(Received for publication, April 30, 1997)
From the Membrane cofactor protein (MCP) is a widely
distributed complement regulatory protein that is expressed on the
basolateral surface of polarized epithelial cells. The basolateral
targeting of the BC1 isoform of MCP was analyzed by generating deletion mutants and point mutants within the cytoplasmic tail of 16 amino acids. A sequence of four amino acids, FTSL, was found to be
indispensable for the basolateral transport of MCP. This tetrapeptide
has two unique features compared with the targeting motifs of other
basolateral proteins: (i) it contains a phenylalanine rather than a
tyrosine at position 1; (ii) it is located at the very COOH-terminal
end. Replacement of the phenylalanine or the leucine by an alanine resulted in a nonpolarized delivery to the cell surface. On the other
hand, substitution of a tyrosine for the phenylalanine did not affect
the basolateral transport of MCP. The latter mutant, however, was
efficiently internalized, whereas the wild type protein was not subject
to endocytosis. Our results indicate that the targeting signal
YXX-large aliphatic that is involved in various sorting
events has been modulated in MCP in such a way that it allows
basolateral transport but not endocytosis.
Polarized epithelial cells can be divided into morphological and
functional subdivisions. Cellular compartmentalization and specific
directional transport of cellular components are responsible for the
creation of an apical and a basolateral plasma membrane domain that are
separated by junctional complexes (for review, see Ref. 1). One of the
best characterized epithelial cell lines is the Madin-Darby canine
kidney cell line in which newly synthesized apical and basolateral
proteins are sorted at the trans-Golgi network by
segregation into different vesicles for direct transport to their
respective membrane domain (2-5). In other polarized cells,
e.g. hepatocytes, apical proteins are first delivered to the
basolateral side where they are sorted and transcytosed to the apical
membrane (6). In recent years, a correlation between the cytoplasmic
tail of a protein and the basolateral targeting has been established
(for review, see Ref. 7). For some basolateral sorting signals
involving a critical tyrosine residue, a close relationship to
determinants for coated pit localization has been described (8-10).
However, some other proteins, e.g. the polymeric
immunoglobulin receptor or the low density lipoprotein receptor,
possess unrelated targeting and endocytosis signals (11, 12). In
contrast to the basolateral sorting, the apical targeting is not well
characterized. For apically secreted proteins the importance of
N-glycans has been demonstrated (13). However, for
membrane-bound apical proteins the involvement of the carbohydrate moiety is still speculative. Proteins anchored by
glycosylphosphatidylinositol (GPI)1 are normally delivered
to the apical plasma membrane (14, 15). Recently, it has been shown
that the distribution of GPI is nonpolarized, indicating that the
sorting machinery for nonprotein-linked GPIs and for GPI-anchored
proteins is different (16).
Membrane cofactor protein (MCP; CD46) is a type I membrane protein and
is expressed on all nucleated human cells tested. MCP functions as a
cofactor for the plasma serine protease factor I by binding to
complement factors C3b and C4b deposited on self tissue (17). By
promoting the proteolytic degradation of these factors, it protects the
cell from complement-mediated damage. In addition, MCP serves as a
receptor for measles virus (18, 19). For virus binding, the
N-glycans of MCP are of critical importance (20-22). The
extracellular portion of MCP consists of four cysteine-rich short
consensus repeats (SCRs), three of which contain sites for
N-glycosylation. The SCRs are followed by a serine,
threonine, and proline-rich region (STP), the site of O-glycosylation. By alternative splicing multiple MCP
isoforms arise which contain different combinations of the STP regions A, B, and C. A commonly expressed isoform contains the BC regions consisting of 29 amino acids. The extracellular portion of MCP is
connected to the hydrophobic transmembrane domain by a short region of
unknown function. The intracellular domain of the protein is divided
into two parts. The membrane proximal portion, designated as the
intracytoplasmic anchor, is present in all MCP isoforms and consists of
10 mainly basic amino acids. The carboxyl-terminal end of MCP,
designated as cytoplasmic tail 1 or 2, is subjected to alternative
splicing like the STP region. In the various isoforms of MCP either of
two different tails was found: one comprising 16 amino acids (tail 1)
and the other consisting of 23 amino acids (tail 2). The nomenclature
of MCP isoforms indicates which segments (A, B, or C) of the STP region
and which cytoplasmic tail (1 or 2) are expressed (for review, see Ref.
17). In a recent paper, we reported that two MCP isoforms with
different tails (MCP-BC1 and MCP-BC2) were directed to the basolateral
surface of polarized epithelial cells. A mutant lacking the cytoplasmic
tail was transported in a nonpolarized fashion. This result indicated
that both tails contain a basolateral sorting signal. We also showed
that the targeting signal of tail 1 is tyrosine-independent in contrast to most basolateral signals identified so far (23).
In this study, we characterized the sorting signal in the cytoplasmic
tail of MCP-BC1 in more detail. By analysis of MCP-BC1 mutants with
deletions in the cytoplasmic tail, an important part of the basolateral
targeting signal was localized in the four carboxyl-terminal amino
acids (FTSL). Studies with point mutants revealed that in addition to
the phenylalanine, the leucine residue at the very COOH-terminal end is
also important. A mutant with the phenylalanine replaced by tyrosine
was also transported to the basolateral surface of polarized epithelial
cells; but in contrast to the wild type protein, it was efficiently
endocytosed.
MDCK cells (strain I) were grown in
Dulbecco's modified essential medium (Life Technologies, Inc.)
supplemented with 10% fetal calf serum (Life Technologies, Inc.), 100 units/ml penicillin, and 100 µg of streptomycin. Tissue
culture-treated 0.4-µm pore size Transwell polycarbonate filters
(Costar Corp., Cambridge, MA) were used for all experiments. Cells were
seeded 5 days before experiments (2 × 105
cells/7.5-mm unit and 2 × 106 cells/24-mm unit). The
polarity was determined by measurement of the transepithelial
resistance using a Millipore ERS apparatus (Bedford, MA). MDCK cells
formed a tight monolayer with an electrical resistance of 1,000-2,500
The construction of the two chimeric MCP proteins used in
this study was described by Lublin and Coyne (24). The DAF-TM consists
of amino acids 1-304 of decay-accelerating factor (DAF, CD55) and
amino acids 270-350 of MCP-BC2, resulting in a DAF molecule anchored
by the MCP transmembrane and cytoplasmic domain. The CD46-GPI (MCP-PI)
consists of amino acids 1-269 of MCP and 307-347 of DAF (numbering
starts from the first amino acid of the mature protein without the
signal peptide), representing a GPI-anchored version of MCP (Fig.
1 upper panel). For our
studies both chimeras were subcloned into the EcoRI site of
the stable expression vector pH
For stable expression, 1.5 × 106 MDCK cells were
transfected with 10 µg of plasmid DNA by electroporation and selected
for neomycin resistance. For each mutant the experiments were performed on uncloned populations of transfectants to minimize the effect of
clonal variations and on at least two subclones with different expression levels (moderate and high expression). MCP expression was
controlled based upon Western blot and immunofluorescence. Although the
expression levels of the different cell populations varied, the
relative amount of each mutant protein delivered to the apical and
basolateral surfaces, respectively, was almost identical in at least
three experiments.
Surfaces of filter-grown MDCK cells were labeled
with the non-membrane-permeating reagent
sulfo-N-hydroxysuccinimidylbiotin (Pierce) as described
recently (23).
For surface immunofluorescence, MDCK
cell clones grown on 7.5-mm Transwell units were fixed on ice with 2%
paraformaldehyde (15 min). The apical and basolateral surfaces were
incubated with mAb J4/48 and a fluorescein isothiocyanate
(FITC)-labeled anti-mouse IgG (DAKO, Denmark) as described earlier
(27). The fluorescence label was visualized with a confocal laser
scanning microscope (LSM Carl Zeiss, Oberkochem, Germany) working with
the blue line of an argon laser.
MDCK cells stably expressing MCP were
grown on coverslips. At 10-50% confluence, surface-expressed MCP was
labeled with a mAb directed against MCP (J4/48; Dianova) diluted 1:20
in PBS-bovine serum albumin. After an incubation for 60 min on ice, the
antibody was removed by washing with PBS+, and the cells
were incubated with cell culture medium for 60 min either at 4 °C or
37 °C to allow endocytosis of the MCP-antibody complex.
Internalization was stopped by rapid cooling on ice. Surface-bound
antibody was detected by an incubation for 60 min on ice with a
rhodamine-conjugated goat anti-mouse Fab fragments (Sigma) at a
dilution of 1:100 in PBS-bovine serum albumin. After washing with
PBS+, the cells were fixed and permeabilized for 5 min at
MDCK cells expressing MCP were
grown on six-well plastic dishes and surface labeled at 4 °C with
sulfo-N-hydroxysuccinimidylbiotin as described earlier (27).
After stopping the biotinylation reaction by adding PBS+
containing 0.1 M glycine and washing with cold
PBS+, prewarmed serum-free cell culture medium was added.
The cells were transferred to a 37 °C incubator for various times.
To stop internalization, the cells were cooled on ice and washed with cold PBS+. Subsequently, the cells were incubated with 500 milliunits Vibrio cholerae sialidase (VCNA; Behring,
Marburg, Germany) for 60 min at 4 °C. This treatment removed all
sialic acid residues on surface MCP, whereas internalized MCP was
protected from the sialidase. After extensive washing with cold
PBS+, the cells were lysed, and MCP was immunoprecipitated
as described earlier (27). Biotinylated MCP was separated on a 12%
sodium dodecyl sulfate-polyacrylamide gel, transferred to
nitrocellulose, and detected as described above. The internalization
rate of MCP was determined by densitometric quantification of the
sialylated and desialylated MCP bands.
We have reported recently that the cytoplasmic tail of MCP
is responsible for the basolateral transport of the isoforms BC1 and
BC2 in polarized epithelial cells (23). To analyze whether the
basolateral targeting signal of MCP is able to redirect an apical
protein to the basolateral domain, two chimeric molecules (Fig. 1,
upper panel) that have been constructed from MCP-BC2 and DAF
(24) were stably expressed in MDCK cells. DAF is known to be localized
on the apical surface of polarized cells (28). The first chimera was a
form of DAF in which the GPI anchor was replaced by the MCP
transmembrane and cytoplasmic domain (DAF-TM). The second chimera
represents a form of MCP which lacked both the transmembrane and the
cytoplasmic domain and was anchored by GPI (MCP-PI). To analyze the
transport of these proteins, MDCK cells stably expressing either of the
chimeras were grown on permeable filter supports. Either the apical or
the basolateral surface proteins were labeled by adding the
non-membrane-permeating reagent sulfo-N-hydroxysuccinimidylbiotin to the respective chamber.
The cells were lysed, and MCP and DAF were isolated by
immunoprecipitation (mAb J4/48 for MCP, anti-CD55 for DAF). The samples
were divided into two aliquots. One was used for a Western blot
analysis to ensure that there was no difference in the total amount of
protein in the cells labeled from two different sides (not shown). The second aliquot was separated on a 12% sodium dodecyl
sulfate-polyacrylamide gel, blotted to nitrocellulose, and the
biotin-labeled proteins were detected with streptavidin/peroxidase. As
shown in Fig. 2, MCP and MCP chimeras
were distributed in a different way on epithelial cells. MCP-BC1
representing a wild type isoform is almost exclusively expressed on the
basolateral surface as described recently (23). DAF-TM was also found
predominantly on the basolateral side indicating that the MCP COOH
terminus comprising the transmembrane and the cytoplasmic domain is
able to direct an apical protein to the basolateral membrane of
polarized cells. In contrast to DAF-TM, an efficient biotinylation of
the MCP-PI molecules was only obtained after labeling the cells from
the apical side. Replacement of the COOH terminus of MCP by a GPI
anchor resulted in the apical expression of the protein. This finding
is in agreement with the apical localization of other GPI-anchored
proteins.
Our previous work indicated that
the basolateral sorting signal of the BC1 isoform of MCP is localized
in the 16 amino acids of the cytoplasmic tail. Studies on a deletion
mutant lacking the six membrane-proximal amino acids (TYLTDE) indicated
that the targeting signal is not dependent on the only tyrosine of the
cytoplasmic tail (23). Here, we analyzed two additional deletion
mutants lacking either six amino acids in the central portion
(BC1-d7-12) or four amino acids at the very COOH-terminal end of the
cytoplasmic tail (BC1-d13-16). The amino acid sequences of the
cytoplasmic portion of these and all mutants described in this paper
are shown in Fig. 1 (lower panel). The distribution of the
BC1 deletion mutants was analyzed by domain-specific surface biotinylation of stably expressing MDCK cells grown on filters. The
result is shown in Fig. 2. As demonstrated recently (23), the MCP form
lacking the cytoplasmic tail (tail-minus) was almost equally
distributed on the apical and the basolateral membranes. The deletion
mutants lacking either the first six amino acids or the following six
amino acids (BC1-d1-6 and BC1-d7-12) were transported like the wild
type protein (MCP-BC1). Therefore, the first 12 amino acids of the
cytoplasmic tail are dispensable for the correct basolateral sorting of
MCP. In contrast, the mutant lacking the four COOH-terminal amino acids
of the tail (BC1-d13-16) was transported like the tail-minus mutant.
This finding indicates that the basolateral sorting of MCP is dependent
on the amino acids FTSL at the carboxyl terminus of the protein.
Aromatic amino acids, especially tyrosine, are involved in
the targeting of several basolateral proteins. As phenylalanine is the
only aromatic amino acid in the FTSL motif of MCP, we analyzed the
importance of this residue. We established MDCK cells stably expressing
MCP-BC1 with the phenylalanine at position 13 of the cytoplasmic tail
replaced either by tyrosine (13F/Y) or by alanine (13F/A). Mutant MCP
forms were analyzed by surface biotinylation of filter-grown cells. The
result of domain-specific biotinylation is shown in Fig.
3. Like the wild type protein (Fig. 2,
MCP-BC1), 13F/Y was found mainly on the basolateral membrane. This
finding indicates that phenylalanine in the basolateral targeting
signal of MCP can be replaced by tyrosine without changing the
direction of the transport. In contrast, substitution of an alanine for the phenylalanine altered the polarized transport dramatically. 13F/A
was found to be equally distributed on both sides of the cells, similar
to the BC1-d13-16 mutant. To confirm the different distribution of the
13F/Y and 13F/A mutants, the filter-grown cells were also analyzed by
indirect immunofluorescence using a confocal laser scanning microscope.
After cell fixation with paraformaldehyde, the nonpermeabilized cells
were incubated from both the apical and the basolateral side with mAb
J4/48 and a FITC-conjugated anti-mouse immunoglobulin. Horizontal
sections of the apical, the center, and the basal portions of the cells are shown in Fig. 4 (upper
panel). In addition, a vertical section (side view) of the cells
is shown in the bottom panel of Fig. 4. Almost no
fluorescence signals were detected in the apical section of MDCK cells
expressing BC1-13F/Y. The section through the center of the cells
showed a honeycomb pattern that in a more diffuse manner was also seen
in the basal section. In the side view, a cup-like pattern typical for
basolateral proteins was observed. In contrast, strong fluorescence
signals were found in all sections of MDCK cells expressing BC1-13F/A.
In the vertical profile, BC1-13F/A was found to encircle the cells
completely. The confocal immunofluorescence analysis confirmed the
result that replacement of the phenylalanine by a tyrosine has no
effect on the targeting of MCP to the basolateral surface of polarized cells, whereas the replacement by an alanine resulted in the loss of
polarized surface expression.
To analyze further the basolateral targeting of
MCP, three mutants with changes in the three COOH-terminal amino acids
(TSL) were generated. MCP lacking all three amino acids (d14-16) or only lacking the leucine (d16) and a MCP form with a substitution of an
alanine for the leucine (16L/A) were stably expressed in MDCK cells and
analyzed by surface biotinylation and confocal microscopy of
filter-grown cells. As shown in Fig. 5,
the three mutants were almost equally distributed on the surface of
MDCK cells, indicating that removal or substitution of the leucine in
position 16 of the cytoplasmic tail results in the loss of the
polarized transport. The confocal immunofluorescence micrographs of
BC1-d14-16, BC1-d16, and BC1-16L/A are shown in Fig.
6. Fluorescence signals were detected in
all sections (apical, center, and basal) of MDCK cells expressing any
of the three mutants. This result confirms the result of the
domain-specific biotinylation and indicates that in addition to the
phenylalanine at position 13 the leucine at position 16 is also
essential for the correct sorting of MCP.
MCP with the phenylalanine replaced by tyrosine
(BC1-13F/Y) was transported to the basolateral membrane like the wild
type protein (BC1-wt). In contrast to all other mutants we tested, the
amount of surface-expressed protein BC1-13F/Y was strongly reduced
although the total amount of protein was comparable to BC1-wt. Because
a motif (YXXL) similar to that one generated in this mutant
(YTSL) is described as an endocytosis signal for several proteins (29,
30), the weak surface expression may have resulted from internalization
of BC1-13F/Y. To examine this possibility, we analyzed BC1-13F/Y for
endocytosis by an antibody uptake experiment. MCP at the surface of
living cells was incubated with mAb J4/48 at 4 °C, and the cells
were either kept on ice for 60 min or warmed to 37 °C to allow
endocytosis to occur. Surface-bound antibodies were detected by
incubation of the living cells with a rhodamine-conjugated second
antibody at 4 °C. After permeabilization of the cells, internalized
MCP-J4/48 complexes were detected with a FITC-conjugated second
antibody. In Fig. 7, the result of the
double immunofluorescence staining (surface, intracellular) is shown
for wild type MCP (BC1-wt) and the tyrosine mutant (BC1-13F/Y). With
cells maintained at 4 °C neither wild type nor mutant MCP was
detected intracellularly. After incubation at 37 °C, most of the
surface-bound J4/48 was still detectable on the surface of MDCK cells
expressing BC1-wt. The intracellular staining only showed a faint
fluorescence, indicating that almost no or only a small amount of the
protein was internalized during an incubation period of 60 min at
37 °C. In contrast, on the surface of cells expressing BC1-13F/Y no
fluorescence signals were seen after incubation at 37 °C. Almost all
MCP was endocytosed and could be detected by intracellular staining. To
confirm the internalization of BC1-13F/Y, we performed a sialidase
protection assay. MCP-expressing cells were surface labeled with biotin
and chased for various periods at 37 °C to allow internalization of proteins. The extent of endocytosis was measured by the proportion of
biotinylated protein that became inaccessible to extracellular VCNA
added at 4 °C at the end of the chase period. After digestion with
VCNA, cells were lysed, and MCP was immunoprecipitated and separated on
a 12% sodium dodecyl sulfate-polyacrylamide gel. Biotinylated proteins
were detected after transfer to nitrocellulose by
streptavidin/peroxidase. MCP on the cell surface was sensitive to VCNA
treatment. The release of sialic acids resulted in an increased
electrophoretic mobility (
Our results demonstrate that the cytoplasmic tail 1 of MCP
contains a basolateral targeting signal. MCP constructs lacking the
COOH terminus were affected in a different way. Substitution of a GPI
anchor for the transmembrane domain (MCP-PI) resulted in the apical
delivery of the chimeric protein. This finding was not unexpected
because transport to the apical membrane is a general feature of
GPI-anchored proteins (14, 15). In contrast to GPI-anchored MCP, a
mutant MCP lacking the cytoplasmic tail but retaining the transmembrane
domain as well as the intracytoplasmic anchor was not redirected to the
apical membrane but was transported to the cell surface in a
nonpolarized fashion. This may indicate the lack of any targeting
signal. For several other basolateral glycoproteins, it has been
reported that deletion of the cytoplasmic tail results in the transport
to the apical plasma membrane (7). N-Glycans have been
suggested to be involved in the apical transport of glycoproteins (13).
If carbohydrates indeed serve as apical targeting signals, one would
have to postulate that the tail-minus mutant of MCP has retained a weak
basolateral sorting signal that counteracts this apical targeting
signal resulting in nonpolarized transport. However, experimental
evidence for a role of N-glycans as apical targeting signal
is available so far only for secretory proteins (31), not for
transmembrane proteins. In this context it should be noted that
nonpolarized transport has been observed not only for the MCP mutant
but also for other basolateral proteins after inactivation of the
basolateral targeting signal or after deletion of the cytoplasmic tail,
e.g. for the asialoglycoprotein receptor (32) and the G
protein of vesicular stomatitis virus (30).
Using deletion analysis, we have shown that the main targeting
information of MCP-BC1 is contained within the amino acids FTSL at the
COOH terminus of the protein. This sorting signal has similarity to the
targeting determinants of several other basolateral proteins that
follow the general motif YXX-large hydrophobic, e.g. the vesicular stomatitis virus G protein (30), the
human nerve growth factor receptor (9), and the asialoglycoprotein receptor (32). The basolateral proteins described so far contain a
tyrosine as critical aromatic amino acid, and their sorting signals are
located within the cytoplasmic tail. The targeting motif of MCP
compared with other basolateral proteins has two unique features: (i)
it contains a phenylalanine at position 1 of the tetrapeptide; (ii)
it is exposed at the end of the cytosolic domain. In agreement with the
general motif, we found that neither phenylalanine nor leucine can be
replaced by alanine without affecting the polarized transport of MCP.
On the other hand, substitution of a tyrosine for the phenylalanine did
not abolish the transport to the basolateral surface, indicating that
phenylalanine and tyrosine can have the same function in the sorting
event from the trans-Golgi network to the basolateral plasma
membrane. Furthermore, our results demonstrate that a basolateral
sorting signal can be recognized also when it is located in a terminal
position.
All basolateral receptor proteins studied so far are endocytosed. Some
of them are transcytosed to the apical cell membrane (33). The majority
of endocytotic receptors are recycled to the plasma membrane and
finally degraded in lysosomes with half-lives of 6-60 h (34).
Lysosomal and Golgi proteins that are found transiently on the
basolateral membrane are rapidly endocytosed and delivered to lysosomes
and the trans-Golgi network, respectively (29, 35). Three
types of signals for receptor-mediated endocytosis are known. Among
these are a dileucine motif (36) and a terminal KKXX motif
(37). The majority of all known internalization signals contain the
motif aromatic-XX-large hydrophobic. In most cases the
aromatic residue is a tyrosine, and therefore the signal overlaps with
the basolateral targeting signal mentioned above, e.g. in the lysosomal acid phosphatase (10) and in the asialoglycoprotein receptor (32). We found that MCP-BC1 is endocytosed only to a very low
extent. In the antibody uptake experiment a very small amount of MCP
was visible intracellularly, whereas no endocytosed protein was
detectable in the sialidase protection assay. This may be caused by a
lower sensitivity of the sialidase assay or clustering of MCP in coated
pits after antibody binding, resulting in an enhanced internalization.
Such a low internalization rate has not been described for any other
well characterized basolateral protein. In this respect, MCP resembles
the apically expressed influenza hemagglutinin, which has been shown to
be excluded from coated pits and which is internalized 40 times more
slowly than is the bulk of the plasma membrane (38). The structural
basis for the inefficient endocytosis of HA is, in addition to the lack of a tyrosine-dependent signal in the cytoplasmic tail, a
structural feature within the transmembrane domain (39). Whether the
transmembrane domain of MCP contributes to the inefficient
internalization of this protein remains to be shown. The cytoplasmic
tail of MCP-BC1 obviously does not contain an endocytosis signal,
although a tyrosine is present at position 2 of the tail sequence. The
FTSL sequence that is important for the basolateral targeting is not
sufficient for endocytotic uptake. The reason for this is not the
terminal position of this tetrapeptide but the aromatic amino acid at
position 1. By replacing the phenylalanine residue by a tyrosine, we
created an efficient endocytosis signal. About 90% of BC1-13F/Y was
endocytosed within 50 min. The internalization did not reach a plateau
after 10-15 min, as is commonly observed with proteins that recycle to
the plasma membrane, suggesting that the protein is delivered to
lysosomes and degraded. This assumption is consistent with the observed
decrease in the total amount of MCP during longer internalization
times. In agreement with the rapid endocytosis, we detected only small
amounts of BC1-13F/Y on the cell surface when we analyzed the
steady-state distribution.
MCP provides an interesting example of how the targeting motif can be
modulated to favor transport to the plasma membrane over endocytosis.
The phenylalanine residue of the COOH-terminal FTSL sequence allows
basolateral transport of MCP but avoids endocytosis. This is optimal
for the physiological function of MCP, i.e. the binding of
the complement factors C3b and C4b on the cell surface to prevent
complement activation. As complement activation of the alternative
pathway occurs continually, protection of the autologous cells from
complement-mediated lysis requires the constitutive expression of
protective proteins on the cell surface. Additionally, if MCP engages a
ligand that is covalently bound to other membrane constituents,
internalization would likely not be possible. Therefore, there need to
be signals that take the protein to its proper location and prevent it
from being internalized. The FTSL motif is an effective way to meet
this requirement because it allows MCP to stay for extended periods of
time on the basolateral surface, i.e. on the membrane domain
that faces the serosal compartment. The correlation of MCP expression
levels and cytoprotection was demonstrated by Oglesby et al.
(41): mouse cells transfected with MCP were protected from complement
in a dose-dependent manner. An example of the effect of low
MCP surface expression is provided by measles virus-infected cells.
Infection by certain strains of measles virus causes a rapid
down-regulation of MCP, and this has been shown to result in an
increased susceptibility of the cells for complement-mediated lysis
(42).
We acknowledge gratefully the technical
assistance of A. Heiner.
Institut für Virologie,
Philipps-Universität Marburg, D-35037 Marburg, Germany, the
§ Division of Rheumatology,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENT
REFERENCES
Cell Culture
× cm2. The different patterns of surface proteins on
the apical and basolateral membrane of the polarized cell lines were
controlled by surface biotinylation.
Apr-1-neo (25). The construction of
the four cDNAs with larger deletions in the cytoplasmic tail of the
isoform BC1 of MCP (tail-minus, d1-6, d7-12, and d13-16) was
described by Liszewski et al. (26). For the generation of
the cDNA mutants 13F/Y, 13F/A, d14-16, d16, and 16L/A, MCP isoform
BC1 was subcloned into the replicative form DNA of M13mp18.
Oligonucleotide-directed mutagenesis based on the phosphorothioate
method using single-stranded DNA of M13mp18 was performed with a
commercial kit (Amersham Corp.). M13mp18-MCP phages with the desired
mutation were selected by sequencing the single-stranded DNA with a
Sequenase kit according to the instructions of the manufacturer
(Perkin-Elmer). The mutated MCP cDNA was excised from the phage
replicative form DNA and ligated into the EcoRI site of
pH
Apr-1-neo. The amino acid sequences of the cytoplasmic tail of all
MCP mutants are shown in Fig. 1 (lower panel).
Fig. 1.
Diagram of MCP-DAF chimeras and amino acid
sequences of the cytoplasmic domains of MCP isoform BC1 mutants.
In the upper panel a diagram of the MCP, DAF-TM, and the
MCP-PI chimeras is shown. The ectodomain of MCP consists of the four
SCRs, the STP B and C, and a short region of unknown function
(U). The TM domain is followed by an intracytoplasmic anchor
(IA) and the cytoplasmic domain (CT). The DAF-TM
comprises the four SCRs (SCR*) and the O-linked carbohydrate
domain (OD) of DAF (AS 1-304) followed by the TM domain,
the intracytoplasmic anchor, and the cytoplasmic domain of MCP-BC2 (AS
270-350). The MCP-PI comprises the four SCRs and the STP B of MCP (AS
1-269) followed by a GPI that results from the cleavage of the DAF-GPI
anchoring hydrophobic signal encoded in amino acids 307-347 of DAF. In
the lower panel the end of the TM domain, the
intracytoplasmic anchor, and the cytoplasmic domains of MCP isoform BC1
wild type (BC1-wt), and mutants are shown in a single letter amino acid
code.
[View Larger Version of this Image (27K GIF file)]
20 °C with methanol/acetone (1:1). Internalized antibodies were
detected with FITC-labeled rabbit anti-mouse IgG (DAKO) at a dilution
of 1:500 in PBS-bovine serum albumin. To avoid nonspecific binding to
the rhodamine-labeled goat Fab fragments bound to the cell surface, the
FITC-labeled antiserum was preabsorbed with goat-IgG agarose (Sigma).
After the double immunofluorescence staining, the samples were mounted
in Mowiol and 10% triethylenediamine. Conventional epifluorescence was
performed with an Axiophot microscope (Zeiss). Pictures were taken with
Kodak Tmax film (3200 ASA) exposed for identical times for both
fluorochromes.
The Cytoplasmic Domain of MCP Contains a Basolateral Targeting
Signal
Fig. 2.
Polarized surface distribution of MCP
chimeric proteins and MCP deletion mutants expressed in MDCK
cells. MDCK cells grown on filters expressed different forms of
MCP: MCP isoform BC1 (BC1-wt), a DAF molecule with MCP anchor (DAF-TM),
a GPI-anchored MCP (MCP-PI), MCP lacking the cytoplasmic tail
(tail-minus), MCP-BC1 lacking the first six amino acids (d1-6) of the
cytoplasmic tail, lacking six amino acids in the mid (d7-12) or
lacking the last four amino acids (d13-16), respectively, of the
cytoplasmic tail. After surface biotinylation either from the apical
(a) or basolateral (b) side, the cells were
lysed, and MCP or DAF, respectively, was immunoprecipitated. Samples
were separated on a 12% polyacrylamide gel under nonreducing
conditions and blotted to nitrocellulose. Surface-biotinylated,
immunoprecipitated proteins were visualized with
streptavidin/peroxidase.
[View Larger Version of this Image (28K GIF file)]
Fig. 3.
Surface expression of MCP-BC1 with an
exchange of the phenylalanine in the cytoplasmic tail. MCP-BC1
cDNA with the phenylalanine in position 13 of the cytoplasmic tail
replaced by a tyrosine (13F/Y) or by an alanine (13F/A) was expressed
in filter-grown MDCK cells. After domain-specific biotinylation from the apical (a) or the basolateral (b) side,
respectively, the cells were lysed, and MCP was immunoprecipitated by
mAb J4/48. Samples were analyzed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis and visualized after
transfer to nitrocellulose by incubation with
streptavidin/peroxidase.
[View Larger Version of this Image (61K GIF file)]
Fig. 4.
Confocal immunofluorescence microscopy of
MCP-BC1 with an exchange of the phenylalanine in the cytoplasmic
tail. MDCK cells expressing MCP-BC1 with a tyrosine (13F/Y) or an
alanine (13F/A) instead of a phenylalanine at position 13 of the
cytoplasmic tail were grown on filters. After fixation with 2%
paraformaldehyde, the apical and basolateral surfaces were incubated
with mAb J4/48 and a FITC-conjugated second antibody. Analysis was
performed with a laser scanning microscope. Confocal immunofluorescence micrographs of horizontal focal planes through the apical surface, the
center, the basal surface, and a vertical profile (side view) through
the monolayers are shown.
[View Larger Version of this Image (95K GIF file)]
Fig. 5.
Cell surface distribution of MCP-BC1 with
mutations in the last three amino acids of the cytoplasmic tail.
MDCK cells expressing MCP isoform BC1 lacking the last three amino
acids of the cytoplasmic tail (d14-16), lacking the last amino acid (d16), or having the leucine at position 16 replaced by an alanine (16L/A), were analyzed for polarized surface distribution by
domain-specific biotinylation: apical (a) or basolateral
(b).
[View Larger Version of this Image (49K GIF file)]
Fig. 6.
Analysis of the surface expression of MCP-BC1
with changes in the last three amino acids of the cytoplasmic tail by
confocal microscopy. MDCK cells expressing MCP isoform BC1 lacking the last three amino acids of the cytoplasmic tail (BC1-d14-16), lacking the last amino acid (BC1-d16), or having the leucine at position 16 replaced by an alanine (BC1-16L/A) were grown on filters. The apical and basolateral side was stained with mAb J4/48 and a
FITC-conjugated anti-mouse immunoglobulin. Confocal immunofluorescence micrographs of horizontal planes through the apical surface, the center, and the basolateral surface are shown.
[View Larger Version of this Image (117K GIF file)]
SA). Internalized protein was resistant to
the enzyme treatment and retained its sialic acids (+SA). In Fig.
8, a clear difference between BC1-wt and BC1-13F/Y can be observed. The protein bands were quantified by densitometric scanning to determine the endocytosis rate. In the case
of BC1-wt, even after 50 min at 37 °C only desialylated protein was
detectable, indicating that no internalization had occurred, and all of
the protein was sensitive to VCNA. In contrast, BC1-13F/Y was found to
become VCNA-resistant with increasing incubation time at 37 °C.
After 15 min 50% of MCP was still sialylated. The amount internalized
increased to more than 90% after an incubation period of 50 min. With
longer endocytosis times, the total amount of BC1-13F/Y decreased,
probably because of degradation of the internalized protein.
Fig. 7.
Uptake of antibodies bound to MCP-BC1 wild
type and BC1-13F/Y mutant. MDCK cells expressing MCP-BC1 wild
type (BC1-wt) or MCP-BC1 with the phenylalanine at position 13 in the
cytoplasmic tail replaced by a tyrosine (BC1-13F/Y) were grown on
coverslips. After incubation of the living cells with mAb J4/48 on ice,
cells were either incubated at 4 °C or at 37 °C. Surface-bound
antibodies were stained with a rhodamine-conjugated anti-mouse serum
(surface). After permeabilization of the cells, internalized antibodies
were stained with a FITC-conjugated anti-mouse serum
(intracellular).
[View Larger Version of this Image (59K GIF file)]
Fig. 8.
Endocytosis of MCP-BC1 wild type and
BC1-13F/Y mutant analyzed by a sialidase protection assay. MDCK
cells expressing MCP-BC1 wild type (BC1-wt) or MCP-BC1 with the
phenylalanine at position 13 in the cytoplasmic tail replaced by a
tyrosine (BC1-13F/Y) were grown to confluence. After surface
biotinylation, the cells were incubated at 37 °C for 0, 5, 15, 30, or 50 min. Cells were treated at 4 °C without () or with (+) VCNA
for 60 min. After lysis of the cells, MCP was immunoprecipitated. The
samples were separated on a 12% polyacrylamide gel and transferred to
nitrocellulose. Biotinylated sialylated (+SA) and
desialylated (
SA) MCP were detected by
streptavidin/peroxidase.
[View Larger Version of this Image (45K GIF file)]
*
This work was supported by Deutsche Forschungsgemeinschaft
Grant He 1168/3-3 (to G. H.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Institut für
Virologie, Philipps-Universität Marburg, Robert-Koch Strasse 17, D-35037 Marburg, Germany. Tel.: 49-6421-28-5360; Fax: 49-6421-28-5482. E-mail: herrler{at}mailer.uni-marburg.de.
1
The abbreviations used are: GPI,
glycosylphosphatidylinositol; MCP, membrane cofactor protein; SCR,
short consensus repeat; STP, serine, threonine, proline-rich region;
MDCK, Madin-Darby canine kidney; DAF, decay accelerating factor; TM,
transmembrane; mAb, monoclonal antibody; FITC, fluorescein
isothiocyanate; PBS, phosphate-buffered saline; VCNA, Vibrio
cholerae sialidase; wt, wild type; SA, sialic acid(s).
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.