of functional TJs may be important for maintaining epithelial integrity during morphogenetic events such as formation of pseudostratified epithelia, organ regeneration, and tissue repair.

Basolateral targeting by leucine-rich repeat domains in epithelial cells.

The asymmetric distribution of proteins to basolateral and apical membranes is an important feature of epithelial cell polarity. To investigate how basolateral LAP proteins (LRR (for leucine-rich repeats) and PDZ (for PSD-95/Discs-large/ZO-1), which play key roles in cell polarity, reach their target membrane, we carried out a structure-function study of three LAP proteins: Caenorhabditis elegans LET-413, human Erbin and human Scribble (hScrib). Deletion and point mutation analyses establish that their LRR domain is crucial for basolateral membrane targeting. This property is specific to the LRR domain of LAP proteins, as the non-LAP protein SUR-8 does not localize at the basolateral membrane of epithelial cells, despite having a closely related LRR domain. Importantly, functional studies of LET-413 in C. elegans show that although its PDZ domain is dispensable during embryogenesis, its LRR domain is essential. Our data establish a novel paradigm for protein localization by showing that a subset of LRR domains direct subcellular localization in polarized cells.

Protein 4.1N Is Required for Translocation of Inositol 1,4,5-Trisphosphate Receptor Type 1 to the Basolateral Membrane Domain in Polarized Madin-Darby Canine Kidney Cells

Protein 4.1N was identified as a binding molecule for the C-terminal cytoplasmic tail of inositol 1,4,5-trisphosphate receptor type 1 (IP(3)R1) using a yeast two-hybrid system. 4.1N and IP(3)R1 associate in both subconfluent and confluent Madin-Darby canine kidney (MDCK) cells, a well studied tight polarized epithelial cell line. In subconfluent MDCK cells, 4.1N is distributed in the cytoplasm and the nucleus; IP(3)R1 is localized in the cytoplasm. In confluent MDCK cells, both 4.1N and IP(3)R1 are predominantly translocated to the basolateral membrane domain, whereas 4.1R, the prototypical homologue of 4.1N, is localized at the tight junctions (Mattagajasingh, S. N., Huang, S. C., Hartenstein, J. S., and Benz, E. J., Jr. (2000) J. Biol. Chem. 275, 30573-30585), and other endoplasmic reticulum marker proteins are still present in the cytoplasm. Moreover, the 4.1N-binding region of IP(3)R1 is necessary and sufficient for the localization of IP(3)R1 at the basolateral membrane domain. A fragment of the IP(3)R1-binding region of 4.1N blocks the localization of co-expressed IP(3)R1 at the basolateral membrane domain. These data indicate that 4.1N is required for IP(3)R1 translocation to the basolateral membrane domain in polarized MDCK cells.

Simultaneous Expression of hCNT1-CFP and hENT1-YFP in Madin-Darby Canine Kidney Cells: LOCALIZATION AND VECTORIAL TRANSPORT STUDIES

To test the hypothesis that human concentrative and equilibrative nucleoside transporters (hCNT1 and hENT1) are present on the apical and basolateral membrane, respectively, we constructed a Madin-Darby canine kidney (MDCK) cell line that simultaneously and stably expresses recombinant hCNT1 and hENT1 gene products tagged with CFP and YFP fluorescent proteins, respectively. Using a confocal microscope, both hCNT1-CFP and hENT1-YFP were found to be distributed uniformly on the plasma membrane of undifferentiated MDCK cells. Upon differentiation of the MDCK cells on Transwell filter inserts, hCNT1-CFP was visualized exclusively on the apical membrane, whereas hENT1-YFP appeared predominantly on the basolateral membrane. As differentiation proceeded, there was an increase in alkaline phosphatase activity, and activity of hENT1 in the apical compartment decreased while hCNT1 activity remained constant. These results suggest that, on differentiation, hENT1 is sorted to the basolateral membrane. This was confirmed when the hCNT1-mediated uptake of [(3)H]uridine from the apical compartment of the differentiated cells was found to be approximately 20-fold higher and that for hENT1 was approximately 4-fold lower than the corresponding uptake from the basal compartment. As observed in vivo, the net transport of [(3)H]adenosine was from the apical to the basal compartment, whereas that for (14)C-deoxyadenosine was from the basal to the apical compartment. In summary, we have shown for the first time that hCNT1 and hENT1 are expressed in polarized MDCK cells on the apical and basolateral membrane, respectively, allowing vectorial transport in both directions depending on the relative activity (ratio of maximal transporter activity to affinity) of each transporter for their substrates.