Glycosylation is important for binding to human calcitonin receptors

Human calcitonin receptor (hCTR) subtypes contain three or four potential Asn-linked glycosylation sites in their extracellular amino termini. The role of glycosylation in hCTR function has not been identified, but it has been suggested that inhibition of glycosylation does not affect binding or signaling. To determine the role of glycosylation in hCTR biology, we studied the effects of inhibition of glycosylation and of substitution of Asn residues that are potential glycosylation sites. Native and mutated hCTRs were studied after transient expression in monkey kidney COS-1 cells. Tunicamycin, administered as part of a treatment protocol that inhibited glycosylation of all expressed receptors, decreased salmon calcitonin (sCT) binding affinities and signaling potencies at hCTRs with three or four potential glycosylation sites. In hCTR3, which contains three potential glycosylation sites at positions 26, 78, and 83, site-specific substitution of Asn-26 by Ala had no effect on sCT binding affinity or potency, whereas substitution of Asn-78 or Asn-83 lowered sCT affinity and potency. A mutant hCTR3 in which all three Asn residues were substituted with Ala exhibited no high-affinity sCT binding and potencies of several calcitonin analogues that were more than 100-fold lower than that of native hCTR3. Our data show that glycosylation is important for high-affinity binding and potency of calcitonin analogues at hCTRs.     

Human copper transporter hCTR1 mediates basolateral uptake of copper into enterocytes: implications for copper homeostasis

Copper is essential for human growth and survival. Enterocytes mediate the absorption of dietary copper from the intestinal lumen into blood as well as utilizing copper for their biosynthetic needs. Currently, the pathways for copper entry into enterocytes remain poorly understood. We demonstrate that the basolateral copper uptake into intestinal cells greatly exceeds the apical uptake. The basolateral but not apical transport is mediated by the high affinity copper transporter hCTR1. This unanticipated conclusion is supported by cell surface biotinylation and confocal microscopy of endogenous hCTR1 in Caco2 cells as well as copper influx measurements that show saturable high affinity uptake at the basolateral but not the apical membrane. Basolateral localization of hCTR1 and polarized copper uptake are also conserved in T84 cells, models for intestinal crypt cells. The lateral localization of hCTR1 seen in intestinal cell lines is recapitulated in immunohistochemical staining of mouse intestinal sections. Biochemical and functional assays reveal the basolateral localization of hCTR1 also in renal Madin-Darby canine kidney cells and opossum kidney cells. Overexpression of hCTR1 in Madin-Darby canine kidney cells results in both apical and basolateral delivery of the overexpressed protein and greatly enhanced copper uptake at both cell surfaces. We propose a model of intestinal copper uptake in which basolateral hCTR1 plays a key role in the physiologically important delivery of copper from blood to intracellular proteins, whereas its role in the initial apical uptake of dietary copper is indirect.     

The internalization signal and the phosphorylation site of transferrin receptor are distinct from the main basolateral sorting information.

Wild-type human transferrin receptor (hTfR), like endogenous canine receptor, is expressed almost exclusively (97%) at the basolateral membrane of transfected Madin-Darbey canine kidney (MDCK) cells. We investigated the role of two distinct features of the hTfR cytoplasmic domain, namely the endocytic signal and the unique phosphorylation site, in polarized cell surface delivery. Basolateral location was not altered by point mutation of Ser24-->Ala24, indicating that phosphorylation is not involved in vectorial sorting of hTfR. The steady state distribution of hTfR was partially affected by a deletion of 36 cytoplasmic residues encompassing the internalization sequence. However, 80% of the receptors were still basolateral. As assessed by pulse-chase experiments in combination with biotinylation, newly synthesized wild-type and deletion mutant receptors were directly sorted to the domain of their steady state residency. Although both receptors could bind human transferrin, endocytosis of the deletion mutant was strongly impaired at either surface. These data indicate that the predominant basolateral targeting signal of hTfR is independent of the internalization sequence.     

Asymmetric distribution of muscarinic acetylcholine receptors in Madin-Darby canine kidney cells

We have characterized the muscarinic AChreceptors (mAChRs) expressed in Madin- Darby canine kidney (MDCK)strain II epithelial cells. Binding studies with themembrane-impermeable antagonist N-[³H]methylscopolaminedemonstrated that mAChRs are ~2.5 times more abundant on thebasolateral than on the apical surface. Apical, but not basolateral,mAChRs inhibited forskolin-stimulated adenylyl cyclase activity inresponse to the agonist carbachol. Neither apical nor basolateralmAChRs exhibited detectable carbachol-stimulated phospholipase Cactivity. Carbachol application to the apical or the basolateralmembrane resulted in a threefold increase in intracellularCa²⁺ concentration, which wascompletely inhibited by pertussis toxin on the apical side andpartially inhibited on the basolateral side. RT-PCR analysis showedthat MDCK cells express the M₄ and M₅ receptor mRNAs. These datasuggest that M₄ receptors reside on the apical and basolateral membranes of polarized MDCK strain IIcells and that the M₅ receptor mayreside in the basolateral membrane of a subset of cells.

     

Cytoplasmic juxtamembrane domain of the human EGF receptor is required for basolateral localization in MDCK cells

Although it is well established that epidermal growth factor receptors (EGFRs) are asymmetrically expressed at the basolateral plasma membrane in polarized epithelial cells, how this process is regulated is not known. The purpose of this study was to address the mechanism of directed EGFR basolateral sorting using the Madin-Darby canine kidney (MDCK) cell model. The first set of experiments established sorting patterns for endogenous canine EGFRs. The polarity of the canine EGFR was not quantitatively affected by differences in electrical resistance exhibited by the MDCK I and MDCK II cell strains. In both cases, greater than 90% of total surface EGFRs was localized to the basolateral surface. Canine EGFRs sort directly to the basolateral membrane from the trans-Golgi network with a halftime of approximately 45 min and have an approximate t_1/2 of 12.5 h once reaching the basolateral surface. Human holoreceptors expressed in stably transfected MDCK cells also localize to the basolateral membrane with similar efficiency. To identify EGFR sequences necessary for basolateral sorting, MDCK cells were transfected with cDNAs coding for cytoplasmically truncated human receptor proteins. Human EGFRs truncated at Arg-651 were localized predominantly at the apical surface of filter-grown cells, whereas receptors truncated at Leu-723 were predominantly basolateral. These results suggest that the cytoplasmic juxtamembrane domain contains a positive basolateral sorting determinant. Moreover, the EGFR ectodomain or transmembrane domain may possess a cryptic sequence that specifically interacts with the apical sorting machinery once the dominant basolateral sorting signal is removed. Further elucidation of the precise loacation of these signals will enhance our basic understanding of regulated plasma membrane sorting, as well as the functional consequences of inappropriate EGFR expression associated with certain pathophysiologic and malignant states. © 1995 Wiley-Liss, Inc.     

Regulation of EGF signaling by cell polarity in MDCK kidney epithelial cells.

Although the presence of a dominant basolateral sorting signal ensures that the majority of newly synthesized epidermal growth factor (EGF) receptors are delivered directly to the basolateral surface in polarized epithelial cells, a fraction of the receptors are also delivered to the apical surface. Similar to most basolateral membrane proteins, the EGF receptor has an additional signal(s) that selectively targets molecules lacking a dominant basolateral signal to the apical surface. Although the physiological relevance of signal hierarchy is not known, alternative targeting may occur in different epithelial cell types or during development. The goal of this study, therefore, was to determine the effect of membrane domain location on EGF receptor function, focusing on EGF-induced MAP kinase signaling and DNA synthesis. Whereas ligand responsiveness was restricted to the basolateral domain in Madin-Darby canine kidney (MDCK) cells expressing a normal complement of receptors, apical ligand was effective if apical receptor density was increased by overexpression of an exogenous wild-type human gene. Unexpectedly, cells expressing apically localized, cytoplasmically truncated receptors, which behave as dominant negative mutations in other cell types, were also responsive to apical EGF. The cytoplasmically truncated molecules appear to have at least two effects: first, to increase the local concentration of ligand at the apical cell surface; and second, to facilitate activation of the relatively few native EGF receptors normally located at the apical surface. These results indicate that cell context is a critical determinant of receptor mutant protein phenotype.     

Bidirectional transcytosis determines the steady state distribution of the transferrin receptor at opposite plasma membrane domains of BeWo cells

Trophoblast-like BeWo cells form well-polarized epithelial monolayers, when cultured on permeable supports. Contrary to other polarized cell systems, in which the transferrin receptor is found predominantly on the basolateral cell surface, BeWo cells express the transferrin receptor at both apical and basolateral cell surfaces (Cerneus, D.P., and A. van der Ende. 1991. J. Cell Biol. 114: 1149-1158). In the present study we have addressed the question whether BeWo cells use a different sorting mechanism to target transferrin receptors to the cell surface, by examining the biosynthetic and transcytotic pathways of the transferrin receptor in BeWo cells. Using trypsin and antibodies to detect transferrin receptors at the cell surface of filter-grown BeWo cells, we show that at least 80% of newly synthesized transferrin receptor follows a direct pathway to the basolateral surface, demonstrating that the transferrin receptor is efficiently intracellularly sorted. After surface arrival, pulse-labeled transferrin receptor equilibrates between apical and basolateral cell surfaces, due to ongoing transcytotic transport in both directions. The subsequent redistribution takes over 120 min and results in a steady state distribution with 1.5-2.0 times more transferrin receptors at the basolateral surface than at the apical surface. By monitoring the fate of surface-bound 125I-transferrin, internalized either from the apical or basolateral surface transcytosis of the transferrin receptor was studied. About 15% of 125I-transferrin is transcytosed in the basolateral to apical direction, whereas 25% is transcytosed in the opposite direction, indicated that the fraction of receptors involved in transcytosis is roughly twofold higher for the apical receptor pool, as compared to the basolateral pool. Upon internalization, both apical and basolateral receptor pools become redistributed on both surfaces, resulting in a twofold higher number of transferrin receptors at the basolateral surface. Our results indicate that in BeWo cells bidirectional transcytosis is the main factor in surface distribution of transferrin receptors on apical and basolateral surfaces, which may represent a cell type-specific, post-endocytic, sorting mechanism.     

An in vitro study of the sub-cellular distribution of nicotinic receptors

Nicotinic and serotoninergic 5HT3 receptors share important sequence identities except for their cytoplasmic loop. Both ends of this loop display conserved 3D helical structures with distinct primary sequences. We decided to check whether these two helices named F and G play a role in the sub-cellular distribution of different nicotinic receptors. We systematically exchanged each helix with the equivalent sequence of neuronal nicotinic and alpha4, beta2 and alpha7 subunits in the functional chimeric alpha7-5HT3 receptor used as a model system. The new chimeras were expressed in vitro in polarized epithelial cells from pig kidney. We quantified synthesis and export of the receptors to the cell surface by measuring alpha-bungarotoxin binding sites. Immunogold labelling was used, at the electron microscope level, to determine the amount of each chimera present at either domain, apical and/or basolateral, of these cells. We noticed that in epithelial cells the majority of alpha-bungarotoxin binding sites remained sequestered in the cytoplasm as already observed in neurons in vivo. The majority of the pentamers present at the cell surface were located at the apical domain. Our results suggest that helix F and G differently regulate assembly and export to the cell surface of alpha-bungarotoxin binding receptors.     

Polarized endocytosis by Madin-Darby canine kidney cells transfected with functional chicken liver glycoprotein receptor

We have studied the expression of the chicken hepatic glycoprotein receptor (chicken hepatic lectin [CHL]) in Madin-Darby canine kidney (MDCK) cells, by transfection of its cDNA under the control of a retroviral promotor. Transfected cell lines stably express 87,000 surface receptors/cell with a kd = 13 nM. In confluent monolayers, approximately 40% of CHL is localized at the plasma membrane. 98% of the surface CHL is expressed at the basolateral surface where it performs polarized endocytosis and degradation of glycoproteins carrying terminal N-acetylglucosamine at a rate of 50,000 ligand molecules/h. Studies of the half-life of metabolically labeled receptor and of the stability of biotinylated cell surface receptor after internalization indicate that transfected CHL performs several rounds of uptake and recycling before it gets degraded. The successful expression of a functional basolateral receptor in MDCK cells opens the way for the characterization of the mechanisms that control targeting and recycling of proteins to the basolateral membrane of epithelial cells.     

The basolateral sorting signals of the thyrotropin and luteinizing hormone receptors: an unusual family of signals sharing an unusual distal intracellular localization, but unrelated in their structures

The mechanisms of the basolateral targeting of G protein-coupled receptors remain largely unknown. Mutagenesis experiments have allowed us to identify the basolateral sorting signals of the TSH and LH receptors expressed in Madin-Darby canine kidney cells and thyroid follicular FRT cells. Unexpectedly these signals (amino acids 731-746 and 672-689, respectively) share an unusual localization in the distal part of the intracellular domain of the receptors at a marked distance from the membrane. When grafted onto the p75-neurotropin receptor, these signals redirect this normally apically expressed protein to the basolateral cell surface. They are independent of the endocytosis signal. The basolateral sorting signals of TSH, LH, and FSH receptors do not exhibit primary sequence homology with each other or with any other known signal. Furthermore, circular dichroism studies show that the three signals exhibit distinct secondary structures. The TSH receptor has a stable helical structure, the LH receptor has both helix and beta-sheet structures, and the FSH receptor sorting signal has a main random coil structure. This means that even in closely-related receptors different secondary structures can be found for basolateral signals unrelated to internalization signals. This observation contrasts with what is known about basolateral signals related to internalization signals for which a common beta-turn structure has been described. Deletion of the basolateral sorting signals results in apical targeting of the receptors, suggesting the existence of apical sorting information. However, a soluble form of the TSH receptor, which harbors all N- and putative O-linked oligosaccharides, is secreted in a nonpolarized fashion. This implies that apical sorting information must be located elsewhere, either in the transmembrane or in the intracellular domains of the receptor.     

Deletion of the cytoplasmic domain of the polymeric immunoglobulin receptor prevents basolateral localization and endocytosis

We deleted the cytoplasmic domain of the polymeric immunoglobulin receptor. When expressed in fibroblasts, the truncated receptor, like the wild-type, reaches the cell surface, can bind ligand, and is cleaved to secretory component. Unlike the wild-type, it is not endocytosed. When expressed in polarized Madin-Darby canine kidney cells, the mutant receptor is transported from the Golgi apparatus directly to the apical surface and cleaved to secretory component. In contrast, the wild-type receptor travels from the Golgi to the basolateral surface and is then endocytosed and sent to the apical surface. These results suggest that the cytoplasmic domain of the receptor is necessary for both basolateral localization and endocytosis.     

Retargeting the coxsackievirus and adenovirus receptor to the apical surface of polarized epithelial cells reveals the glycocalyx as a barrier to adenovirus-mediated gene transfer

Lumenal delivery of adenovirus vectors (AdV) results in inefficient gene transfer to human airway epithelium. The human coxsackievirus and adenovirus receptor (hCAR) was detected by immunofluorescence selectively at the basolateral surfaces of freshly excised human airway epithelial cells, suggesting that the absence of apical hCAR constitutes a barrier to adenovirus-mediated gene delivery in vivo. In transfected polarized Madin-Darby canine kidney cells, wild-type hCAR was expressed selectively at the basolateral membrane, whereas hCAR lacking the transmembrane and/or cytoplasmic domains was expressed on both the basolateral and apical membranes. Cells expressing apical hCAR still were not efficiently transduced by AdV applied to the apical surface. However, after the cells were treated with agents that remove components of the apical surface glycocalyx, AdV transduction occurred. These results indicate that adenovirus can infect via receptors located at the apical cell membrane but that the glycocalyx impedes interaction of AdV with apical receptors.     

Differential trafficking of transforming growth factor-beta receptors and ligand in polarized epithelial cells

Epithelial cells in vivo form tight cell-cell associations that spatially separate distinct apical and basolateral domains. These domains provide discrete cellular processes essential for proper tissue and organ development. Using confocal imaging and selective plasma membrane domain activation, the type I and type II transforming growth factor-beta (TGFbeta) receptors were found to be localized specifically at the basolateral surfaces of polarized Madin-Darby canine kidney (MDCK) cells. Receptors concentrated predominantly at the lateral sites of cell-cell contact, adjacent to the gap junctional complex. Cytoplasmic domain truncations for each receptor resulted in the loss of specific lateral domain targeting and dispersion to both the apical and basal domains. Whereas receptors concentrate basolaterally in regions of direct cell-cell contact in nonpolarized MDCK cell monolayers, receptor staining was absent from areas of noncell contact. In contrast to the defined basolateral polarity observed for the TGFbeta receptor complex, TGFbeta ligand secretion was found to be from the apical surfaces. Confocal imaging of MDCK cells with an antibody to TGFbeta1 confirmed a predominant apical localization, with a stark absence at the basal membrane. These findings indicate that cell adhesion regulates the localization of TGFbeta receptors in polarized epithelial cultures and that the response to TGFbeta is dependent upon the spatial distribution and secretion of TGFbeta receptors and ligand, respectively.     

Basolateral sorting of human poliovirus receptor alpha involves an interaction with the mu1B subunit of the clathrin adaptor complex in polarized epithelial cells

Poliovirus receptor (hPVR/CD155) is a cell surface glycoprotein that belongs to the immunoglobulin superfamily but its natural function remains unknown. Two membrane-bound isoforms, hPVRalpha and hPVRdelta, are known to date, and they differ only in the amino acid sequence of their cytoplasmic domains. To gain an insight into the possible function of the cytoplasmic domains, we examined the localization of introduced hPVRalpha and hPVRdelta in polarized epithelial cells deficient of native hPVRs. Basolateral sorting of hPVRalpha was observed in Madine-Darby canine kidney cells expressing mu1B, but not in LLC-PK1 porcine kidney cells deficient in mu1B. Distribution of hPVRdelta, however, occurred both on the apical and basolateral plasma membranes of these two cell lines. Basolateral sorting of hPVRalpha was also seen in LLC-PK1 cells that expressed an intact exogenous mu1B, but not in the cells that expressed a mutant mu1B lacking binding ability to tyrosine-containing signals. These results indicate that mu1B is involved in the distribution of hPVRalpha to the basolateral membrane. Comparative distribution analysis of hPVRalpha using a series of mutants with truncations and substitutions in the cytoplasmic tail demonstrated that determinant for the basolateral sorting resided in the tyrosine-containing motif of the cytoplasmic tail. Furthermore, yeast two hybrid analysis strongly suggested that the tyrosine motif directly interacted with mu1B protein. Thus, basolateral sorting of hPVRalpha appears to involve the interaction with mu1B through a tyrosine motif existing in the cytoplasmic domain.     

A hepatocyte-specific basolateral membrane protein is targeted to the same domain when expressed in Madin-Darby canine kidney cells

Different mechanisms for polarized sorting of apical and basolateral plasma membrane proteins appear to be operative in different cell types. In hepatocytes, all proteins are first transported to the basolateral surface, where sorting (probably signal-mediated) of apical proteins then takes place. In contrast, in Madin-Darby canine kidney (MDCK) cells, proteins are directly transported from the trans-Golgi network to their appropriate plasma membrane domain. In order to study the differences in the sorting requirements of the two cell types, we have expressed a hepatocyte-specific basolateral membrane protein, the asialoglycoprotein receptor H1, in MDCK cells. H1 was found to be specifically transported to the basolateral domain also in this heterologous system, suggesting that either the same basolateral targeting signal is operative in both cell types or, more likely, that basolateral transport occurs "by default," i.e. without the requirement for a sorting signal.     

Multiple regions within the coxsackievirus and adenovirus receptor cytoplasmic domain are required for basolateral sorting

The coxsackievirus and adenovirus receptor (CAR) mediates attachment and infection by coxsackie B viruses and many adenoviruses. In human airway epithelia, as well as in transfected Madin-Darby canine kidney cells, CAR is expressed exclusively on the basolateral surface. Variants of CAR that lack the cytoplasmic domain or are attached to the cell membrane by a glycosylphosphatidylinositol anchor are expressed on both the apical and basolateral surfaces. We have examined the localization of CAR variants with progressive truncations of the cytoplasmic domain, as well as with mutations that ablate a potential PDZ (PSD95/dlg/ZO-1) interaction motif and a putative tyrosine-based sorting signal. In addition, we have examined the targeting of two murine CAR isoforms, with different C-terminal sequences. The results suggest that multiple regions within the CAR cytoplasmic domain contain information that is necessary for basolateral targeting.