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Endocrinology Vol. 143, No. 4 1291-1301
Copyright © 2002 by The Endocrine Society

RECEPTORS

Differential Recognition of a Tyrosine-Dependent Signal in the Basolateral and Endocytic Pathways of Thyroid Epithelial Cells

Concetta Lipardi, Germana Ruggiano, Lorena Perrone, Simona Paladino, Laure Monlauzeur, Lucio Nitsch, André Le Bivic and Chiara Zurzolo

Centro di Endocrinologia ed Oncologia Sperimentale del Centro Nazionale delle Ricerche-Dipartimento di Biologia e Patologia Cellulare e Molecolare (C.L., G.R., L.P., L.N., C.Z.), Università degli Studi di Napoli "Federico II," 80131 Napoli, Italy; and Institut de Biologie du Développement de Marseille (L.M., A.L.), Université de la Méditerranée, Faculté des Sciences de Luminy, Marseille, France

Address all correspondence and requests for reprints to: Chiara Zurzolo, Dipartimento di Biologia e Patologia Cellulare e Molecolare, II Facoltà di Medicina, Via Pansini 5, 80131 Napoli, Italy. E-mail: . zurzolo{at}ds.unina.it


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Trafficking of receptors is of crucial importance for the physiology of most exocrine and endocrine organs. It is not known yet if the same mechanisms are used for sorting in the exocytic and endocytic pathways in the different epithelial tissues. In this work, we have used a deletion mutant of the human neurotrophin receptor p75hNTR that is normally localized on the apical membrane when expressed in Madin-Darby canine kidney cells. This internal 57-amino acid deletion of the cytoplasmic tail leads to a relocation of the protein from the apical to the basolateral membrane and to rapid and efficient endocytosis. These events are mediated by a signal localized within 9 amino acids of the mutated cytoplasmic tail that is strictly dependent on a tyrosine residue (Tyr-308). We have analyzed the basolateral sorting efficiency and endocytic capacity of this signal in Fischer rat thyroid (FRT) cells, in which basolateral and endocytic determinants have not yet been identified. We found that this targeting signal can mediate efficient transport to the basolateral membrane also in FRT cells with similar tyrosine dependence as in MDCK cells. In contrast to MDCK cells, this Tyr-based signal was not able to mediate coated pits localization and endocytosis in FRT cells. These data represent the first characterization of basolateral/endocytic signals in thyroid epithelial cells. Furthermore, our results indicate that requirements for tyrosine-dependant basolateral sorting signals are conserved among cell lines from different tissues but that the recognition of the colinear endocytic signal is tissue specific.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN ORDER TO perform their function, epithelial cells such as thyroid cells have a polarized organization. Tight junctions separate their plasma membrane into two distinct domains, the apical (AP) domain and the basolateral (BL) domain (1, 2). These domains have striking differences in their protein and lipid composition and, subsequently, also in their specific biological functions (3, 4). The apical domain of thyrocytes faces the follicular lumen where the thyroglobulin is secreted and stored before used as a substrate for iodination and hormonogenesis (5, 6). The basolateral domain is in contact with the bloodstream where thyrocytes take up iodide and release thyroid hormones (7, 8, 9). The maintenance of asymmetric protein distribution in the two domains of the plasma membrane is achieved by the continuous and differential traffic of newly synthesized proteins through distinct transport vesicles from the trans-Golgi network (TGN) to the plasma membrane (10). Trafficking can occur via direct or indirect pathways depending upon cell type and/or the properties of the protein itself (11). Primary thyrocytes and Fischer rat thyroid (FRT) cells are good models to study the polarized organization of thyroid cells and thus its impact on the biological functions of the thyroid gland.

In Madin-Darby canine kidney (MDCK) cells, the most used cell model in cell polarity, traffic to the BL membrane is mediated by discrete amino acid sorting signals localized in the cytoplasmic domains of different proteins (11, 12, 13, 14). These signals consist of short amino acid sequences, often active only at a critical distance from the membrane (15, 16). A tyrosine residue, surrounded by charged amino acids, is often critical for BL sorting (17). Furthermore, stretches of acidic amino acids (18), a glycine residue (19), or the presence of di-leucine motifs can increase the targeting efficiency of this type of signal (18, 20). More recently, PDZ binding motifs, formed by C-terminal sequences, have also been shown to mediate BL localization of many growth factor receptors in polarized epithelial cells (21). Besides the PDZ domain, all the above mentioned sorting signals have been found to be important for BL-directed traffic and also for other post-Golgi destinations, as well as in the endocytic pathway (17). Currently, the most popular classification divides these signals into two classes: the first one includes signals related to clathrin-coated pits-determinants that generally, but not always, rely on the same tyrosine residue that is necessary for BL sorting and endocytosis, the second group includes signals that do not coincide with endocytic determinants, even though they might depend on a specific tyrosine residue (see Refs. 14 and 22 and references cited within).

It has not been resolved whether the different types of BL determinants so far found mediate sorting by a common mechanism or whether there exists common machinery for the recognition of BL and endocytic signals. Four different tetrameric complexes of adaptor proteins, AP-1, AP-2, AP-3, and AP-4 have been found to interact with the cytoplasmic signals responsible for the sorting of proteins at the TGN or the plasma membrane via coated vesicles, but they do not seem to function in basolateral sorting (23). Very recently, it has been shown in LLC-PK1 epithelial cells that basolateral sorting is mediated in part by an epithelial cell specific AP1 complex with a µ1B subunit instead of µ1A (24). Furthermore, besides the different use of targeting pathways by different epithelial cell lines (11, 12, 25, 26), the tissue-specific localization patterns of different proteins within the plasma membrane of different cell types must also be considered (2, 3, 27, 28, 29). Another question therefore arises as to whether other epithelial cells recognize signals identified in MDCK cells.

FRT cells (28, 30) show important tissue-specific differences in the localization of membrane proteins (26) when compared with MDCK cells. Because the polarity of most BL transmembrane proteins is conserved in FRT cells, it is likely that they recognize cytosolic BL signals like MDCK cells do. The polarity of many apical proteins, however, is not conserved in FRT cells, suggesting that they have different sorting mechanisms. An investigation of their sorting capacities is therefore necessary before they can become a model for thyroid membrane sorting. To address these questions, we have analyzed the membrane localization and endocytosis of different p75 human neurotrophin receptor (p75hNTR) mutants (31) in transfected FRT cells. These mutants contain a deletion of 57 amino acids within the cytoplasmic tail that is responsible for their BL sorting and endocytosis (32). Mutational analysis of the cytoplasmic tail of the receptor revealed that in MDCK cells both BL localization and endocytosis are mediated by a discrete signal of 9 amino acids which is dependent upon a specific tyrosine residue (Y308) (33). We show here that this signal is also responsible for BL sorting in FRT cells, and that the Y308 residue is critical. However, in contrast to MDCK cells, this signal does not mediate coated pits localization and/or endocytosis. These data therefore indicate that although recognition of BL sorting signals is conserved in thyroid epithelial cells, endocytosis is differentially regulated in FRT cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Cell culture reagents were purchased from Life Technologies, Inc. (Grand Island, NY). Protein A-Sepharose was from Pharmacia Fine Chemicals (Uppsala, Sweden). Sulfosuccinimidyl-6-(biotinamide) hexanoate (NHS-LC-biotin), sulfo-N-hydroxyl-succinimido-biotin (NHS-SS-biotin), and streptavidin-agarose were purchased from Pierce Chemical Co. (Rockford, IL). All other reagents were obtained from Sigma (St. Louis, MO).

Cell culture and antibodies
FRT cells were grown in Coon’s modified Ham’s F12 medium with 5% FBS. When grown on filters, 2 x 106 cells were seeded on Transwell chambers (0.45 mm pore size, 24-mm diameter, Costar, Data Packaging, Cambridge, MA) and cultured for 5 d with changes of medium every other day. Monolayer integrity was confirmed by measuring transepithelial resistance using a Millicell-ERS meter and electrode (Millipore Corp., Bedford, MA). Antibodies were as follows: mouse monoclonal antibody ME 20.4 against the ectodomain of human neurotrophin receptor, p75hNTR, monoclonal antibody antirat transferrin receptor Clone OX 26 (Crawley Down, Sussex, UK), polyclonal antibody against calf intestinal alkaline phosphatase (Rockland, Gilbertsville, PA), rabbit affinity purified antibody to whole molecule mouse IgG (Cappel, Organon Teknika Corp., West Chester, PA). Gold conjugated to monoclonal antibody (5- and 10-nm diameter) were from BioCell Research Laboratories (Cardiff, UK). Mouse anti-{alpha} adaptin (AP-2) was from Sigma.

Constructs, transfection, and clonal selection
PS, PS321, PS315, PS315 Y-A, and PS315 Y-F mutant cDNAs were described earlier (32, 33). PLAP-PS321 cDNA is a chimeric gene containing most of the ectodomain of PLAP (from amino acids Met-1 to Ala-496) and the transmembrane domain and 21 amino acids from the cytoplasmic domain of PS321 (33) subcloned in the pcB6 vector using XbaI and EcoRI restriction sites. FRT cells were transfected using the DNA-calcium phosphate procedure as described (28). Clones expressing the neomycin resistance marker were selected in medium containing G418 (0.5 mg/ml). Clones were isolated with penny cylinders or by limit dilution and screened for expression of PS hNTR mutants by indirect immunofluorescence.

Steady-state biotinylation
Cells grown on filters were incubated for 30 min in DMEM without cysteine and pulsed overnight in the same medium containing 1 mCi/ml of 35S cysteine [Amersham Pharmacia Biotech (Buckinghamshire, UK); 1000 Ci/mmol]. Proteins present either on the apical or the basolateral plasma membrane domains were biotinylated at 4 C using sulfosuccinimidyl-6-(biotinamide) hexanoate (NHS-LC-biotin) as we have previously described (34). Biotinylated proteins were immunoprecipitated with ME 20.4 against anti hNTR (1:200) and subsequently precipitated with streptavidin agarose-beads. Proteins were then run on 10% SDS-PAGE and revealed by fluorography.

Biotin targeting assay
Cells grown on filters were starved for 30 min in DMEM without cysteine, labeled 20 min in the same medium containing 1 mCi/ml of 35S cysteine, and chased for various lengths of time. After surface labeling with NHS-LC-biotin, followed by lysis and double precipitation against anti p75hNTR antibody and streptavidin beads, as previously described (34), the proteins were separated by SDS-PAGE and detected by fluorography.

Biotin internalization assay
Cells grown on filters were incubated for 30 min in DMEM without cysteine and pulsed overnight in the same medium containing 1 mCi/ml of cysteine. Filters were biotinylated using sulfosuccinimidyl-6-(biotinamide) hexanoate (NHS-SS-biotin) from the apical or basolateral plasma membrane domains. Sample filters were incubated at 37 C for 1 h while control filters were kept at 4 C. After washing all filters with ice-cold PBS containing 0.1 mM CaCl2 and 1 mM MgCl2 (PBS CM), reduction with gluthatione (50 mM) was carried out twice from the apical or basolateral domain at 4 C as described (34). Subsequent lysis, double precipitation with antibody and streptavidin beads, and SDS-PAGE were performed as described above.

Surface binding and internalization of NGF
Cells grown on filters were incubated for 1 h at 37 C in 5% CO2 with DMEM containing 10 ng/ml NGF labeled with Na 125I (specific activity of 100 mCi/ml) using the chloramine-T procedure. Cells were then washed four times with cold 1% BSA in PBS CM. Surface binding of NGF was determined by washing the cells with 0.5 ml ice-cold 0.2 M acetic acid containing 0.5 M NaCl two times for 5 min on ice (32). The two washes were pooled and counted. Internalized fractions were determined by counting the filters with a {gamma}-counter (Beckman Coulter, Inc., Palo Alto, CA).

Time course internalization by indirect immunofluorescence
Cells grown on glass coverslips or filters were washed twice with PBS CM, and preincubated at 4 C with primary antibodies: mAb (ME 20.4) against p75hNTR (1:200), pAb against PLAP (1:300), and mAb against TfR (1:10). After the incubation at 37 C for various times to allow for endocytosis to occur cells were cooled on ice. Subsequently, they were fixed in 2% paraformaldehyde in PBS CM for 20 min at room temperature. Aldehyde groups were quenched with 50 mM NH4Cl in PBS CM for 10 min. After cell permeabilization with 0.075% gelatin and 0.2% saponin in PBS CM, receptors were stained using fluorescein-conjugated or rhodamine-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA), at a dilution of 1:50 for 30 min. Control cells were not permeabilized to identify the receptors on the surface. The cells were washed with PBS CM, mounted using PBS/glycerol (1:1), analyzed, and photographed with an Axiophot microscope (Carl Zeiss, Jena, Germany). Cells were also observed in the confocal microscope (Carl Zeiss LMS 410). Serial sections (20 optical sections, 0.7 µm each) were analyzed using LSM Unit software (Carl Zeiss).

Electron microscopy
Cells grown on glass coverslips were incubated with a monoclonal antibody against p75hNTR (1:200) or with the monoclonal antibody against TfR (1:100) for 2 h at 4 C in Areal medium (mF12 +10% FBS + 10 mM HEPES-sodium bicarbonate). After three washes with cold 10% FBS in PBS CM, cells were incubated with antimouse antibody conjugated to 5-nm gold (1:30) for 1 h at 4 C in Areal medium. After washing with cold 10% FBS in PBS CM, cells were incubated for 15 min at 37 C to allow internalization of PS321. Cells were fixed in 0.5% paraformaldehyde and 2.5% glutaraldehyde (Polyscience, Warrington, PA) in 0.1 M cacodylate buffer at pH 7.3 for 15 min and in 1% OsO4 (Sigma) in the same buffer for 15 min. After rinsing in water, cells were stained for 1 h with 1% aqueous uranyl acetate. Finally, after dehydration in a graded series of ethanols, cells were harvested by scraping with a rubber policeman and embedded in Poly/Bed 812 (Polyscience). Thin sections were stained with uranyl acetate and lead citrate (35, 36) and examined with a Philips 400 T electron microscope.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
FRT cells recognize the Tyr-dependent BL sorting signal in the cytoplasmic tail of PS hNTR mutants
To determine whether the basolateral sorting signal contained in PS, a basolateral P75NTR mutant recognized by FRT cells, we transfected this cell line with cDNAs encoding three different PS deletion mutants: PS, PS321, and PS315. These three mutants have, respectively, 98, 19, and 13 amino acids remaining of the cytoplasmic tail (Fig. 1AGo). We characterized the surface distribution of the mutant receptors by domain selective biotinylation after metabolic labeling of stably expressing clones previously selected by indirect immunofluorescence (data not shown). For each construct, several clones with different levels of expression were selected and analyzed for their polarity. No difference could be detected between the clones (data not shown), and thus a representative clone for each construct was used in all subsequent studies. Biotinylated proteins were detected by SDS-PAGE followed by autoradiography after double precipitation with a specific monoclonal antibody raised against the ectodomain of p75hNTR and with streptavidin beads. As in MDCK cells, all PS hNTR proteins were localized preferentially on the BL surface of FRT cells (Fig. 2AGo). Quantification of fluorograms from two independent experiments by densitometry indicated that between 85 and 95% of the total surface mutant proteins were basolaterally localized in each clone (data not shown). We also found that amounts of the BL-localized PS mutant proteins were always higher compared with that of the PS321 and PS315 mutants as was the case in MDCK cells (33). PS321 and PS315 mutants had similar polarity ratios, showing that the cluster of negatively charged residues (EEVE), present only within the tail of PS321, was not able to increase the levels of BL expression, even though such a cluster was found to be important for BL sorting of other transmembrane proteins (18).



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  		Figure 1. Schematic representation and surface localization of PS hNTR mutants in transfected FRT cells. A, PS, PS deleted cDNA containing the entire COOH-terminal tail with two tyrosine residues (Tyr-308, Tyr-340), a cluster of acidic amino acids (EEVE), three di-leucines and a PDZ binding motif. PS321, PS cDNA with stop codon replacing codon for amino acid 321. PS315, PS cDNA with stop codon replacing codon for amino acid 315. Both PS321 and PS315 contain only one tyrosine at position 308. PS315 Y-F, point mutation of Tyr-308 into phenylalanine. PS315 Y-A, point mutation of Tyr-308 into alanine. B, PLAP-PS321, PLAP ectodomain cDNA (empty box) fused to transmembrane and cytoplasmic tail of PS321 cDNA. C, Sequence of the cytoplasmic tail of PS321. All PS hNTR mutants have been created by an internal deletion of 57 amino acids within the cytoplasmic tail of wild-type p75hNTR, from Cys-250 to Gly-306. This deletion leads to a new primary sequence within the tail, in which Asp-248 and Ser-249 are followed by Leu-307 and Tyr-308.

 


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  		Figure 2. Steady-state distribution and delivery to plasma membrane of PS, PS321 and PS315 mutants and of PLAPsec and PLAP-PS321 mutants in transfected FRT cells. A, Cells grown on filters for 5 d were labeled with 35S cysteine overnight. Surface expressed PS hNTR mutants were biotinylated from the apical (A) or basolateral (B) side and immunoprecipitated as described in Materials and Methods. Immuno- and streptavidin-precipitated proteins were revealed by 10% SDS-PAGE and autoradiography. B, Filter grown cells were pulsed with 35S cysteine for 20 min at 37 C and then biotinylated from either the AP or BL domains at various times of chase. After cell lysis, biotinylated proteins were double precipitated with specific antibody and streptavidin-agarose beads, and detected by 10% SDS-PAGE and fluorography. While PS321 and PS315 mutants are expressed with an 85–90% BL polarity, the PS mutant is almost exclusively localized on the BL surface. C, FRT cells expressing PLAPsec and PLAP-PS321 grown on filters for 5 d were labeled with 35S cysteine overnight. Apical (A) and basolateral (B) media of cells expressing the ectodomain of PLAP were separately immunoprecipitated. Cells expressing PLAP-PS321 were biotinylated, double precipitated using a polyclonal antibody against PLAP and streptavidin beads, and detected by 10% SDS-PAGE and fluorography. PLAPsec is preferentially secreted from the AP surface, whereas PLAP-PS321 is mainly localized on the BL surface. The amino acids of the COOH-terminal tail of PLAP-PS321 mutant are shown in Fig. 1CGo.

 
To detect the arrival of newly synthesized PS321 and PS315 at the cell surface, we used a biotin targeting assay (34). Cells grown on filters were pulsed briefly with 35S cysteine, and biotinylated from the AP or BL side after 0, 30, and 100 min of chase. Biotinylated proteins were immunoprecipitated with the specific antibody, reprecipitated with streptavidin-agarose beads and analyzed by SDS-PAGE (Fig. 2BGo). By comparing the surface aliquots to the total of newly synthesized proteins (data not shown), we calculated that the BL-delivered proteins were always more than the AP-delivered amounts indicating that both mutants were directly delivered to the BL domain after intracellular sorting. We have previously shown that a deletion mutant (XI), with only 5 amino acids remaining of the cytoplasmic tail (up to Ser-249), was apically localized in FRT cells (37). These data suggested that the sequence (Ser-249 to Arg-314) created by this deletion (see Fig. 1CGo) was recognized as a direct BL sorting signal in FRT cells like in MDCK cells (32). We have analyzed the plasma membrane distribution of a chimeric protein (PLAP-PS321), stably transfected in FRT cells, to confirm that this sequence was necessary and sufficient to deliver a protein to the BL domain of FRT cells (Fig. 1BGo). This chimera contains the ectodomain of placental alkaline phosphatase (PLAPsec, which is apically secreted in FRT cells, Fig. 2CGo) and the transmembrane domain and cytoplasmic tail of PS321 (33). We found that more than 80% of the PLAP-PS321 expressed on the plasma membrane was restricted to the BL domain (Fig. 2CGo).

Because in MDCK cells, Y308 was found to be essential for BL localization of PS315 (33), we studied the plasma membrane localization of PS315 Y-A and PS315 Y-F, which contain substitutions of Y308 with, respectively, alanine and phenylalanine (Fig. 1AGo), in stably transfected FRT cells. As expected, both mutants lost their BL localization and were found enriched on the AP surface (Fig. 3AGo). Quantification of two different experiments showed that 90% of the total membrane proteins were localized on the AP domain (data not shown). To rule out the possibility that AP localization of these mutants resulted from an indirect delivery, by which the proteins were first sorted to the BL domain, we performed a biotin-targeting assay (Fig. 3BGo). The data clearly showed that the AP localization of both mutants was achieved via direct targeting to the apical surface, indicating that Y308 was essential for BL sorting in FRT cells.



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  		Figure 3. Steady-state distribution and delivery to plasma membrane of PS Tyr mutants in transfected FRT cells. A, After overnight 35S cysteine labeling, filter grown cells were biotinylated from the two different domains, lysed and double precipitated with anti p75hNTR antibody and streptavidin beads. Precipitates were run on 10% SDS-PAGE and visualized by fluorography. B, Cells grown on filters were labeled for 20 min with 35S cysteine. At different times of chase surface proteins were biotinylated from the AP or the BL sides, and after cell lysis were precipitated with antibody against p75hNTR and streptavidin beads. Samples were run on 10% SDS PAGE and detected by fluorography. In contrast to PS315, both Phe and Ala mutants of Y-308 are apically sorted.

 
PS hNTR mutants are poorly endocytosed by FRT cells
Because the p75 hNTR BL localization signal in MDCK cells overlaps with an internalization signal located within the same stretch of amino acids of the cytoplasmic tail (33), we tested the endocytic capacity of the wild-type (WT) receptor and its BL mutants in FRT cells. We performed a biotin assay using NHS-SS-biotin, a glutathione-cleavable analog of biotin (34). Cells expressing WT and different PS hNTR mutants were labeled overnight with 35S cysteine, biotinylated on their AP and BL surfaces and allowed to internalize for 1 h at 37 C. They were then treated with glutathione to remove biotin from the proteins still on the surface, whereas all internalized proteins were protected from the glutathione treatment. Cell lysates were subsequently double precipitated using the specific antibody and streptavidin beads (Fig. 4AGo). Similarly to what was previously found in MDCK cells (32), the WT protein was endocytosed poorly from the AP (7%) or the BL (1.5%) domains in FRT cells. Differently from MDCK cells, however, we found that the three BL mutants (PS, PS321 and PS315) behaved similarly and did not show any significant increase in internalization from either domain compared with the WT. In fact, the internalized fraction from either the BL or AP domain was never more than 10% of the total surface protein. This result was very different to what has been found in MDCK cells, where PS mutants showed high endocytic rates (~90–95%) (32) (see Fig. 4BGo).



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  		Figure 4. Endocytosis of PS hNTR mutants in the absence (A) and presence (B) of NGF in transfected FRT cells. A, Cells grown to confluence on filters were labeled overnight with 35S cysteine and biotinylated from the AP or BL surface with NHS-SS-biotin, reducible by treatment with GSH. Control samples for biotinylation efficiency (GSH-, 0 C) were left at 0 C. Control samples for glutathione efficiency (GSH+, 0 C) were left on ice and exposed to GSH treatment at 0 C to reduce biotin at the cell surface. Experimental samples (GSH+, 37 C) were incubated for 60 min at 37 C to allow the receptor to be internalized and then treated with GSH to reduce the biotin remaining at the cell surface. After cell lysis, biotinylated proteins were recovered by double precipitation with specific antibody and streptavidin beads, separated by SDS-PAGE and visualized by fluorography. The gels presented are representative of at least triplicate experiments. Neither the WT nor the mutants are efficiently internalized in FRT cells. B, Quantitative analysis of endocytosis of the WT and PS315 mutant proteins vs. the total protein present on the surface in MDCK (black bars) and FRT (gray bars) cells. After binding at 4 C with 125I-NGF, cells were incubated at 37 C for 60 min. The surface bound NGF was determined by counting the acetic acid wash, whereas the internalized fraction was detected by counting the cells. Results are expressed as percent of total endocytosis corrected for nonspecific binding in untransfected FRT cells. Columns represent averages of three independent experiments. Bars indicate SDs. Also in these conditions both the WT and the PS315 mutants are poorly internalized in FRT cells, whereas PS315 is highly endocytosed in MDCK cells.

 
Because this assay was performed in the absence of nerve growth factor (NGF), we hypothesized that the absence of the ligand could have an effect on the level of internalization of the receptor. We therefore analyzed the ability of hNTR and the PS315 mutants to internalize bound NGF both in MDCK and FRT cells. After binding of 125I-NGF on ice, cells grown on filters were allowed to endocytose NGF at 37 C for 1 h. Surface and internalized fractions of the ligand were determined by counting the radioactivity associated with acid sensitive and resistant 125I-NGF, as previously described (32, 38) (Fig. 4CGo). The results confirmed the data obtained with the biotin endocytosis assay, in that both the WT and PS315 mutants had a very low internalization rate in FRT cells (14–22%), which was again very different to the very high internalization rate of PS315 using the same assay in MDCK cells (32). Furthermore, as we showed already (33), in MDCK cells the levels of internalization were dependent on the presence of Y308. These experiments therefore indicated that the typical BL/endocytic signal identified in MDCK cells was able to mediate only BL sorting and not endocytosis in FRT cells.

The lack of endocytosis of the PS hNTR mutants is not due to a general internalization defect of FRT cells
The lack of endocytosis of PS hNTR mutants in FRT cells could either be due to a defect in the recognition of the signal within their cytosolic tail as an endocytic signal, or to a more general internalization defect of FRT cells. To explore these different possibilities, we studied by indirect immunofluorescence the endocytic pathway of the endogenous transferrin receptor (TfR) of FRT cells, comparing it with the PS321 hNTR mutant. Cells grown on filters were allowed to bind specific antibodies against the two proteins at 4 C and were then immediately shifted to 37 C to allow antibody internalization for different times. After each time point, cells were fixed, permeabilized, treated with fluorescein- or rhodamine-labeled secondary antibodies, and examined by immunofluorescence microscopy. As shown in Fig. 5Go, A–D, we found that, although more slowly than in MDCK cells, TfR was efficiently internalized by FRT cells and after 15 min was present inside the cytoplasm within organelles that had the typical appearance of endosomal compartments (39). On the contrary, the PS hNTR mutant remained at the cell surface for the entire length of the experiment (Fig. 5Go, E–H), as predicted from our biochemical data. Similar staining was found for all other PS hNTR mutants (data not shown). These data suggested that the general internalization capacity of FRT cells was not impaired, but rather that the lack of endocytosis of the PS mutants was due to the inability of FRT cells to recognize the BL signal as an endocytic signal.



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  		Figure 5. Kinetics of internalization of endogenous TfR and transfected PS321 mutant in FRT cells. After binding at 4 C with the monoclonal antibody against p75hNTR (right panel) or with a monoclonal antibody against TfR (left panel), cells were incubated at 37 C for 1, 10, 20, and 40 min to allow internalization of the receptor. Cells were then fixed and permeabilized. Antimouse fluorescein-labeled and rhodamine-labeled secondary antibodies were used to visualize respectively TfR (A, B, C, and D) and PS321 mutant (E, F, G, and H). Bar, 10 µm. While TfR is found intracellularly after an incubation of 20 min, the PS NTR mutant remains mainly on the surface at all incubation times.

 
The ectodomain of PS hNTR mutants is not responsible for lack of internalization of p75 mutants in FRT cells
Another possible explanation for the lack of internalization of PS hNTR mutants was that, although carrying a functional endocytic signal, these proteins became trapped at the surface of FRT cells by the binding of the ectodomain to its coreceptor TrKA or to other factors. It was previously shown that the interaction between p75hNTR and TrKA via their extracellular domains leads to immobilization of these receptors on the surface, thus enhancing the binding affinity for NGF (39, 40). Indeed, we found that FRT cells express TrKA and that it colocalizes with PS hNTR mutants on the BL surface (data not shown). Therefore, to rule out the possibility that the ectodomain of hNTR mutants was responsible for the absence of internalization of the PS hNTR mutants in FRT cells, we analyzed the endocytic ability of the chimeric protein PLAP-PS321, in which the hNTR ectodomain was substituted by the PLAP ectodomain (Fig. 1AGo). We studied the kinetics of internalization of this fusion protein together with TfR by double immunofluorescence in a time course experiment similar to the one described in Fig. 5Go. Cells expressing both TfR and the PLAP-PS321 fusion protein were grown on coverslips to subconfluence, labeled with specific antibodies against TfR and PLAP, and then allowed to internalize for various times at 37 C. We found that PLAP-PS321 was preferentially localized on the surface and not intracellularly at all times of chase (Figs. 6Go, D–F), similarly to what we had shown for PS321 (Fig. 5Go). On the contrary, we found that in the same cells most of the TfR was localized in intracellular vesicular compartments, where PLAP-PS321 was absent. These data showed that FRT cells were able to internalize TfR independently of the condition of growth used (filters or coverslips) in the different experiments. They also show that in similar conditions the signal contained in the cytoplasmic tail of PS mutants was not able to mediate efficient internalization of the fusion protein PLAP-PS321. However, to directly compare the kinetics of internalization of PS321 (Fig. 5Go) and PLAP-PS321 in polarized cells and to definitively rule out the possibility that PLAP-PS321 was present in intracellular compartments, we analyzed the localization of PLAP-PS321 and TfR by confocal microscopy. We found that after binding with specific antibodies and incubation at 37 C for 30 min PLAP-PS321 remains on the surface, whereas TfR is mainly present intracellularly (Fig. 7AGo). To quantify these morphological data, we have analyzed the efficiency of the internalization of PLAP-PS321 protein by performing a biotin endocytic assay, similarly to what we have shown for all PS mutants in Fig. 4AGo. These experiments demonstrated that PLAP-PS321 was poorly endocytosed (6.2%, AP + BL) compared with the total protein expressed at the cell surface (Fig. 7BGo). In conclusion, because the swapping of ectodomains between PLAP and NTR did not affect internalization, these data indicated that the ectodomain of p75hNTR was not responsible for the absence of internalization of the PS hNTR mutants. The lack of active internalization of PS hNTR mutants could also be the consequence of a defect in AP-2 expression because this adaptor complex is involved in endocytosis. Total extracts of MDCK and FRT cells were analyzed by SDS-PAGE gel and AP-2 was detected after electrotransfer to nitrocellulose. Both cell lines showed equivalent levels of AP-2 and, while MDCK cells showed a doublet, only one band was observed in FRT cells (Fig. 7CGo). Thus, a low level of AP-2 in FRT cells was not responsible for the slow endocytosis of PS hNTR mutants.



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  		Figure 6. Double kinetics of internalization of endogenous TfR and transfected PLAP-PS321 mutant in FRT cells. After binding at 4 C with monoclonal antibody against TfR (A–C) and polyclonal antibody against PLAP (D–F), FRT cells expressing PLAP-PS321 were incubated at 37 C for 1, 10, and 20 min to allow internalization of the two receptors. Cells were then fixed and permeabilized. Both receptors were visualized in the same field using either antimouse rhodamine-labeled secondary antibody or antirabbit fluorescein-labeled secondary antibody. Bar, 10 µm. PLAP-PS321 is poorly internalized and never colocalizes with the intracellular staining of TfR but rather stays on the cell surface as small clusters of fluorescence.

 


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  		Figure 7. Analysis of internalization of PLAP-PS321 by confocal microscopy and biotinylation assay. A, Cells grown on filters were incubated with specific antibodies against PLAP and TfR at 4 C. Cells were then incubated at 37 C for 30 min to allow the internalization of the two receptors and subsequently fixed, permeabilized and incubated with rhodamine and fluorescein-labeled secondary antibodies. The samples were observed with a confocal microscope. Images were collected in Z and X-Y sections. B, Cells grown to confluence on filters were labeled overnight with 35S cysteine and biotinylated from the AP or BL surface with NHS-SS-biotin. Experimental samples (GSH+, 37 C) were incubated for 60 min at 37 C to allow receptor internalization. Treatment with GSH (GSH+) was performed to reduce the biotin present on the surface. After lysis, cells were immunoprecipitated against PLAP antibody and reprecipitated against streptavidin. C, Western blot of AP-2 in total extracts of MDCK and FRT cells. Thirty micrograms of total proteins were loaded on SDS-PAGE gels and revealed with an antibody specific for AP-2 {alpha}-subunit.

 
The cytosolic signal of PS hNTR mutants is not recognized as a coated-pit localization signal by FRT cells
All these data suggested that the lack of endocytosis of the PS hNTR mutants was due to an inability of FRT cells to recognize the BL cytosolic signal as an internalization signal. To explore at what level the endocytic signal was impaired in FRT cells and in particular to analyze whether they were able to localize the PS hNTR mutant proteins to clathrin coated pits, we studied the localization of PS321 at the cell surface using an immunogold assay. After scraping from the dishes, monolayers were fixed and incubated with a monoclonal antibody against p75hNTR. Subsequently, they were postfixed, treated with secondary antibodies conjugated with colloidal gold and processed for electron microscopy. We found that PS321 was selectively excluded from clathrin-coated pits and was mainly localized along the BL surface (Fig. 8Go, A–C). Indeed, by performing a morphometric analysis, we calculated that the PS321 mutant was very poorly localized to coated pits and small vesicles close to the surface (0.4% for each µm of the plasma membrane) (Table 1Go). We observed a similar behavior for all other PS mutants (data not shown). Therefore, these data strongly indicated that in FRT cells the low internalization rate of endocytosis of the PS hNTR mutants was due to the inability of the BL signal present in the cytoplasmic tail of these proteins to localize them into clathrin-coated pits. In contrast, using the same technique, we could observe that TfR accumulated in coated invaginations and vesicles (Fig. 8Go, D–F), proving that they are indeed functional structures.



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  		Figure 8. A–C, Immunogold localization of PS321 at the cell surface in transfected FRT cells. FRT cells expressing PS321 were fixed with paraformaldehyde and glutaraldehyde and processed for immunolabelling with monoclonal antibody against p75hNTR (see Materials and Methods). Bound IgG was visualized with 5 nm gold conjugated secondary antibody. Arrowheads indicate PS321 mutant on the surface. Arrow shows clathrin-coated vesicles lacking PS321 mutant protein. Morphometric analysis is shown in Table 1Go. D–F, Immunogold localization of TfR using 10 nm gold conjugated secondary antibody. Arrowheads indicate TfR associated with coated pits of coated vesicles (arrows). Bar, 100 nm.

 

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  		Table 1. Quantitation of PS321 mutant distribution at the plasma membrane in FRT cells by immunogold labeling

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this work, we have investigated for the first time the recognition of a basolateral signal/endocytic signal by thyroid cells. For this we have studied the surface expression and intracellular routing of different mutants of the low affinity NGF receptor (p75hNTR) (Fig. 1Go) in transfected FRT cells. These mutants (PS, PS321, and PS315) contain a functional BL sorting signal overlapping with an endocytic signal within a 9 amino acid region of the cytoplasmic tail (Fig. 1Go, A and C), which has been previously characterized in MDCK cells (32, 33).

Thyroid epithelial cells (FRT) recognize a tyrosine-based basolateral signal
We found that, like renal epithelial MDCK cells, FRT cells were able to recognize the basolateral sorting signal present in the tail of PS hNTR mutants and that this signal was strictly dependent upon the same Tyr residue (Y308). In fact, the new sequence created by the 57-amino acid deletion in the cytoplasmic tail of the PS hNTR mutants (Fig. 1CGo) could itself mediate BL sorting of these proteins in FRT cells, as shown by the BL localization of PS315. Furthermore, mutations of Tyr-308 were responsible for the AP localization of two mutants in which this tyrosine was replaced by phenylalanine or alanine (PS315 Y-F and PS315 Y-A mutants) (Fig. 3Go, A and B). These data indicate that a common machinery recognizes the Tyr-based signal in the cytosolic tail of the PS NTR mutants in both MDCK and FRT cells. Addition of this motif to an apically sorted protein (PLAP) causes relocation of the fusion protein to the BL membrane in MDCK (33) and in FRT cells (Fig. 2CGo). This indicates that this BL signal is dominant over the characterized apical signal localized in the ectodomain of p75hNTR (41) and over a putative apical signal present in the ectodomain of PLAP. We observed that the PS mutant was better sorted to the BL plasma membrane (>95%) compared with others (PS321 and PS315 mutants, for example). Indeed, this was also the case in MDCK cells (33), and we showed recently that there is additional basolateral sorting information at position 322/323 (di-leucine motif LL) necessary for efficient basolateral expression in intestinal cells and MDCK cells (42). This additional motif is likely to be recognized also by FRT cells. The basolateral localization of the PS hNTR mutants in FRT cells was the result of an intracellular sorting step because we showed that both PS321 and PS315 mutants were directly delivered to the BL plasma membrane of FRT cells using a biotin-based targeting assay (Figs. 2BGo and 3BGo).

Despite the identification of different BL sorting signals, little is known about the machinery that recognizes these sequences in the TGN and/or endosomes in polarized epithelial cells. The signal present in the cytoplasmic tail of the PS hNTR mutants is part of a subset of Tyr-based signals that are able to mediate both rapid internalization and BL sorting, suggesting that they can be recognized differently at more than one site in the cell (17, 18). The molecular basis for this ability of Tyr-based signals to direct proteins to distinct compartments is unknown. There are four adaptor complexes (AP-1–4) known to play a role in recruiting membrane proteins into transport vesicles (for review, see Ref. 43). Two well-characterized adaptor complexes, AP-1 and AP-2, mediate association of clathrin with the TGN and the plasma membrane, respectively (23). Differential recognition of the Tyr-based signals by the AP-1 and AP-2 complexes could determine at least in part the targeting specificity of the signals (17). While interaction with AP-2 mediates rapid internalization from the plasma membrane, interaction with AP-1 is probably involved in lysosome targeting (17). Recently, it was suggested that clathrin- and AP-1-coated endosomal (44) and TGN (45) tubules could be involved in the BL sorting process in polarized cells. Furthermore, it has been shown recently that an AP1 complex containing a µ1B chain is necessary for basolateral targeting in MDCK and LLCPK1 cells (24, 46). Two other adaptor complexes (AP-3 and 4) have been characterized and localized to the region of the TGN (44, 47, 48). Whereas AP-3 is involved in sorting to vacuoles and lysosomes (45), there is no evidence yet for a function of AP-4 in basolateral transport and sorting.

Thyroid epithelial cells (FRT) do not recognize efficiently a tyrosine-based endocytic signal
We have studied for the first time the ability of FRT cells to internalize a protein (P75 hNTR) carrying an endocytic signal identified in another epithelial cell line (MDCK cells). For this, the internalization rate of different PS hNTR mutants in FRT cells was measured. In contrast to MDCK cells (32), we found that either in the presence or absence of the NGF ligand, no PS hNTR mutant was actively internalized in FRT cells (Fig. 4Go, A, and B). The PS mutants and the WT p75 showed similar levels of endocytosis suggesting that their endocytosis was not signal mediated. The poor internalization of the PS hNTR mutants could be due to immobilization of the proteins at the plasma membrane as a result of interactions of their ectodomains with TrKA, which in FRT cells colocalizes with the PS mutants on the BL surface (data not shown). This is, however, unlikely because a mutant (PLAP-PS321) in which the ectodomain of PS321 was replaced by the one from PLAP was not actively endocytosed despite its lack of interaction with TrKA.

We found by morphometric analysis that the PS321 mutant was selectively excluded from coated pits (Table 1Go), indicating that the deficient step in endocytosis might be recruitment into coated pits. We could rule out a general defect of FRT cells, however, since we found that they were able to mediate both localization of TfR in coated pits and coated vesicles (Fig. 8Go, D–F), and endocytosis (Figs. 5–7GoGoGo), albeit with slower kinetics compared with MDCK cells (not shown). Different explanations can be proposed for the lack of internalization of PS hNTR mutants in FRT cells. One remote possibility is that internalization of these proteins is not mediated by the AP-2 complex, which is involved in the endocytosis of TfR, but by another as yet unknown adaptor that is missing in FRT cells. Another explanation could be that FRT cells have lower levels of AP-2 than MDCK cells and as a consequence endogenous receptors compete with PS mutants for AP-2 binding and subsequent internalization. But this is unlikely because there was no significant difference between the two cell lines when we quantified the presence of AP-2. There is, however, a qualitative difference between the forms expressed in MDCK and FRT cells, but to date we do not know the nature of this difference and whether it is responsible for the slow internalization of hNTR mutants in FRT cells. It is also possible that the internalization signal of PS hNTR mutants may not be able to interact with the AP-2 complex because of a posttranslationnal modification of the functional tyrosine-based motif. Indeed, it has been shown that engagement of plasma membrane proteins within clathrin-coated pits is regulated by the phosphorylation of both tyrosine-based signals and the AP-2 complex (17). For example, phosphorylation of the critical tyrosine residue in the tail of the CTLA4 T cell coreceptor has been shown to abrogate interaction with the µ2 chain of the AP-2 complex (49) and to allow binding to other SH2-containing signal transduction molecules. Furthermore, phosphorylation of other residues outside the signal could modify the local conformation context, making the signals more or less accessible for the interaction with AP-2. For example, internalization of some BL plasma membrane proteins carrying di-leucine motifs can be stimulated by the phosphorylation of a neighboring Ser residue, activating its binding to AP-2 and subsequent internalization (50). It is worth noting that in PS hNTR mutants Y308 is surrounded by serines that can be potentially phosphorylated (see Fig. 1CGo).

Because FRT cells do not have caveolae (51), another possible explanation for the absence of internalization of PS hNTR mutants in FRT cells is that this receptor is internalized via caveolae. Indeed, in NIH 3T3 cells, the WT p75hNTR has been found associated with caveolin and localized in caveolae (52). However, we do not favor this hypothesis because the sequence of the PS signal shows clear homology with other signals that can mediate internalization via coated pits (33). Furthermore, in Caco-2 cells that are also devoid of caveolae (53), endocytosis of the PS hNTR mutant is not abolished (Le Bivic, A., unpublished results) suggesting that caveolae do not play a direct role in endocytosis of the protein. A further hypothesis that was recently put forward is that a crucial step for mediating internalization of many receptors is their dimerization (54). This event would put two internalization signals adjacent to each other; therefore, recognition of the dimer would increase the avidity of the binding relative to the monomer. We have evidence that in FRT cells the p75hNTR does not form such dimers, whereas in MDCK cells it does (data not shown).

In conclusion, the data reported here strongly indicates that in FRT cells different molecular machineries are required for the recognition of the apparently overlapping BL and endocytic signals present in the cytoplasmic tail of PS hNTR. It remains to be determined if this is a specificity of epithelial thyroid cells vs. other well-studied epithelial models such as MDCK cells. In vivo experiments will help clarify this issue and will shed light on the tissue specific regulation of endocytosis in relation to tissue function.


   Acknowledgments
 
We thank Franco D’Agnello and Mario Berardone for the art work.


   Footnotes
 
This work was supported by grants from Associazione Italiana per la Ricerca sul Cancro (AIRC), Centro Nazionale delle Ricerche-progetto finalizzato Biotecnologie, Galileo, Progetti di Ricerca di Luteresse Nazionale and EC Grants No. BIO4-CT-98-6055 and HPRN-CT-2000-00077 (to C.Z.) and by Centre National de la Recherche Scientifique, Association Française pour la Recherche sur le Cancer, Association Française de Lutte contre la Mucoviscidose and a Galileo program (A.L.B.).

Abbreviations: AP, Apical; BL, basolateral; FRT, Fischer rat thyroid; GSH, glutathione; hNTR, human neurotrophin receptor; MDCK, Madin-Darby canine kidney; NGF, nerve growth factor; PLAP, placental alkaline phosphatase; TfR, transferrin receptor; TGN, trans-Golgi network; TX-100, Triton X-100; WT, wild-type.

Received July 26, 2001.

Accepted for publication December 10, 2001.


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