(Received for publication, December 26, 1995)
From the
Both in vivo and in vitro the distribution of
the resident plasma membrane adhesion protein, CD44, is restricted to
the basolateral domain of polarized epithelial cells, suggesting a role
in interepithelial interactions. To determine how this localization
might be regulated, a range of CD44 cytoplasmic domain mutations were
generated and a minimal 5 amino acid sequence,
His-Leu-Val-Asn-Lys
, was identified which
when deleted results in expression of CD44 on the apical microvillal
membrane. Further mutagenesis throughout this regions pinpointed a
critical di-hydrophobic motif, Leu
/Val
. The
ability of wild type but not mutant CD44 cytoplasmic domains to
redirect an apically targeted protein, placental alkaline phosphatase,
to the basolateral plasma membrane demonstrates that this sequence can
function as a dominant localization signal. This
His
-Lys
sequence is spatially
separate from other CD44 regulatory elements and as discussed here, a
comparison with known basolateral sorting sequences identified in other
transmembrane proteins suggests that a distinct mechanism operates to
retain resident plasma membrane proteins in their correct plasma
membrane subdomains.
CD44 was originally identified as an abundant 80-100-kDa transmembrane glycoprotein on a variety of cell types. In recent years, much of the interest in CD44 has stemmed from experiments demonstrating that it is a principle receptor for the important extracellular matrix glycosaminoglycan, hyaluronan, and that inappropriate expression can result in increased tumor growth and metastasis (reviewed in Refs. 1 and 2). However, although enhanced levels of CD44 expression and/or alterations in CD44 splicing are frequently found in epithelial tumors, little is known about the function and regulation of this adhesion protein in epithelial cells. In stratified epithelia such as the skin, abundant CD44 is found in the basal and spinous layers co-localized with its ligand, hyaluronan(3, 4, 5) , and an interaction between CD44 and hyaluronan mediates cell:cell adhesion between keratinocytes(6, 7) . A different picture emerges in simple epithelia such as that in the gastrointestinal tract where CD44 is found only on the dividing cells within the crypts of Lieberkühn. In these cells abundant CD44 is found on the lateral plasma membranes, but no intercellular hyaluronan is detected(3) . This raises the possibility that in simple epithelia CD44 may have a role in hyaluronan-independent intercellular interactions.
Polarized epithelia have two compositionally and
morphologically distinct cell surface domains reflecting their
disparate functions (reviewed in (8) and (9) ). The
upper apical microvillal surface provides a protective role by acting
as a barrier between the external and internal environments and it
contains the necessary transporters for the uptake of small molecules.
The basolateral surface is involved in cell:cell and cell:matrix
adhesion and also contains plasma membrane proteins involved in signal
transduction and nutrient uptake. Generation and maintenance of these
two domains is required for the correct functioning of polarized cells
and involves the continuous sorting and correct plasma membrane
insertion of newly synthesized proteins. In addition, the ability of
mature proteins presented at the plasma membrane to be endo- and
transcytosed must be strictly regulated (reviewed in (10, 11, 12, 13) ). In the studies
described here we have employed the well characterized MDCK cell line
which can be grown to confluence to generate a functional polarized
monolayer. In these cells CD44 is localized exclusively to the lateral
plasma membrane (14) supporting the in vivo data for a
role of CD44 in interepithelial interactions. As has been demonstrated
for a number of other transmembrane
receptors(15, 16, 17) , the basolateral
localization of CD44 is regulated by the cytoplasmic domain in that the
truncation of this domain results in a tailless (T-) ()molecule which is localized to the apical microvillal
membrane(14) . However, unlike these well characterized
transmembrane receptors, CD44 is a long lived resident plasma membrane
protein that is not subject to rapid endo- or
transcytosis(14, 18, 19, 20, 21, 22) .
This manuscript describes experiments identifying a specific sequence
motif within the CD44 cytoplasmic domain that regulates the correct
localization of this protein in polarized epithelial cells. These
results provide insight into the regulation of this important adhesion
protein which has been strongly implicated to play a role in epithelial
disease progression. Furthermore, these studies provide clues as to the
mechanisms controlling the trafficking and stabilization of resident
membrane proteins in polarized cells.
To generate the
Gly
-Pro
mutant, the same
restriction strategy as described above was employed. Following the PvuII digestion the 255-bp fragment was discarded and the 90-
and 66-bp fragments were purified. pUC19/WT CD44 was restricted with MboII producing a 256-bp fragment, which was isolated and
purified. This 256-bp fragment was then treated with the Klenow
fragment of DNA polymerase 1 and dNTPs to fill in overhanging ends and
then restricted with BstXI generating two fragments of 196 and
60 bp. The 196-bp fragment was retained and ligated along with the 90-
and 66-bp PvuII fragments into the BstXI linearized
pSR
-neo/Ala
CD44 vector to generate a 20-amino acid
deletion from Gly
-Pro
.
The
following oligonucleotides (see Table 1) were used for loop out
or point mutagenesis with WT CD44 in the pSELECT vector (Promega) as a
template. C15 for Gly
-Ser
, C14
for
Lys
-Val
, C12 for
His
-Ser
, C11 for
Glu
-Thr
, C21 for
His
-Lys
, C22 for
Gln
-Ser
, C19 for
Ala
, C25 for Ala
, C23 for
Ala
/Ala
. All mutants generated in the
pSELECT vector were excised and subcloned into the pSR
-neo
eukaryotic expression vector, as described previously(24) .
The Ala, Ala
, and Ala
mutants were generated by a two round polymerase chain reaction
mutagenesis strategy, using C24, C18, and C20 mutagenic
oligonucleotides, respectively, as primers. The first round was primed
with C18, C20 or C24, and C4, the template used was pUC19/CD44. The
product was purified, denatured by heating, and used with primer C3 in
a second round polymerase chain reaction using the same template. The
product was then isolated, purified, Klenow-treated, and ligated into
end-polished BamHI restricted pSR
-neo expression vector.
The identity of all mutant clones was confirmed by restriction
endonuclease digestion and DNA sequencing.
Figure 1: Deletion mutations in the cytoplasmic domain of human CD44. Mutations were generated as described under ``Materials and Methods.'' Amino acid numbers are as according to Stamenkovic et al.(27) .
Figure 2:
Distribution of CD44 cytoplasmic domain
deletion mutants in polarized MDCK cells. Clonal lines of MDCK cells
expressing Gly
-Pro
(a,
c),
Ser
-Thr
(b, d)
Gly
-Ser
(e),
Lys
-Val
(f),
His
-Ser
(g),
Glu
-Thr
(h),
His
-Lys
(i), and
Glu
-Ser
(j) human CD44
were grown to confluence on Transwell filters. Cells were fixed,
permeabilized, and incubated with the anti-human CD44 mAb, E1/2,
followed by rhodamine-conjugated anti-mouse Ig and visualized by
confocal microscopy. a and b show horizontal xy
sections through the cells. c-j show vertical xz
sections taken in 0.2-µm steps through the cells with the apical
plasma membrane of the cells orientated topmost. Bar, 10
µm.
To confirm and quantify the
distribution of these CD44 cytoplasmic deletion proteins in epithelial
cells, clonal MDCK cell transfectants were cultured on duplicate
Transwell filters, the integrity of the monolayer was assessed by
[H]inulin testing, and then cells were
surface-biotinylated from either the apical or basolateral side. In
agreement with the confocal data, it was found that in those mutants
with a WT CD44 localization pattern, less than 13% of the CD44 was
accessible to biotinylation on the apical plasma membrane, and
conversely, in those mutants whose cell surface localization resembled
T- CD44, greater than 88% of the CD44 could be labeled by apical
biotinylation (Fig. 3).
Figure 3:
Cell
surface biotinylation of CD44 deletion mutants in polarized MDCK cells.
Clonal lines of MDCK cells expressing WT (a), T- (b),
Gly
-Pro
(c),
Ser
-Thr
(d)
Gly
-Ser
(e),
Lys
-Val
(f),
His
-Ser
(g),
Glu
-Thr
(h),
His
-Lys
(i), or
Glu
-Ser
(j) human CD44
were grown to confluence on duplicate Transwell filters and
biotinylated either from the apical (A) or from the
basolateral (B) sides. Transfected CD44 was immunoprecipitated
with mAb E1/2, resolved on a 10% polyacrylamide gel, and blotted onto
nitrocellulose. Biotinylated protein was visualized using horseradish
peroxidase-streptavidin and the ECL reagent. The percentage of
biotinylated CD44 expressed apically is shown. Blots were exposed to
x-ray film for 1 min. Molecular size markers are in
kilodaltons.
Figure 4:
Point mutations within the HLVNK
basolateral localization sequence define critical amino acids. A series
of alanine mutations were generated in the
His-Lys
sequence as described under
``Materials and Methods'' (A). Clonal lines of MDCK
cells expressing alanine point mutants were generated, and the
distribution of transfected protein was examined by confocal
microscopy. B shows vertical xz sections taken through
polarized monolayers of transfected MDCK cells cultured on Transwell
filters where human CD44 has been detected using mAb E1/2 as described
in the legend to Fig. 2. Bar = 10 µm. C shows biotinylation of CD44 on the apical and basolateral
membranes as described in the legend to Fig. 3and the percent
of transfected CD44 detected on the apical plasma membrane. The three
molecular size markers represent 116, 84, and 58 kDa. ND = not done.
Figure 5: Distribution of PLAP/CD44 chimeric proteins in MDCK cells. A, chimeras composed of the PLAP extracellular domain (striped), CD44 transmembrane domain (shaded), and different CD44 cytoplasmic domain mutants (open) were generated as described under ``Materials and Methods.'' G418-resistant mixed MDCK cell populations expressing chimeric proteins were generated, and the distribution of transfected protein was examined by confocal microscopy. B shows horizontal xy (upper) vertical xz (lower) sections taken through polarized monolayers of transfected MDCK cells cultured on Transwell filters (see Fig. 2) using a polyclonal antiserum directed against PLAP followed by a Texas Red-conjugated anti-rabbit antiserum (Vector Laboratories). Bar = 10 µm. C shows the biotinylation of chimeric protein on the apical and basolateral plasma membranes as described in Fig. 3and the percent of chimeric protein detected on the apical plasma membrane. Molecular size markers represent 116, 84, and 58 kDa.
Both the endogenous canine CD44 and transfected WT human CD44 are localized to the basolateral plasma membrane of polarized MDCK epithelial cells. This distribution could result from (a) sorting of CD44 in the trans-Golgi network (TGN) followed by direct delivery to the basolateral membrane, (b) delivery of CD44 to the apical plasma membrane followed by rapid and efficient transcytosis to the basolateral plasma membrane, or (c) nonspecific delivery to both plasma membranes followed by stabilization of CD44 at the basolateral membrane and rapid degradation/transcytosis of apical CD44. This latter pathway is unlikely to operate in polarized cells, as we demonstrate here that the transmembrane/cytoplasmic domain of CD44 is capable of redirecting an apically targeted protein. This indicates that CD44 encodes a dominant sorting signal.
Mutational analysis of
a number of basolaterally localized cell surface receptors has
demonstrated the presence of dominant signals contained within their
cytoplasmic domains that mediate their direct delivery from the TGN to
the basolateral plasma membrane. For many of these proteins, such as
the nerve growth factor receptor(30, 31) ,
asialoglycoprotein receptor(32) , and low density lipoprotein
receptor (33) , there is a functional and spatial overlap of a
basolateral sorting signal with a signal required for the recruitment
of the receptors into coated pits on the plasma membrane and subsequent
endocytosis. Characteristically these overlapping signals contain an
aromatic (usually tyrosine)-X-X-large hydrophobic motif.
Secondary structure analysis suggests that such motifs can form
-turns that presumably interact with intracellular machinery
(reviewed in (34) ). In some cases the overlapping basolateral
sorting motifs and endocytosis motifs can be distinguished by
mutagenesis. For example endocytosis of lysosomal acid phosphatase is
critically dependent on the tyrosine residue in the YRHV motif, but
basolateral sorting remains efficient if this residue is substituted
for a phenylalanine(35) . This suggests that the same motif
interacts with different sets of cellular machinery which have
individual sequence and secondary structure requirements. A second
group of basolateral sorting determinants as identified in the low
density lipoprotein receptor(33) , transferrin
receptor(17) , and polymeric immunoglobulin receptor (16) are functionally and spatially separate from endocytosis
determinants. There is no clear sequence consensus within this second
group of targeting signals, although interestingly, a soluble peptide
representing the polymeric immunoglobulin receptor basolateral signal
has been shown to adopt a
-type turn in solution (36) .
In this study we have identified a minimal 5 amino acid basolateral
localization motif within the CD44 cytoplasmic domain. In principle
this motif could function either as a TGN sorting signal for the direct
delivery of CD44 to the basolateral membrane or as an internalization
motif required for the transcytosis of apically expressed CD44. Our
experiments do not distinguish between these two possibilities;
however, a transcytosis signal is considered unlikely for the following
reasons. Transcytosis would require CD44 to be recruited into the
coated pits, and yet in studies by Bretscher et al.(18) , it was demonstrated that CD44 (H63 in their
nomenclature) is excluded from these plasma membrane subdomains.
Supporting data for the stable residency of CD44 on the plasma membrane
comes from metabolic labeling of CD44 in MDCK cells, demonstrating that
it has a long (>24 h) half-life in these cells (14) and that
labeled anti-CD44 mAbs remain bound to CD44 on the cell surface for
>100 h without becoming
internalized(19, 20, 21) . Finally, antibody
labeling of permeabilized cells indicates that there is not a
significant pool of intracellular CD44(14, 24) . In
addition there is no precedent for resident basolateral transmembrane
proteins being routed via the apical plasma membrane in mature
polarized monolayers such as those examined here. Together, these data
suggest that the His-Lys
motif
functions to sort CD44 in the TGN for subsequent delivery to the
basolateral plasma membrane. Unfortunately, the long lived nature of
CD44 combined with its extensive post-translational glycosylation
prevents the necessary metabolic pulse labeling combined with cell
surface biotinylation which would be required to directly test this. As
a consequence, the sequence motif identified in these experiments
cannot be unambiguously classified as a sorting sequence, and we
therefore refer to it as a basolateral localization motif.
The
His-Lys
basolateral localization
motif has no sequence similarity with the well characterized
basolateral sorting signals described above. Alanine scan mutagenesis
of this region reveals a critical dependence on the leucine residue
(Leu
) and partial dependence on the neighboring valine
residue (Val
). Recently there have been several reports
demonstrating that Leu-Z di-hydrophobic motifs (where Z is usually Leu, Ile, or Val) can play a important role
intracellular protein targeting (reviewed in (37) ). In all of
these cases, this LZ motif is associated with the endosomal
targeting of proteins from the TGN or plasma membrane. The only
exception is the Fc receptor, FcRII-B2, which has a critical dependence
on an LL motif for basolateral sorting in epithelial
cells(38, 39) ; however, unlike CD44 this motif also
mediates recruitment of FcRII-B2 into the endocytic pathway. Moreover,
both internalization and sorting of FcRII-B2 is partially dependent on
an upstream Y residue in the context of YSLL. The CD44 cytoplasmic
domain does not contain any tyrosine residues, and its single
phenylalanine residue is not conserved between species (reviewed in (22) ). The only other aromatic amino acid within the CD44
cytoplasmic domain is His
that lies adjacent to the
Leu
/Val
motif, and mutation of this
histidine has no effect on CD44 localization (see Fig. 4).
It
is not known what secondary structure the CD44 LV di-hydrophobic motif
forms. Sandoval et al.(40) have demonstrated that a
critical LI motif within the cytoplasmic tail of the lysosomal protein,
LIMP II, is held in an extended configuration and does not adopt a
-turn. Thus it remains to be determined whether the basolateral
sorting cellular machinery which recognizes
-turn motifs could
also interact with di-hydrophobic motifs, such as those found in
FcRII-B2 and CD44. The second issue raised from these studies is how
the cellular machinery distinguishes between basolaterally sorted
proteins such as FcRII-B2 that are readily endocytosed and those such
as CD44 that remain as resident plasma membrane proteins. In this
respect, studies on the vesicular stomatitis virus glycoprotein are of
interest. Vesicular stomatitis virus glycoprotein utilizes a classic
Y-X-X-aliphatic motif, YTDI, to mediate its basolateral
sorting in MDCK cells, but this glycoprotein remains expressed at the
plasma membrane and is not endocytosed(41, 42) . If
common machinery is used for basolateral sorting, then resident
basolateral proteins such as vesicular stomatitis virus glycoprotein
and CD44 must either be missing additional components necessary for
efficient endocytosis and/or be subject to retention mechanisms that
would prevent recruitment into the coated pits.
The cytoplasmic tail
of CD44 is highly conserved between species (reviewed in (22) )
and, as demonstrated here, is necessary for the correct plasma membrane
localization of this molecule. In addition, this intracellular domain
of CD44 can regulate ligand binding as cells expressing truncated CD44
mutants exhibit reduced or abolished ability to bind
hyaluronan(43, 44, 45, 46) .
Although there is dispute in the literature as to whether ligand
binding is regulated by a discrete domain, there is general agreement
that truncation of the CD44 cytoplasmic domain upstream of the
basolateral localization sequence identified here does not alter its
ability to associate with hyaluronan (44, 46, 47, 48) . Similarly, this
basolateral localization sequence appears to be functionally and
spatially distinct from the transmembrane domain of CD44 that regulates
the Triton X-100 solubility of this receptor (14, 49, 53) and from the two phosphorylation
sites, Ser and Ser
(24) . There are
reports in the literature that di-hydrophobic motifs might function in
the context of upstream phosphorylation events, for example to enhance
the internalization of CD4(50) . However, mutation of the
Ser
and Ser
phosphorylation sites does not
disrupt the basolateral localization of CD44 (24) nor reduce
the half-life of the protein (data not shown). This WT distribution of
the CD44 phosphorylation mutants also strengthens the suggestion that
CD44 is not rapidly transcytosed from the apical membrane given that at
least in some cases transcytosis is known to be a phosphorylation
dependent mechanism(51) . To understand how these different
domains of CD44 regulate function, it will be necessary to identify
both cytoplasmic and membrane components with which these individual
receptor domains interact. Finally, the lateral plasma membrane
distribution of CD44 in simple epithelial cells suggests a role for
CD44 in interepithelial interactions. There is increasing evidence that
this interaction may operate in a hyaluronan-independent manner, and
therefore identification of novel ligands will be crucial in
elucidating mechanisms controlling this important transmembrane
receptor.