©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Cytoplasmic Tyrosine Is Essential for the Basolateral Localization of Mutants of the Human Nerve Growth Factor Receptor in Madin- Darby Canine Kidney Cells (*)

Laure Monlauzeur (1), Ayyapan Rajasekaran (2), Moses Chao (2), Enrique Rodriguez-Boulan (2), André Le Bivic (1)(§)

From the (1) From Laboratoire de Génétique et Physiologie du Développement, Unité Mixte de Recherche 9943, Faculté des Sciences de Luminy, 13288 Marseille, Cedex 09 France and the (2) Department of Cell Biology and Anatomy, Cornell University Medical College, New York, New York 10021

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Deletion of 58 internal amino acids from the C-terminal cytoplasmic domain of p75 human nerve growth factor receptor (hNGFR) changes its localization from apical to basolateral in transfected Madin-Darby Canine Kidney (MDCK) cells (Le Bivic, A., Sambuy, Y., Patzak, A., Patil, N., Chao, M., and Rodriguez-Boulan, E. (1991) J. Cell Biol. 115, 607-618). The mutant protein, PS-NGFR, also shows a dramatic increase in its ability to endocytose NGF and to recycle through basolateral endosomes. We report here the site-directed mutagenesis analysis of PS-NGFR to localize and characterize its basolateral and endocytic sorting signals. Both signals reside in the proximal part of the PS cytoplasmic tail, between positions 306 and 314. Transferring the cytoplasmic tail (19 residues) and transmembrane domain of a truncated PS mutant to the ectodomain of the placental alkaline phosphatase, an apical glypiated ectoenzyme, redirected it to the basolateral membrane and the endocytic compartments. A tyrosine at position 308, present in this short cytoplasmic segment, was mutated into phenylalanine or alanine. The resulting mutants were expressed predominantly on the apical membrane of MDCK cells. Their ability to endocytose NGF was reduced with the alanine mutant showing the stronger diminution. The PS mutant contains a short cytoplasmic sequence necessary both for basolateral targeting and endocytosis, and the requirement for tyrosine at position 308 is crucial for basolateral targeting.


INTRODUCTION

Epithelial cells carry out a variety of vectorial transport and secretory processes that depend on the polarized distribution of proteins into distinct apical and basolateral domains, segregated by tight junctions. Research in recent years has started to unravel the biogenetic mechanisms responsible for this polarity (Mostov et al., 1992, Rodriguez-Boulan and Powell, 1992, Simons and Wandinger-Ness, 1990). Sorting of apical and basolateral proteins into separate routes occurs intracellularly (Le Bivic et al., 1989, 1990; Matlin and Simons, 1984; Misek et al., 1984; Rindler et al., 1985), in the trans-Golgi network (Rodriguez-Boulan et al., 1992; Simons and Wandinger-Ness, 1990), by incorporation into distinct sets of apical and basolateral vesicles (Rindler et al., 1984; Rodriguez-Boulan et al., 1984; Wandinger-Ness et al., 1990).

The discovery of sorting information in the luminal domain of apical proteins (Brown et al., 1989; Lisanti et al., 1989; Mostov et al., 1987; Roth et al., 1987; Stephens et al., 1986) led to the early hypothesis that the incorporation of proteins into the apical pathway was signal-mediated, whereas basolateral proteins were transported to the cell surface by bulk flow (Simons and Wandinger-Ness, 1990). However, in the last 3 years, discrete sorting signals have been identified in the cytoplasmic domain of basolateral proteins (Mostov et al., 1992; Rodriguez-Boulan et al., 1992). Although they have no clear consensus sequence, a group of basolateral signals structurally and functionally overlaps with endocytic determinants; this occurs in LDLR,() lysosomal glycoprotein 120, and FcR (Hunziker et al., 1991; Hunziker and Fumey, 1994; Matter et al., 1992, 1994), LAP (Prill et al., 1993), vesicular stomatitis virus G protein (Thomas et al., 1993), and in mutant forms of influenza hemagglutinin (Brewer and Roth, 1991) and NGF receptor (Le Bivic et al., 1991). A second, smaller group of basolateral signals appears not to be associated with endocytic activity; these include a 17-amino acid domain in pIgR (Casanova et al., 1991), a second signal in LDLR (Matter et al., 1992), and in the tranferrin receptor (Dargemont et al. 1993).

Basolateral signals with endocytic ability possess, in some instances, tyrosine residues that are critical for both endocytosis and basolateral targeting. An example of this is a mutant influenza hemagglutinin in which a tyrosine residue was substituted for cysteine 543 in the 11 amino acid tail (Brewer et al., 1991). On the other hand, the basolateral/endocytic signal of LAP contains a tyrosine residue that is strictly required for endocytosis but not for basolateral delivery (Prill et al., 1993). Certain endocytic signals adopt characteristic type I b-turns (Collawn et al., 1990). Interestingly, a recent NMR study of the pIgR signal, which has no significant overlap with endocytic determinants, demonstrates the presence of a critical -turn followed by a nascent -helix (Aroeti et al., 1993). Taken together, all of these results suggest the existence of common features in basolateral signals; however, whether the variations observed represent the existence of different types of basolateral sorting mechanisms is still unclear.

The p75 hNGFR is a type I transmembrane glycoprotein with a poor ability to endocytose NGF (Johnson et al., 1986). When expressed by transfection in the dog kidney cell line MDCK, hNGFR is targeted to the apical surface; however, a 58 amino acid internal deletion in the cytoplasmic tail (PS-hNGFR) results in both high NGF endocytosis and basolateral targeting (Le Bivic et al., 1991). We speculated that relocation of a tyrosine residue from a distal location to a more hydrophilic environment in the vicinity of the membrane was responsible for the expression of the new basolateral and endocytic activities (Le Bivic et al., 1991). In order to identify and characterize this signal, we have produced mutants of PS-NGFR and characterized their sorting patterns. These experiments localize the basolateral signal of hNGFR PS to a 9-amino acid stretch, Ser-306 to Ala-314, which encompasses Tyr-308. This motif is sufficient to redirect placental alkaline phosphatase (PLAP), an apical membrane protein (Brown et al., 1989), to the basolateral membrane and to the endosomal/lysosomal network. Basolateral targeting is strictly dependent on Tyr-308, as shown by phenylalanine substitution of this residue.


MATERIALS AND METHODS

Reagents

Cell culture reagents were purchased from Life Technologies, Inc. Affinity-purified antibodies (rabbit anti-mouse IgG) and tetramethylrhodamine isothiocyanate-conjugated antibodies were from Biosys (Pasteur Institute, Paris). Protein A-Sepharose was from Pharmacia Fine Chemicals (Uppsala, Sweden). Sulfosuccinimidyl-6-(biotinamido)hexanoate (NHS-LC-biotin) and streptavidin-agarose were purchased from Pierce Chemical Co. Products for molecular biology were from Boehringer Mannheim Biochemical (Mannheim, Germany). All other reagents were from Sigma.

Cells and Antibodies

MDCK II cells were grown and transfected as described (Le Bivic et al., 1991). For experiments, cells were grown on Transwell chambers (Costar) as described (Le Bivic et al., 1991). Mouse monoclonal antibody ME20.4 against human NGF receptor was produced as ascites and used as described in the text. Rabbit polyclonal antibody against human PLAP was from Accurate Chemical and Scientific Corp (Westbury, NY). Mouse monoclonal antibody against MDCK lysosomal associated membrane protein type 1 was characterized previously (Nabi et al., 1991), and mouse monoclonal antibody against human transferrin receptor cytoplasmic tail was provided by Dr. I. Trowbridge (La Jolla, CA).

Transfection and Clonal Selection

Cells were transfected using the DNA-calcium phosphate procedure (Graham and Van der Eb, 1973). Resistant colonies growing in the presence of 0.5 mg/ml G418 were isolated with cloning rings and screened for hNGFR expression by indirect immunofluorescence. Indirect immunofluorescence was performed as described (Rodriguez-Boulan, 1983).

Cell surface biotinylation was performed as described (Sargiacomo et al., 1989) modified according to a recent report (Gottardi and Caplan, 1993). Pulse-chase, immune, and streptavidin precipitation surface delivery experiments were carried out as shown previously (Le Bivic et al., 1989, 1991). I-NGF binding was carried out according to Bernd(1986) as described previously (Le Bivic et al., 1991).

Constructs

Full length (WT) and PS hNGF receptor cDNA were obtained as described (Hempstead et al., 1990). Mutants of PS hNGFR were prepared by polymerase chain reaction. Polymerase chain reaction primers on the 3` side contained the HindIII site after the mutation to facilitate subcloning into the expression vector pMV7. Mutant PS 321 was obtained using the primer CGC AAG CTT CTA CTC CAC CTC CTC; mutant PS315 using the primer GCT AAG CTT CTA GGC TGG GGG CAG GCT CCA; mutant PS 315 Y F using the primer GTT AAG CTT CTA GGC TGG GGG CAG GCT GCT AAA GAG GCT GTT CCA, and PS 315 Y A using the primer GTT AAG CTT CTA GGC TGG GGG CAG GCT GCT GCC GAG GCT GTT CCA. Polymerase chain reaction 5` primer was GGC GAA TTC GCC GCG GCC AGC TCC GGC containing the EcoRI site. To produce the chimera PLAP-PS 321, a pGEM PLAP plasmid (Berger, 1988) was digested by NaeI and EcoRI to obtain a 1544-base pair fragment coding for most of the ectodomain of PLAP (from 1 to Ala-496). A pGEM PS 321 plasmid was digested by BstEII, and the resulting plasmid was blunted with the Klenow enzyme followed by an EcoRI digestion. This plasmid with PS cDNA coding for residues 215-321 containing the transmembrane and 21 amino acids from the cytoplasmic domain was ligated to the 1544 base pairs from PLAP. Plasmids containing the right size fragment were selected, and the cDNA (1.7 kilobases) was subcloned into pMV7 using EcoRI/HindIII. All the mutants were sequenced using the Sanger technique with the Pharmacia T7 kit.

RESULTS

The Basolateral and Endocytic Signals Are Localized in the Same Segment of the Cytoplasmic Tail of PS

We have previously shown that a 57-amino acid deletion in the cytoplasmic tail of p75 hNGFR changes its cellular distribution from apical to basolateral and promotes its endocytic ability in MDCK cells (Le Bivic et al., 1991). We posited that an endocytic/basolateral signal had been created at the level of a tyrosine residue (Tyr-308) upon relocation to a different environment in the PS hNGFR mutant. To further explore this possibility, we designed two mutants of PS hNGFR in which the cytoplasmic tail was shortened to 19 or 13 amino acids proximal to the transmembrane domain by inserting a stop codon at positions 321 or 315 (see Fig. 1). These constructs were permanently expressed in MDCK II by transfection and selection with neomycin. For each construct, a number of clones was isolated and analyzed for expression of the protein by indirect immunofluorescence. In cells permeabilized with saponin, both PS321 and PS315 gave a typical basolateral staining, with a punctate intracellular staining similar to the pattern obtained with the original PS mutant (not shown). Several positive clones were analyzed for each construct, and all gave an identical expression pattern. Staining in the absence of saponin only revealed a faint apical labeling (not shown). In contrast, cells expressing WT p75 hNGFR gave a strong apical staining pattern in the presence or absence of saponin (Le Bivic et al., 1991).


Figure 1: Scheme of hNGFR cDNA mutants. A, boxes represent the signal sequence and the transmembrane domain (TM). WT, full length cDNA; PS, deletion from amino acid 249 to amino acid 306. PS321, PS cDNA with a stop codon replacing codon for amino acid 321. PS315, PS cDNA with a stop codon replacing codon for amino acid 315. PS315 Y F, point mutation of Tyr-308 into Phe-308. B, chimera with the ectodomain of PLAP (amino acids 1 to 497) (empty box) linked to amino acid 215 of PS321. C, amino acid sequence of the cytoplasmic tail of PS321, PS315, PS315 Y F and Y A.



To quantitate the polarized distribution of PS321 and PS315, transfected cells were grown to confluency in Transwells and were metabolically labeled with [S]cysteine overnight. Cells were chased for 2 h in the presence of cold cysteine and then biotinylated on the apical or on the basolateral surface. Cells were extracted, and wild type and mutant p75 hNGFR forms were immunoprecipitated, released from the beads, and reprecipitated with streptavidin beads as described before (Fig. 2, A and B) (Le Bivic et al., 1991). In agreement with our previous results using surface immunoprecipitation, surface WT p75 hNGFR was found mostly apical while surface PS was predominantly basolateral (Le Bivic et al. 1991). Surface pools of PS321 and PS315 were found mainly at the basolateral surface. PS321 and PS315 have thus retained the basolateral information contained in the PS mutant. In order to show that this basolateral sorting information was indeed present in the cytoplasmic tail of PS and not the result of a conformational change induced in the ectodomain of p75 resulting in the loss of an apical signal, a chimeric protein was designed. The ectodomain of PLAP, a glycophosphoinositide-anchored apical membrane glycoprotein, was fused to the cytoplasmic and transmembrane domain of PS321. When expressed in MDCK cells, this PLAP/PS321 construct directed the synthesis of a precursor protein with an apparent molecular mass of 70 kDa that was processed more slowly than hNGFR mutants into a 75-80-kDa mature product (Fig. 3). The slower maturation of the chimera was likely due to the ectodomain of PLAP, as the kinetics were very similar to those described for PLAP and for other PLAP chimeras expressed in MDCK cells (Casanova et al., 1991). By indirect immunofluorescence in the presence of saponin, PLAP/PS321 was localized to the basolateral membrane and to intracellular vesicles, a pattern very similar to the PS mutants (not shown). Little labeling was seen on the apical surface by indirect immunofluorescence labeling without saponin treatment (not shown). Surface distribution of PLAP/PS321 was also investigated using the double precipitation technique. The majority of surface PLAP/PS321 was detected on the basolateral membrane (Fig. 2, A and B) with a ratio comparable to that of PS321, indicating that basolateral information was indeed present in the short cytoplasmic domain. All the transfected clones used in this study were controlled for the correct polarization (>80%) of an apical endogenous marker, gp114 (Le Bivic et al. 1990), and found to be normally polarized (not shown).


Figure 2: A, surface expression of hNGFR mutants in MDCK cells. Cells were grown on filters and metabolically labeled overnight with [S]cysteine, then chased for 2 h with an excess of cold cysteine. Surface expressed hNGFR mutants were biotinylated from the apical (A) or the basolateral (B) side. After cell lysis, mutant proteins were immunoprecipitated and reprecipitated with streptavidin beads. Precipitates were analyzed on 8% SDS-polyacrylamide gel electrophoresis and visualized by fluorography. B, quantification of apical and basolateral surface expression at steady state of hNGFR mutants. Black bars represent apical expression while hatched bars are for basolateral expression (n = 3).




Figure 3: Processing of hNGFR mutants in MDCK cells. Clones of MDCK cells expressing different mutants of PS were grown on filters and pulsed for 20 min with 1 mCi/ml [S]cysteine and chased for the time indicated. After immunoprecipitation, PS mutants were analyzed on 8% SDS-PAGE and fluorography. , mature form; , precursor form.



Surface delivery of newly synthesized mutants was followed as described in Le Bivic et al.(1991). Cells grown on filters were metabolically labeled for 20 min and then chased for 30 or 60 min in the presence of the monoclonal antibody ME-204 either in the apical or the basolateral medium at 37 °C. Mutant PLAP/PS321 was chased for 2 h in normal medium before adding the antibody to allow for normal processing by the Golgi complex (see Fig. 3). After several washes at 4 °C bound antibodies were precipitated by protein A-Sepharose beads, and the remaining supernatant was immunoprecipitated with fresh antibody to estimate the amount of unaccessible hNGFR. Results are shown in Fig. 4. WT hNGFR was mainly delivered to the apical side while PS, PS321, PS315, and PLAP/PS321 were predominantly delivered to the basolateral side of MDCK cells, with values close to steady state levels.


Figure 4: Surface delivery of hNGFR mutants in MDCK cells. Cells grown on filters were pulsed for 20 min with [S]cysteine and then chased for 30 or 60 min. in the presence of ME20.4 (1/100) in the apical (black bars) or the basolateral (hatched bars) medium at 37 °C. After several washes at 4 °C, surface-bound antibody was precipitated with protein A-Sepharose, and the cell extract was immunoprecipitated by the addition of fresh ME20.4. Results are expressed as a percentage of surface antigen corrected for the total amount of antigen at each time point. Experiments were done in duplicate.



To measure the endocytic capacity of PS mutants, cells grown on filters were allowed to endocytose I-NGF on the apical or the basolateral side for 1 h at 37 °C. Surface-bound NGF was released by acid wash and compared to internalized NGF (Fig. 5). WT p75 mediated endocytosis of NGF inefficiently, as previously shown (Le Bivic et al., 1991). In contrast, PS and PS315 were very active in NGF endocytosis, suggesting that the endocytic and basolateral signals overlap. NGF endocytosis was consistently greater from the basolateral than from the apical side, an observation we already reported for other p75 constructs expressed in MDCK cells (Le Bivic et al., 1991). Since endocytosis of PLAP/PS321 could not be measured by a ligand binding assay, we performed double immunolocalization using two markers of the endocytic pathways such as transferrin receptor (Hopkins et al., 1990) and lysosomal associated membrane protein type 1 (Nabi et al., 1991) (not shown). We found that internal PLAP/PS321 partially colocalized with both markers indicating that it is found in the endocytic/lysosomal pathway.


Figure 5: NGF internalization in MDCK cells expressing hNGFR mutants. Confluent monolayers grown on filters were incubated 1 h at 37 °C with I-NGF added to the apical or the basolateral side, then washed at 4 °C with phosphate-buffered saline/bovine serum albumin. Filters were acid-washed to determine surface-bound I-NGF, and radioactivity still associated with the filter was counted as internalized I-NGF. Results are corrected for nonspecific binding on untransfected MDCK cells. Results are expressed as percent of total cell-associated radioactivity from the apical side (hatched bars) or basolateral side (empty bars). Results are expressed as the mean of three independent experiments.



Tyrosine 308 Is Crucial for Basolateral Targeting

In a previous study, we have expressed a p75 mutant named XI that had a stop codon after the fifth amino acid of the cytoplasmic tail corresponding to Ser-306 (see Fig. 1). Since this mutant was expressed on the apical membrane of MDCK cells, we concluded that the stretch of residues that spanned from Ser-306 to Ala-314 was essential for basolateral targeting. Because of the critical role of tyrosine residues in endocytosis and basolateral targeting, we carried systematic mutagenesis analysis of a tyrosine included in this segment. Tyr-308 was mutated into a phenylalanine or an alanine, and the PS315 Y F or Y A mutants were expressed in MDCK cells. Pulse-chase analysis of the mutants showed that they were processed at the same rates (Fig. 6). By indirect immunofluorescence with or without saponin treatment (not shown) on cloned cells, both PS315 Y F and Y A showed a strong apical labeling resembling the apical pattern observed in cells expressing WT p75. Surface PS315 Y F and Y A were mostly apically expressed as detected by the double precipitation technique (Fig. 7, A and B). This result showed that Tyr-308 is indeed very important for the recognition of the basolateral targeting motif by cytoplasmic components of the basolateral sorting machinery. This was confirmed by a targeting assay performed as for previous mutants (Fig. 4). Both PS315 Y A and Y F were delivered to the apical side very efficiently as opposed to PS315.


Figure 6: Processing of hNGFR mutants in MDCK cells. Clones of MDCK cells expressing different mutants of PS were grown on filters and pulsed for 20 min with 1 mCi/ml [S]cysteine and chased for the time indicated. After immunoprecipitation, PS mutants were analyzed on 8% SDS-polyacrylamide gel electrophoresis and fluorography. , mature form; , precursor form.




Figure 7: A, surface expression of Tyr-308 mutants in MDCK cells. Cells were grown on filters and metabolically labeled overnight with [S]cysteine then chased for 2 h with an excess of cold cysteine. Surface-expressed hNGFR mutants were biotinylated from the apical (A) or the basolateral (B) side. After cell lysis, mutant proteins were immunoprecipitated and reprecipitated with streptavidin beads. Precipitates were analyzed on 8% SDS-polyacrylamide gel electrophoresis and visualized by fluorography. B, quantification of apical and basolateral surface expression at steady state of hNGFR mutants. Black bars represent apical expression, while hatched bars are for basolateral expression (n = 3).



We next sought to evaluate the endocytic capacity of the Tyr-308 mutants. For this purpose, cells were grown on filters and allowed to endocytose I-NGF for 1 h at 37 °C. After extensive washes at 4 °C, surface and internal radioactivity was discriminated by acid exposure of the cell surface (Fig. 8). Both PS315 Y F and Y A still mediated NGF endocytosis from the basolateral side, albeit with a lower capacity than PS315. PS315 Y F was also capable of endocytosing NGF from the apical membrane with a capacity intermediary between WT and PS315. PS315 Y A, however, mediated NGF endocytosis inefficiently from the apical side suggesting that endocytosis from the apical and the basolateral side are governed by different mechanisms (Riezman, 1993). These results suggest that in the cytoplasmic tail of PS315 the basolateral sorting signal and the endocytic signal do not have exactly the same requirements at the molecular level while being found in the same stretch of amino acids.


Figure 8: NGF internalization in MDCK cells expressing Tyr-308 mutants. Confluent monolayers grown on filters were incubated for 1 h at 37 °C with I-NGF added to the apical or the basolateral side, then washed at 4 °C with phosphate-buffered saline/bovine serum albumin. Filters were acid-washed to determine surface-bound I-NGF, and radioactivity still associated with the filter was counted as internalized I-NGF. Results are corrected for nonspecific binding on untransfected MDCK cells. Results are expressed as percent of total cell-associated radioactivity from the apical side (hatched bars) or basolateral side (empty bars). Results are expressed as the mean of three independent experiments.



DISCUSSION

The Basolateral Targeting Signal Present in PS Is Located between the Transmembrane Domain and Alanine 314

We have performed site-directed mutagenesis analysis to characterize a basolateral sorting signal created by an internal deletion in the cytoplasmic tail of p75 hNGFR. Progressive C-terminal truncations up to Ala-314 preserved the basolateral sorting signal indicating that the residues between the transmembrane domain and Ala-314 contained all the information necessary for basolateral targeting. Furthermore, since in a previous study we showed that a mutant XI truncated after Ser-306 was localized to the apical membrane (Le Bivic et al., 1991), all the results taken together indicate that residues essential for basolateral targeting must be located between Ser-306 and Ala-314. This indeed confirms the hypothesis that a sorting signal had been created at the site of the deletion in the PS mutant. The information contained in this signal is sufficient to redirect an apical marker such as PLAP (PLAP's ectodomain is apically secreted) to the basolateral membrane by linking it to the transmembrane and the proximal part of the cytoplasmic domain of PS. The sequence containing the amino acid stretch from the membrane to Ala-314 thus acted as a dominant autonomous basolateral sorting signal.

Role of Tyr-308 in Basolateral Targeting and Endocytosis

Tyrosine residues are known to be part of basolateral signals in the LDLR (Matter et al., 1992), pIgR (Aroeti et al. 1993), tyrosine mutant of hemagglutinin (Brewer et al., 1991), LAP (Prill et al., 1993), and vesicular stomatitis virus G (Thomas et al., 1993). Their role in endocytosis is also well documented (for review, see Vaux (1992)). Changing Tyr-308 into Phe-308 or Ala-308 completely reversed the polarity of PS315 (from 81% basolateral to 87 or 89% apical). This result argued srongly in favor of a crucial role for Tyr-308 in basolateral targeting. PS315 Y F mutant, however, was still able to mediate endocytosis at a higher level than PS315 Y A suggesting that the endocytic and basolateral signals colocalized in the same stretch of amino acids, but were not exactly identical. A similar observation was made with the proximal basolateral determinant of the LDLR depending on a tyrosine that was colinear with the coated pit localization signal (Matter et al., 1992). It was also the case for LAP in which the two signals are colinear but differed in their requirement for a tyrosine residue (Prill et al., 1993). It was, however, puzzling that in LAP the tyrosine was necessary for endocytosis but not for basolateral targeting since changing it to phenylalanine did not modify LAP surface expression but greatly reduced endocytosis (Prill et al., 1993). We observed a different behavior for PS315 suggesting that differences in structure between the two protein signals might explain the requirement for tyrosine in sorting. The role of tyrosine in endocytosis has been documented in the LDLR where it could be substituted by a phenylalanine or a tryptophan (Davis et al., 1987). It was not the case, however, for tyrosine mutant of hemagglutinin, indicating that a permissive environment must exist for conservative changes between tyrosine and phenylalanine.

Basolateral Sorting Signals

So far, several basolateral sorting signals have been identified in MDCK cells. These motifs have little in common at the amino acid level, but several features appear to be recurrent. First, they are all located in the cytoplasmic domain, and most of them are predicted to form a -turn structure similar to the one described for coated pit localization determinants (Collawn et al., 1990). It is the case for the pIgR (RNVD), LDLR (NPVY), LAP (PPGY), and the basolateral signal in tyrosine mutant of hemagglutinin is thought to have the structure of a loop (Ktistakis et al., 1991). In PS315, the sequence NSLY shows some homology with the other signals described with a cluster of structure-breaking residues (arginine, asparagine, serine), a possible interaction between the side chain of the asparagine, and the hydroxyl group of the tyrosine followed by other structure-breaking residues (serine, proline). This interaction between the side chains of tyrosine and asparagine have been suggested in the NPVY sequence and is believed to stabilize the -turn crucial for endocytosis (Vaux, 1992). This -turn is also stabilized by interactions between the side chain of asparagine and the aromatic ring reducing the need for a hydroxyl group. That tyrosine can be replaced by a phenylalanine without blocking totally endocytosis in both PS315 and LDLR suggests a common structural organization. In the case of LAP, the structure forming the -turn is composed of the sequence QPPDY with a possible interaction between the side chain of glutamine and the hydroxyl group of the tyrosine. Breaking this interaction by substituting a phenylalanine results in the destabilization of the -turn and loss of endocytosis (Prill et al., 1993). It is worth pointing out that, in that case, basolateral targeting is maintained suggesting that it might not be dependent on a tight turn. The dileucine basolateral targeting motif that has been identified in the FcRBII receptor (Hunziker and Fumey, 1994; Matter et al., 1994) might bring some light to this point. Finally, Matter et al.(1994) have suggested that a cluster of negatively charged residues might be necessary for basolateral targeting (Matter et al., 1992, 1994). Although such a cluster (EEVE) was found in PS321, 9 amino acids away from Tyr-308, its deletion in PS315 had no effect on basolateral targeting, indicating that it does not play a role in PS. Further work will be necessary to establish the exact configuration of a basolateral signal assuming that their apparent diversity covers a common secondary structure recognized by the cellular sorting machinery.


FOOTNOTES

*
This work was supported by CNRS Unité Mixte de Recherche Grant 9943, Association pour la Recherche sur le Cancer Grant 6421, INSERM Grant 930203, and an Association Franaise de Lutte contre la Mucoviscidose grant (to A. L. B.), a North Atlantic Treaty Organization Collaborative Research Program, and National Institutes of Health Grant 34107 (to E. R. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed. Fax: 33-91-26-97-48.

The abbreviations used are: LDLR, light density lipoprotein receptor; hNGFR, human nerve growth factor receptor; MDCK, Madin-Darby canine kidney; PLAP, placental alkaline phosphatase; NGF, nerve growth factor; pIgR, polymeric immunoglobulin receptor; WT, wild type; LAP, lysosomal acid phosphatase; PS315 Y F, point mutation of Tyr-308 Phe-308; PS315 Y A, point mutation of Tyr-308 Ala-308.


ACKNOWLEDGEMENTS

We would like to thank C. Mirre, M. H Delgrossi, and M. Garcia for their constant help, P. Chavrier, J. Boretto, and R. Gristina for helping with DNA sequencing. We also liked to thank G. Rougon for helpful discussions.


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