From the
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.
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,
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
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.
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).
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.
(
)
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).
-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.
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).
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.
F, point mutation of Tyr-308
Phe-308; PS315 Y
A, point mutation of Tyr-308
Ala-308.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.