(Received for publication, August 3, 1995; and in revised form, September 12, 1995)
From the
The cytoplasmic domains of many membrane proteins have short sequences, usually including a tyrosine or a di-leucine, that function as sorting signals. P-selectin is an adhesion receptor for leukocytes that is expressed on activated platelets and endothelial cells. Its 35-residue cytoplasmic domain contains signals for sorting into regulated secretory granules, for endocytosis, and for movement from endosomes to lysosomes. The domain has a membrane-distal sequence, YGVFTNAAF, that resembles some tyrosine-based signals. We studied the effects of deletions and mutations in the cytoplasmic tail of human P-selectin on its internalization in clathrin-coated pits of transfected Chinese hamster ovary cells. Mutations and deletions in the putative tyrosine-based motif did not clearly implicate these residues as critical components of a short internalization signal. Indeed, a construct containing a truncated 18-residue cytoplasmic domain with a single substitution (K761A/H773Stop) was internalized nearly three times as fast as wild-type P-selectin; this construct contained no di-leucine, tyrosine, or other known sorting motif. Substitution of residues throughout the cytoplasmic domain affected the internalization rate of P-selectin. Furthermore, the cytoplasmic domain of P-selectin mediated faster internalization when attached to the extracellular and transmembrane domains of the low density lipoprotein receptor than when attached to the corresponding domains of P-selectin. Thus, we were unable to identify a short internalization signal in the cytoplasmic tail of P-selectin. Residues throughout the cytoplasmic domain, and perhaps the transmembrane sequence to which the domain is attached, affect the efficiency of internalization.
The cellular trafficking of many membrane proteins appears to be
directed by short sorting signals in the cytoplasmic domains (reviewed
in (1) and (2) ). The most studied signal has an
aromatic residue, usually a tyrosine, within a sequence of four to six
amino acids (3, 4, 5, 6) . The
tyrosine is often separated from a hydrophobic residue by two other
amino acids(7, 8) . Tyrosine-based signals mediate
endocytosis of membrane proteins through clathrin-coated
pits(1, 2) and direct sorting of membrane proteins to
a variety of intracellular
compartments(9, 10, 11, 12, 13, 14, 15, 16) .
``Di-leucine'' motifs are a second group of signals that
mediate the internalization and sorting of membrane
proteins(17, 18, 19, 20, 21) .
Some tyrosine-based signals form a
turn(5, 22, 23, 24) , whereas the
structures of di-leucine motifs are unknown. Tyrosine- or
di-leucine-based signals may also function when inserted into the
cytoplasmic domains of other membrane proteins(1, 2) .
Cytoplasmic domains with tyrosine-based signals bind to adaptin
proteins of clathrin coats of the plasma membrane or trans-Golgi
network(25, 26, 27, 28) . However,
direct binding of tyrosine- or di-leucine-based motifs to adaptins has
not been demonstrated.
Membrane proteins of the endoplasmic reticulum have retrieval signals with the consensus sequence KKXX, located at the extreme C termini of their cytoplasmic domains(29) . These signals interact with coatomers(30) , which were originally described on transport vesicles of the Golgi complex(31) . One C-terminal sequence, KKFF, has been shown to mediate internalization of membrane proteins through clathrin-coated pits(32, 33) . These findings suggest that coatomer-mediated retrieval of membrane proteins is mechanistically related to clathrin-dependent sorting. The data also suggest that diverse sequences may create related sorting signals.
The selectins are a family of three type I membrane glycoproteins
that initiate leukocyte adhesion to the vessel wall by interacting with
cell-surface carbohydrates (reviewed in (34) ). The surface
expression of the selectins is tightly regulated, a means to control
the extent of leukocyte recruitment into sites of inflammation.
P-selectin is located in the membrane of secretory granules of
platelets and endothelial
cells(35, 36, 37, 38) . Upon
stimulation of the cells with thrombin or other agonists, P-selectin is
redistributed to the plasma membrane, where it mediates adhesion of
leukocytes(39, 40, 41) . The protein is
rapidly internalized from the surface of activated endothelial
cells(42) . Some of the internalized P-selectin molecules
return to the trans-Golgi network, where they are resorted
into secretory granules(43) . When expressed in CHO ()cells, which lack regulated secretion, P-selectin is
constitutively delivered to the cell surface and then
internalized(44, 45, 46) . P-selectin is also
efficiently targeted from endosomes to lysosomes for degradation; this
mechanism limits recycling of internalized P-selectin to the plasma
membrane(46) .
The 35-residue cytoplasmic tail of human P-selectin contains signals for sorting into secretory granules(44) , for internalization from the plasma membrane(43) , and for movement from endosomes into lysosomes (46) . As shown in Fig. 1, the sequence of the cytoplasmic domain is highly conserved across species(47, 48, 49, 50) . This suggests that the overall structure of the domain must be maintained to mediate the trafficking of P-selectin to multiple destinations. The human cytoplasmic domain has no obvious di-leucine or KKFF signals. However, it has three aromatic residues within a membrane-distal sequence, YGVFTNAAF, that has some similarities to known tyrosine-based internalization signals(1, 2) . When attached to a truncated cytoplasmic domain of the mannose 6-phosphate/insulin-like growth factor-II receptor, the YGVF sequence mediates internalization at 40% of the rate of the endogeneous receptor sequence, YSKV(8) . Deletion of 11 internal residues from the P-selectin cytoplasmic tail, leaving the aromatic residues intact, abrogates endosomal sorting into lysosomes (46) . These data are consistent with a model in which signals for internalization and for endosomal sorting are located in distinct portions of the cytoplasmic domain of P-selectin. However, the results do not exclude a more complex model in which the conformation of the intact cytoplasmic domain creates sorting determinants that differ from those in shorter constructs.
Figure 1:
Conservation of amino acid sequences in
the cytoplasmic domains of P-selectin from different species. The numbers for amino acid positions correspond to the human
sequence. Residues in other species that are identical to the human
counterpart are marked with a dash. The cytoplasmic domain
from each species has 35 amino acids. The sequence data are from the
following references: human(47) , murine(48) ,
rat(49) , and bovine (50) . The ovine sequence has been
deposited in GenBank with the accession number L34270 (S.
A. Burns, E. J. Neufeld, and J. J. Donady, unpublished data). The
canine sequence has been deposited in GenBank
with the
accession number M88170 (A. M. Manning, W. E. Sanders, Jr., G. L.
Kukielka, M. Dore, C. L. Rosenbloom, H. L. Hawkins, L. H. Michael, M.
L. Entman, C. W. Smith, A. L. Beaudet, and D. C. Anderson, unpublished
data).
In this study, we examined the effects of a variety of mutations and deletions in the cytoplasmic domain on the endocytosis of P-selectin in CHO cells. We identified no obvious short internalization signals. Indeed, a truncated construct that has no tyrosine-, di-leucine-, or KKFF-type signals mediated rapid internalization. Residues throughout the cytoplasmic domain contribute to the efficiency of internalization.
In the assay, confluent CHO cells in 12-well plates
were placed on ice for 5 min and washed four times with prechilled
medium (-minimum Eagle's medium, 1% FBS). Then 500 µl of
medium containing 1.5 µg/ml
I-G1 were added to each
well. After 30 min on ice, the cells were washed rapidly four times
with ice-cold medium. To each of two wells, 500 µl of prewarmed 0.1 M acetate, 0.15 M NaCl, pH 4, were added, whereas to
each of the other wells 500 µl of prewarmed medium were added. The
plates were then transferred immediately to a 37 °C water bath
where they were floated without their lids. At 1-min intervals, the
medium containing the released
I-G1 was collected from a
well and replaced with 500 µl of 0.1 M acetate buffer,
0.15 M NaCl, pH 4. After an additional 10-min incubation at 37
°C, the acetate buffer was removed, and the cells were solubilized
with 1 ml of 1 N NH
OH.
In each experiment, the
total I-G1 bound at time 0 was determined as the sum of
the radioactivity of cell lysate (acid-resistant bound
I-G1), medium (spontaneous release of bound
I-G1), and acid eluate (original surface-bound
I-G1). At each subsequent time, the amount of
radioactivity released or internalized was plotted as the percentage of
the total radioactivity bound to the surface at time 0. The amount
remaining on the cell surface was obtained by subtracting the sum of
the released and internalized radioactivity from the initial
surface-bound radioactivity. Spontaneous release of bound
I-G1 was less than 10% of the total radioactivity. In
each analysis, the initial rate of internalization was calculated by
measuring the maximal slope of the uptake curve. This usually followed
a short lag of 15-30 s. In some constructs the lag period
extended up to 2 min, and the lag period in the N782A mutant was
4-6 min. When a mutant construct was analyzed, the wild-type form
was also included in the experiment. The internalization rate of each
mutant was examined in at least two different clones. At least two
separate experiments were performed with each clone, except for the
C766A/H773Stop mutant where one clone was examined four times.
For each time point, half of the cells were treated with 10
mM sodium 2-mercaptoethanesulfonate (MESNA) (Sigma) in 50
mM Tris, 100 mM NaCl, pH 8.5. The other half was
treated with the Tris buffer without MESNA. The solutions were
incubated with the cells for 30 min on ice. The MESNA solutions were
replaced with fresh solution at 10-min intervals. After three washes
with 1 ml of 5 mg/ml iodoacetamide in HBSS, the cells were lysed with
250 µl/well of 1% Triton X-100 in 100 mM sodium phosphate,
pH 8, containing 0.3 unit/ml aprotinin, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml pepstatin, and 5 mg/ml
iodoacetamide. Following centrifugation of each lysate at 10,000
g, the supernatant was transferred to a new
microcentrifuge tube and stored at 4 °C until analysis.
The
amount of internalized biotinylated P-selectin protected from MESNA
cleavage was quantitated by an ELISA consisting of a preclearance step
and a detection step. In the preclearance step, cell lysates were added
to microtiter wells coated with T10, a mAb directed to the platelet
In the detection step, the
precleared lysates were transferred to microtiter wells coated with a
mixture of mAbs to P-selectin or, as a control, to wells coated with
T10. Wells of Immulon I plates (Dynatech Laboratories Inc., Chantilly,
VA) were coated overnight at 4 °C with 100-µl solutions
containing 15 µg/ml T10 or 5 µg/ml each of S12, W40, and G1 in
sodium carbonate buffer, pH 9.2. After two washes with 20 mM Tris-HCl, 140 mM NaCl, 1.5 mM CaCl
,
pH 7.4 (TBS), the coated wells were blocked with 0.1 M sodium
phosphate buffer, pH 8.0, 1% Triton X-100, 1% globulin-free BSA for 2 h
at 37 °C (4 h for the detection wells), and then rinsed with TBS,
1% Triton X-100. Quadruplicate 50-µl aliquots of cell lysate were
added to the preclearance wells. After 2 h at 37 °C, 50 µl of
0.1 M sodium phosphate, pH 8, 1% Triton X-100, 1%
globulin-free BSA were added to each well. Duplicate aliquots of the
diluted cell lysates were transferred to the S12-, W40-, and G1-coated
detection wells or to the control T10-coated detection wells. After 45
min at 37 °C, the contents were aspirated, and the wells were
rinsed five times with TBS, 1% Triton X-100. The immobilized
biotinylated P-selectin was quantitated with an ELISA amplification
system (Life Technologies, Inc.). Briefly, 100 µl of 1 µg/ml
steptavidin alkaline phosphatase were added for 30 min at room
temperature. After two washes with TBS, 1% Triton X-100, and three
washes with TBS, 50 µl of NADPH were added to each well for 15 min
at room temperature. Then, 50 µl of reconstituted amplifier
solution containing alcohol dehydrogenase and diaphorase were added.
After 15 min at room temperature, the reaction was stopped by the
addition of 50 µl of 0.3 M H
SO
.
The absorbence of the product was read at 495 nm on a V
kinetic microplate reader interfaced to
Softmax software (Molecular Devices, Palo Alto, CA).
To establish the linear range of detection for the ELISA, serial dilutions of cell lysate were assayed from biotinylated cells that were not incubated at 37 °C or treated with MESNA. Comparable dilutions were used for the experimental wells; absorbence values within the linear range of the assay were then corrected for the dilution factor. The absorbence values of the control T10-detection wells were less than 10% of the corresponding samples in the S12-, G1-, and W40-coated wells and were subtracted to obtain the specific P-selectin-dependent signals. The specificity of the assay was further determined by demonstrating that preincubation of the detection wells with 2 µg/ml purified platelet P-selectin (55) completely eliminated the specific signal. At each time point, the amount of internalized, MESNA-resistant P-selectin was expressed as a percentage of that detected on cells at time 0 that were not treated with MESNA. Preliminary experiments indicated that the amount of biotinylated P-selectin in untreated cells did not change after incubation of the cells for 10 min at 37 °C. At time 0, treatment with MESNA removed 90-95% of the biotinylated cell surface P-selectin.
In the experiment comparing the internalization rate of wild-type P-selectin with the LDL receptor and the chimeric LLP protein, the biotinylation assay was performed as described previously(46) .
A modified assay was used to
examine the effect of cell acidification on internalization of
P-selectin. This assay exploited the ability of mAb S12 to remain bound
to P-selectin in acidic buffers. Preliminary studies demonstrated that I-S12 bound identically to transfected cells at pH 7.4
and 5.2. Surface-bound S12 was removed by incubating the cells with
Pronase (Calbiochem). Cells in 24-well plates were placed on ice and
washed four times with chilled Ringer's solution containing 1%
FBS.
I-S12 (1 µg/ml in 250 µl of Ringer's
solution, 1% FBS in the presence or absence of 10 mM acetic
acid) was added to each well for 30 min on ice. After four washes with
chilled Ringer's solution, 1% FBS, plates containing one set of
cells were transferred to a 37 °C water bath without their lids,
whereas the other set remained on ice. After 10 min, the cells at 37
°C were returned to ice and washed once with chilled Ringer's
solution, 1% FBS. Both sets of cells were then washed four times with
Ringer's solution without FBS. Half of each set of cells was
treated with 0.3% (w/v) Pronase in Ringer's solution; the other
half was treated with Ringer's solution without Pronase. After 1
h on ice, the solutions were removed, and their radioactivity was
counted in a gamma scintillation counter.
After correcting the
radioactivity of solution containing Pronase (SP) from that in the
absence of Pronase obtained from each series of cells, the percentage
of internalized P-selectin was calculated as: SP (4 °C) - SP
(37 °C)/SP (4 °C) 100%.
Figure 2:
Amino
acid sequences of the cytoplasmic domains of human P-selectin
constructs. The 35 residues of the wild-type cytoplasmic domain have
been assigned to the ST, C1, and C2 domains according to exon-intron
boundaries. The name of each construct is listed at the left of its
schematic diagram. Internal deletions of the indicated residues are
preceded by a . The nomenclature for point mutations is as
follows: S788Stop means that the codon for Ser-778 is replaced by a
stop codon; Y777A means that the codon for Tyr-777 is replaced by a
codon for alanine.
To study the endocytosis of P-selectin independently of its sorting into secretory granules, we used CHO cells, which lack the regulated secretory pathway. In transfected CHO cells, wild-type P-selectin is primarily distributed on the plasma membrane and in endosomes(44, 46) . We expressed in these cells a series of constructs with various mutations and deletions in the cytoplasmic domain of P-selectin (Fig. 2). All constructs contained the extracellular and transmembrane domains of P-selectin. To quantitate the rate of internalization of P-selectin in CHO cells, we modified a method used to study rapid internalization of the mannose 6-phosphate/insulin-like growth factor-II receptor(8) . Cells were incubated at 4 °C with G1, a mAb to P-selectin that is rapidly dissociated from its ligand when exposed to acidic buffers. Following a wash, the cells were quickly warmed to 37 °C to initiate internalization of P-selectin with its bound antibody. At 1-min intervals, the medium was collected to measure spontaneously released antibody, and the cells were incubated with acetate buffer at pH 4 to remove surface-bound antibody. The cells were then lysed to measure the radioactivity of internalized G1 that was protected from the acid-dissociation procedure.
As measured by this assay, wild-type P-selectin was rapidly internalized, reaching a plateau 10 min after warming the cells to 37 °C (Fig. 3A). In contrast, a tail-less construct terminating at Lys-761 was internalized extremely slowly during this period (Fig. 3B). Spontaneous release of bound G1 from both cell lines was low, indicating that the differences in internalization rates were not due to different off-rates for the antibody. There was no increase in trichloroacetic acid-soluble radioactivity in the medium from either cell line during the incubation period, indicating that the antibody was not degraded. Consistent internalization rates were measured for each construct over a 10-20-fold range of expression levels (data not shown).
Figure 3:
Internalization of P-selectin measured by
removal of surface-bound antibody on transfected CHO cells. The
internalization of wild-type (A) or tail-less (B)
P-selectin was measured by the susceptibility of I-labeled mAb G1 to removal with acidic buffer as
described under ``Experimental Procedures.'' The amount of
radioactivity released or internalized was plotted as the percentage of
the total radioactivity initially bound to the surface. The
radioactivity remaining on the cell surface was obtained by subtracting
at each point the sum of the released and internalized radioactivity
from the initial surface-bound radioactivity. Each point represents the
mean of three determinations in A and three determinations in B.
To ensure that binding of antibody did not induce the rapid internalization of P-selectin, we developed an alternative assay in which cell surface proteins were modified at 4 °C with sulfo-NHS-ss-biotin, which can be cleaved with the membrane-impermeant reducing agent, MESNA(53, 54, 63) . At intervals following warming to 37 °C, the cells were returned to ice and incubated with or without MESNA. An ELISA was used to calculate the percentage of internalized biotinylated P-selectin that was resistant to MESNA, relative to the initial surface-biotinylated P-selectin. As measured by this assay, wild-type P-selectin was also rapidly internalized during the first 10 min after warming the cells to 37 °C (Fig. 4). In contrast, the tail-less construct remained primarily on the cell surface. Thus, binding of antibody did not induce the rapid internalization of P-selectin. We routinely used the antibody-binding assay because it was simpler to perform. The internalization rate was determined by measuring the slope of the initial linear portion of the curve, as described under ``Experimental Procedures.''
Figure 4: Internalization of P-selectin measured by removal of surface-bound biotin on transfected CHO cells. Cells were treated with sulfo-NHS-ss-biotin at 4 °C, washed, and then incubated at 37 °C for the indicated times. The cells were then treated with MESNA, lysed, and assayed for biotinylated P-selectin by ELISA as described under ``Experimental Procedures.'' At each time point, the amount of internalized, MESNA-resistant P-selectin was expressed as a percentage of that detected on cells at time 0 that were not treated with MESNA. The data represent the mean ± S.E. of six determinations.
We first tested a
construct with an internal deletion of C1, D762-S772, which fused
the C2 region directly to the ST segment. This construct was
internalized at
90% of the rate of wild-type P-selectin (Fig. 5A). However, a construct in which a stop codon
was substituted for Asn-782 in
D762-S772 was endocytosed at only
30% the rate of wild-type. These data indicate that, in the absence of
C1, the C2 domain mediates internalization, and both halves of C2 are
required for its optimal function.
Figure 5:
Effect of deletions in the cytoplasmic
domain on the internalization of P-selectin. A, effect of
internal deletion of the C1 domain on internalization; B,
effect of C-terminal deletions of the C2 domain on internalization. The
internalization rate of each construct was measured by determining the
slope of the initial linear upward slope of the internalization curve
(example in Fig. 3) as described under ``Experimental
Procedures.'' Each bar represents the mean ± S.E. of 55
determinations with three different clones for wild-type P-selectin, 12
determinations with two different clones for D762-S772, eight
determinations with two different clones for
D762-S772/N782Stop,
24 determinations with three different clones for tail-less, and at
least four determinations with two different clones for each C-terminal
deletion construct.
To examine the role of the C2
region in the context of the entire cytoplasmic domain, we measured the
endocytosis of a series of constructs with C-terminal deletions of
increasing size. Unexpectedly, deletions of C2, including all three
aromatic residues, had little or no effect on the internalization rate (Fig. 5B). A construct lacking the entire C2 region,
H773Stop, was still internalized at 50% the rate of wild-type
P-selectin. Deletion of an additional six residues (P767Stop)
eliminated internalization. Thus, in the absence of C2, the C1 domain
also mediates internalization, and both halves of C1 are required for
its optimal function. The collective results indicate that either C1 or
C2 is capable of mediating endocytosis when fused to the ST segment.
However, the data do not establish whether C1 and C2 contribute
independent internalization signals within the intact cytoplasmic
domain. It should be noted that the sum of the internalization rates of
the C1 (H773Stop) and C2 (
D762-S772) constructs is higher than
that of wild-type P-selectin.
Figure 6: Effect of mutations in the cytoplasmic domain of a construct lacking C2 on internalization of P-selectin. Each bar represents the mean ± S.E. of at least four determinations with two different clones, except for the C766A/H773Stop construct in which four independent determinations were performed on one clone.
Figure 7: Effect of mutations in the intact cytoplasmic domain on internalization of P-selectin. A, mutations in the C1 region; B, mutations in the C2 region. Each bar represents the mean ± S.E. of at least four determinations with two different clones.
We also mutated each residue in C2 from Tyr-777 to Phe-785 within the context of the entire cytoplasmic domain (Fig. 7B). Mutation of Gly-778 to alanine within the putative YGVF motif markedly increased internalization. This result is consistent with previous observations that glycine is poorly tolerated at the second position of four-residue tyrosine-based internalization signals(8) . However, mutation of Phe-780 to alanine also increased internalization; this result is not consistent with the requirement for a hydrophobic residue at the fourth position of tyrosine-based signals(8) . Furthermore, mutation of Asn-782 or Phe-785 significantly inhibited internalization (Fig. 7B), even though deletion of sequences containing these residues had little or no effect on internalization (Fig. 5B).
The data in Fig. 7indicate that the effects of mutations of individual residues in the context of the entire cytoplasmic domain are not always consistent with the effects produced by C-terminal deletions or by point mutations in the context of a cytoplasmic domain lacking the C2 region. Overall, the data do not clearly implicate a specific short sequence as the sole internalization signal. However, the results do suggest that many residues in the cytoplasmic domain contribute to optimal internalization of P-selectin. To further address this possibility, we substituted the seven-residue ST sequence of P-selectin with the six membrane-proximal residues of human L-selectin(65) . This construct was internalized at less than half the rate of wild-type P-selectin, even though the sequences in the C1 and C2 regions remained intact (Fig. 8).
Figure 8: Effect of substituting the seven residues in the ST domain of P-selectin with the six membrane-proximal residues of L-selectin on internalization of P-selectin. The bar for the ST/L-selectin construct represents the mean ± S.E. of seven determinations with two different clones.
Figure 9: The cytoplasmic domain of P-selectin mediates more rapid internalization when attached to the extracellular and transmembrane domains of the LDL receptor than when attached to the corresponding domains of P-selectin. A biotinylation assay was used to compare the internalization rates of wild-type P-selectin, the LDL receptor (LDL-R), and a chimeric protein in which the cytoplasmic domain of LDL-R was was replaced with the cytoplasmic domain of P-selectin (LLP).
The cytoplasm of CHO cells transfected with various P-selectin constructs was acidified by incubation in medium containing acetic acid, which lowered the pH to 5.2. Because this pH affected the binding of mAb G1, we developed an alternative assay in which mAb S12, which binds efficiently to P-selectin at acidic pH, was incubated with cells and then removed with Pronase. The cumulative percentage of internalized P-selectin was measured over a 10-min period. The results of this assay indicated that cytoplasmic acidification markedly inhibited internalization of wild-type P-selectin, of the constructs containing either the C1 or the C2 domains, and of the constructs with point mutations that significantly accelerated the rate of internalization (Fig. 10A). Using the conventional G1-binding assay, we found that incubation of cells in hypertonic medium also prevented the internalization of all forms of P-selectin (Fig. 10B). These data suggest that P-selectin and the examined P-selectin constructs are internalized in clathrin-coated pits.
Figure 10: Effect of cytoplasmic acidification and hypertonic medium on internalization of P-selectin constructs. Transfected CHO cells were treated with neutral or acidic medium (A) or isotonic or hypertonic medium (B). Internalization of the indicated protein after a 10-min incubation at 37 °C was measured with the modified mAb S12 binding assay in A or with the standard mAb G1 binding assay in B, as described under ``Experimental Procedures.'' Each bar represents the mean ± S.E. of at least three experiments.
The cytoplasmic domain of P-selectin contains a signal(s) that mediates rapid endocytosis in clathrin-coated pits. Our results indicate that many residues in the cytoplasmic domain affect, directly or indirectly, the internalization of P-selectin. Deletions or substitutions throughout the cytoplasmic domain either increased or decreased the internalization rate. Constructs with either the C1 domain or the C2 domain fused directly to the ST region mediated internalization in clathrin-coated pits. Although the simplest interpretation of this result is that C1 and C2 each contains an independent internalization signal, the conformations of C1 and C2 in the deletion constructs may be very different from those in the intact cytoplasmic tail. Indeed, the sum of the internalization rates of the deletion constructs exceeded that of wild-type P-selectin. Thus, the data do not establish the presence of independent signals in C1 and C2 in the context of the entire cytoplasmic domain.
The construct containing only the ST and C1 domains (H773Stop) has no di-leucine-based motifs (17) or KKXX-type signals(29, 30, 32, 33) . It has only one aromatic residue, Phe-758, and substitution of this residue with alanine did not affect the internalization rate. Replacement of Lys-761 with alanine in the H773Stop construct resulted in an internalization rate nearly three times that of wild-type P-selectin, similar to the rapid internalization rates of the transferrin and LDL receptors(1, 2) . Therefore, a cytoplasmic sequence lacking any of the described short cytoplasmic signals can mediate very rapid internalization in clathrin-coated pits.
The C2 domain has three aromatic residues within the sequence YGVFTNAAF. The YGVF sequence resembles canonical four-residue tyrosine-based internalization signals where an aromatic residue is separated from a hydrophobic residue by two other amino acids(1, 2, 8) . The YGFV sequence mediates rapid internalization when attached to a truncated cytoplasmic domain of the mannose 6-phosphate/insulin-like growth factor-II receptor(8) . In contrast, the function of this motif is less clear in the intact cytoplasmic domain of P-selectin, because mutation of Phe-780 to alanine actually increased the internalization rate. The NAAF sequence has slight similarity to the NPXY motif described in the LDL receptor(3, 64) . Consistent with the possible importance of this sequence, mutation of either Asn-782 or Phe-785 significantly decreased the internalization rate. In marked contrast, deletion of the NAAF sequence had little effect on internalization. Thus, the mutagenesis studies do not clearly define a short internalization signal based on the aromatic amino acids.
Despite analyzing many constructs, we were unable to assign
internalization signals to specific short amino acid sequences in the
cytoplasmic domain of P-selectin. Our data contrast with many reports
of short tyrosine-, di-leucine-, or C-terminal KKFF-like sequences that
function as signals for internalization and other sorting
functions(1, 2, 29, 30, 32, 33) .
These studies also relied on measurements of internalization or sorting
of membrane proteins that were subjected to site-directed mutagenesis.
It has been suggested that diverse short linear sequences form a family
of related structures that interact with sorting
proteins(1, 2, 67) . Perhaps the cytoplasmic
domain of P-selectin has a short linear signal that is not readily
identified because many other residues contribute to its appropriate
orientation. Alternatively, an internalization motif might be created
by juxtaposition of residues from discontinuous portions of the amino
acid sequence. Even putative linear sorting motifs in other proteins
require further structural definition. Analysis by nuclear magnetic
resonance indicates that some small peptides with tyrosine-based motifs
form turns in aqueous solutions(23, 24) .
However, one peptide containing a tyrosine-based internalization motif
has been shown to form a nascent helix instead of a
turn in
solution(68) . The structure of an amino acid sequence may
differ depending on whether it is studied as an isolated small peptide
or as part of a larger peptide corresponding to the entire cytoplasmic
domain(68) . Furthermore, some linear peptides adopt stable
structures only when bound to a membrane or to a macromolecular
ligand(69, 70) . It should be emphasized that the
actual structures of cytoplasmic domains that contact the membrane
surface, adaptins, or other sorting molecules have not been identified.
Determination of these sites will require analysis of interacting
proteins by co-crystallization and x-ray diffraction or by nuclear
magnetic resonance.
The structural features in the cytoplasmic domain of P-selectin that mediate sorting to secretory granules and movement from endosomes to lysosomes are also unknown. Although deletion of the C1 region eliminates endosomal sorting with little affect on the internalization rate(46) , it is unclear whether C1 presents an endosomal sorting signal in the intact cytoplasmic tail. Mutations in the cytoplasmic domain that differentially affect internalization, sorting to secretory granules, and delivery to lysosomes will indicate that the sorting signals have different structures, but will not clarify the details of the structures.
We found that the cytoplasmic domain of P-selectin mediated more rapid internalization when attached to the extracellular and transmembrane domains of the LDL receptor (the LLP construct) than when attached to the corresponding domains of P-selectin. Since the LDL receptor is not internalized in the absence of its own cytoplasmic domain(64) , the faster internalization of LLP relative to wild-type P-selectin may be due to a different presentation of the P-selectin cytoplasmic tail. The LDL receptor forms oligomers in the membrane, but only in the presence of its own cytoplasmic domain; there is no clear correlation of oligomerization with the rate of internalization(71) . The transmembrane domain of P-selectin promotes its oligomerization in solution, even in nonionic detergents above the critical micellar concentration(72) . It is not known whether P-selectin also oligomerizes in the cell membrane and, if so, whether the oligomers affects the orientation and sorting functions of the cytoplasmic domain. LLP and P-selectin have similar half-lives in CHO cells(46) , but the assay used for these measurements might not distinguish minor differences in endosomal sorting.
We conclude that site-directed mutagenesis does not always identify a short sorting signal in the cytoplasmic domain of a membrane protein. Our data indicate that many residues in the cytoplasmic domain of P-selectin contribute to internalization. The highly conserved sequence of the cytoplasmic domain of P-selectin may be required to create a structure that mediates not only internalization, but also sorting into regulated secretory granules and rapid movement from endosomes to lysosomes.