From the Medical Research Council Laboratory for Molecular Cell Biology and Department of Biochemistry, University College London, Gower Street, London WC1E 6BT, United Kingdom
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ABSTRACT |
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Signals controlling the intracellular targeting of many membrane proteins are present as short sequences within their cytoplasmic domains. P-selectin is a type I membrane protein receptor for leukocytes, acting during the inflammation response. Heterologous expression experiments have demonstrated that its 35-residue cytoplasmic tail contains signals for targeting to synaptic-like microvesicles, dense-cored granules, and lysosomes. We have examined the lysosomal targeting information present within the cytoplasmic tail by site-directed mutagenesis of horseradish peroxidase-P-selectin chimeras followed by transient transfection in H.Ep.2 cells. Assaying lysosomal targeting by subcellular fractionation as well as intracellular proteolysis, we have discovered a novel lysosomal targeting signal, KCPL, located within the C1 domain of the cytoplasmic tail. Alanine substitution of this tetrapeptide reduced lysosomal targeting to the level of a tailless horseradish peroxidase-P-selectin chimera, which was previously found to be deficient in both internalization and delivery to lysosomes. A proline residue within this lysosomal targeting signal makes a major contribution to the efficiency of lysosomal targeting. A diaminobenzidine density shift procedure established that chimeras with an inactivated KCPL sequence are present within transferrin-positive compartments. Such a mutant also displays an increased level of expression at the plasma membrane. Our results indicate that the sequence KCPL within the cytoplasmic tail of P-selectin is a structural element that mediates sorting from endosomes to lysosomes.
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INTRODUCTION |
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A substantial body of data indicates that post-Golgi sorting of
integral membrane proteins is largely dependent upon information contained within the cytoplasmic domains of these proteins (for review,
see References 1-4). This information is usually found in short
stretches of amino acids, "sorting signals," which serve to direct
proteins to a variety of intracellular destinations. The most common
signals can be classified into two groups: Tyr-based and di-Leu
signals. Conformational modeling and two-dimensional nuclear magnetic
resonance spectroscopy (NMR) of peptides encoding these motifs have
revealed that the first group, typically conforming to
Asn-Pro-X-Tyr or Tyr-X-X-Ø sequences
(where X is any amino acid and Ø is a bulky hydrophobic
amino acid), form a "tight turn" (5-7), whereas di-Leu or Leu-Ile
motifs may display an extended, random coil conformation within the
cytoplasmic domain of that protein (8). More recently, a novel category
of targeting signals that lie within an -helix (9, 10) has been
defined. In contrast, other sorting signals are centered on strongly
hydrophilic sequences, comprising clusters of acidic residues (Glu and
Asp) functioning either as independent determinants or in concert with
Tyr-based or di-Leu motifs (11, 12).
The type I membrane protein P-selectin belongs to a family of adhesion molecules. It initiates leukocyte recruitment during the inflammatory response and is involved in hemostasis (13-16). Found in the membrane of regulated secretory organelles of platelets and endothelial cells (16-18), it is redistributed to the plasma membrane after stimulation of these cells with agonists (14, 19, 20). When expressed in cells lacking a regulated secretory pathway, P-selectin is constitutively transported to the cell surface (21-23). After appearance at the plasma membrane, it is rapidly internalized (20-22, 24, 25) and passes through the endocytic pathway to its final destination(s) (25, 26). When expressed in Chinese hamster ovary and PC12 cells, P-selectin is efficiently targeted to lysosomes, and its cytoplasmic tail comprising the C1 and C2 domains is both necessary and sufficient for this trafficking step mediated by the putative lysosomal targeting signal within the C1 domain (21).
Much data derived from a variety of systems suggest that LTS1 and internalization signals often overlap or are co-linear, both for proteins biosynthetically targeted to lysosomes such as resident lysosomal membrane proteins as well as for membrane proteins capable of efficient lysosomal targeting during receptor-mediated endocytosis (see Table I and references cited therein). One interesting exception is the EGFR, in which mutations inactivating lysosomal targeting were found not to affect internalization (27-29). Thus, although an overlap is often evident, there is no simple correlation between internalization and lysosomal targeting, implying that the structural requirements for interacting with the cytoplasmic machinery which facilitates these two trafficking events may be complex.
Since the short cytoplasmic tail of P-selectin has been found to contain a variety of signals for targeting to organelles along the regulated secretory pathway as well as on the endocytic pathway (21, 25, 26), distinguishing different signals from each other is a complex problem. We therefore chose to study targeting in a system that lacks some of the possible destinations. We have begun by analyzing P-selectin targeting to late endocytic compartments in cells in which the endocytic pathway has been extensively characterized (30-34) and that lack regulated secretory organelles.
In this study, we examined the effects of mutations within the cytoplasmic tail on lysosomal targeting of P-selectin in H.Ep.2 cells, which lack regulated secretory organelles. We have uncovered a novel short LTS, KCPL, which is located within the C1 domain and which has no obvious homology to LTSs identified in other proteins. The proline residue within this LTS significantly contributes to the efficiency of lysosomal sorting.
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EXPERIMENTAL PROCEDURES |
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Materials and Reagents-- Rabbit polyclonal anti-HRP was from DAKO, Glostrup, Denmark. Fab fragments of this antibody were generated using a commercial ImmunoPure® Fab preparation kit (Pierce) according to the manufacturer's instructions. Mouse receptor-grade epidermal growth factor (EGF) and human, iron-saturated transferrin (Trn) were purchased from Sigma. Iodination of Fab fragments, EGF, and Trn has been performed according to the modified IODO-GEN method as described elsewhere (35). The specific activity of preparations were: 3 × 104, 1 × 105, and 2 × 104 cpm/ng, respectively. Protein concentration was determined using Coomassie Plus protein assay reagent (Pierce) according to the manufacturer's instructions. Other chemicals were from Sigma and Pharmacia Biotech Inc.
Constructs--
A chimeric cDNA containing the human growth
hormone signal sequence, followed by HRP, and finally the transmembrane
domain and cytoplasmic tail of P-selectin (Fig. 1) was generated as
described previously (26). cDNAs encoding the deletion mutants,
ssHRPP-selectin776, ssHRPP-selectin763, and
ssHRPP-selectinC1, were constructed as detailed in this
report. Both the point mutation of proline 767 and the tetra-alanine
changes were made using the Stratagene QuikChange site-directed
mutagenesis kit. Briefly, cesium chloride-purified pRK34 plasmid
containing the ssHRPP-selectin was added to two
complementary synthetic oligonucleotide primers which contained the
change of interest. After temperature cycling with Pfu DNA
polymerase, a mutated plasmid containing a staggered nick was
generated. The parental DNA was then digested away with DpnI
restriction endonuclease, which is active only on methylated and
hemimethylated DNA. The nicked mutated vector was transformed into
supercompetent Escherichia coli. The sequences generated were confirmed by sequencing. The sequences of the oligonucleotide primers used to generate the mutants are listed below. Only the sense
primer is shown; the antisense primer used is the exact complement.
Cell Culture and Transfection-- The human cell line H.Ep.2 was cultured and transiently transfected as described elsewhere (36). Cells were plated on 150- and 35-mm dishes and in some experiments were grown for 24 h in presence of 100 µM each of pepstatin A and leupeptin prior to use.
HRP Proteolysis Assay-- Cells on 35-mm dishes were placed on ice, washed twice with ice-cold PBS, and subjected to Triton X-114 partitioning as described (37) followed by a standard HRP assay (see below) performed with two phases. Amounts of HRP activity present in the upper, hydrophilic and lower, hydrophobic phases were used to determine the percentage of clipped chimera as a ratio of HRP activity in the upper phase to the total activity in the lysate. To normalize for inter-experimental variations, we subtracted the fraction of HRP proteolysis of tailless ssHRPP-selectin763 chimera (typically about 25%) as background from each data set, since this chimera has been demonstrated previously to be incapable of internalization and, consequently, of delivery to lysosomes instead accumulating on the plasma membrane (26).
Cell Surface Expression Assay--
Cells on 35-mm dishes were
washed with growth medium, placed on ice, and incubated with 2 µg/ml
125I-Fab fragments in growth medium at 4 °C for 1 h. Unbound ligand was removed by three rinses with growth medium. Cells
were then washed with ice-cold PBS and lysed in 1 ml of HRP assaying
buffer (100 mM sodium citrate, pH 5.5, 0.1% Triton X-100)
at 4 °C for 10 min. Lysates were counted in a -counter (Hewlett
Packard) and then spun at 13,000 × g for 5 min in a
refrigerated centrifuge to pellet cell debris. Supernatants were used
for measurement of HRP activity (see below). Cell surface expression
levels were determined as a ratio of the amount of 125I-Fab
fragments to the level of HRP activity for each chimera, to normalize
for variation of expression level. The ratio for ssHRPP-selectin was set at 1 for all experiments, and the
data from all other chimeras have been normalized accordingly.
Endocytosis Assays-- Cells on 150-mm dishes were washed with binding medium (Dulbecco's modified Eagle's medium, 20 mM HEPES, 0.1% bovine serum albumin) and incubated with either 5 ng/ml 125I-EGF at 4 °C for 1 h or 100 ng/ml 125I-Trn at 37 °C for 1 h. Excess ligand was removed by three rinses with cold Dulbecco's modified Eagle's medium. The cells loaded with 125I-Trn were placed on ice and subsequently treated with MES buffer I (25 mM MES, pH 4.0, 150 mM NaCl) and MES buffer II (25 mM MES, pH 7.5, 150 mM NaCl) at 4 °C for 10 min each to remove any cell surface-bound ligand. The cells labeled with 125I-EGF were allowed to internalize ligand for 30 min at 37 °C, followed by a 2.5-min mild acid wash (100 mM sodium acetate, pH 4.5, 500 mM NaCl) at 4 °C to extract 125I-EGF present on the plasma membrane. In both cases, the cells were then thoroughly washed with ice-cold binding medium, washed twice with HB (320 mM sucrose, 10 mM HEPES, pH 7.3), and subjected to subcellular fractionation as detailed below.
Subcellular Fractionation and Quantitation of
Data--
Following two rinses with ice-cold HB, cells were scraped
into 1.5 ml of HB with a rubber policeman and passed 10 times through a
ball bearing homogenizer with a 0.009-mm clearance (made at EMBL,
Heidelberg, Germany). The cell homogenate was spun at
8,500 × g for 5 min and 1.3 ml of postnuclear
supernatant (PNS) was layered on an 11-ml 1-16% preformed linear
Ficoll gradient made in HB. The gradients were centrifuged for 45 min
at 35 ,00 rpm in a SW40Ti rotor (Beckman Instruments, Palo Alto, CA)
and then fractionated in 0.5-ml fractions from the top of the tube
using an Autodensi-Flow IIC (Buchler Instruments, Kansas City, MO), and
radioactivity of fractions was counted. Positions of lysosomes and late
endosomes were identified by measurement of activity of the lysosomal
marker enzyme, N-acetyl--D-glucosaminidase
(NAGA), as described previously (38).
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(Eq. 1) |
DAB Cytochemistry-- A modification of the procedure, originally developed by Courtoy et al. (39) was used. A 4.5 mM solution of DAB in HB was prepared, adjusted to pH 7.3 with 1 N NaOH, and filtered through an 0.22-µm filter (Millipore Corp.). PNS or gradient fractions (700 µl) were mixed with 800 µl of DAB solution and 8 µl of 6% H2O2 and incubated for 15 min at room temperature in the dark. A 700-µl control sample from the same PNS or Ficoll fraction was incubated in parallel with 800 µl of HB alone. Subsequently, the mixtures (1.5 ml) were layered on Ficoll gradients made with HB supplemented with 1 mM imidazole. When the DAB reaction was performed with PNS, the samples were centrifuged on 1-16% Ficoll gradients. Ficoll fractions subjected to DAB reaction were recentrifuged on secondary Ficoll gradients; 7-25% Ficoll gradients were used to analyze the LE/Lys fractions, and 3-16% Ficoll gradients were used for analysis of plasma-membrane vesicles labeled by 125I-EGF.
To quantitate the degree of the density shift of each ligand, the amount of radioactivity remaining within the peak of ligand distribution as a consequence of DAB cytochemistry was divided by the radioactivity in the endosomal peak in the samples not incubated with DAB, expressed in percentages and subtracted from 100%.HRP Assay-- HRP activity was assayed in triplicate as described previously (26).
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RESULTS |
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HRP-P-selectin Chimeras Are Sorted to the Late Endosomes and Lysosomes in H.Ep.2 Cells-- It has been demonstrated previously that the C1 domain of the cytoplasmic tail of P-selectin is involved in the targeting of this protein to lysosomes (21). To further characterize the signal(s) involved in lysosomal targeting, a series of chimeras between P-selectin and the enzymatic reporter HRP (Fig. 1) were constructed, and their lysosomal trafficking in H.Ep.2 cells following transient expression was analyzed. These chimeras contain the transmembrane and cytoplasmic domains of human P-selectin with the lumenal portion replaced by HRP and can be followed through the cell by their enzymatic activity.
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Effect of Different Mutations within the C1 Domain of the Cytoplasmic Tail on Targeting to LE/Lys-- To quantitate the targeting to LE/Lys of wild-type and mutant ssHRPP-selectin chimeras, cDNAs encoding the various chimeras (Fig. 1) were transiently transfected into H.Ep.2 cells. To make sure that the data on targeting for different mutants were comparable, we determined the expression level for each chimera as a ratio of HRP activity to milligrams of total protein. In two independent experiments (Fig. 5A), the greatest difference between expression levels of any mutant and that of ssHRPP-selectin was 3.215 ± 0.025 and 2.17 ± 0.05 (± S.E.).
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Contribution of Proline 767 to Lysosomal Targeting--
The
sequence KCPL is different from motifs known to be involved in sorting
or thought to be critical for internalization. However, it contains a
proline (number 767 within the human sequence; Ref. 15) followed by
leucine. This Pro-Leu pairing is present within tyrosine-based sorting
signals mediating the transport of CD3 to lysosomes (46) and of
HLA-DMb to antigen-processing, lysosome-like compartments (47). In
addition, proline has been identified as a key amino acid within
NPXY-based motifs required for internalization (5). We
have therefore engineered a chimera in which proline 767 is
replaced by alanine; ssHRPP-selectinP767A.
Subcellular fractionation demonstrated that the efficiency of lysosomal
targeting of this mutant was about 35% of the level of
ssHRPP-selectin and was only 2-fold higher then that for
ssHRPP-selectinKCPL (Fig. 5B), suggesting that
proline does indeed make a major contribution to lysosomal targeting.
Surprisingly, this mutation did not result in a dramatic effect on HRP
proteolysis since the clipping of ssHRPP-selectinP767A was
only half that of ssHRPP-selectin (Fig. 5C).
These data suggest the possibility of differential targeting and
degradation within the late endocytic system.
Cell Surface Expression of HRP-P-selectin Chimeras--
Since
we had established that ssHRPP-selectinC1 and
ssHRPP-selectinKCPL are not sorted to LE/Lys, we next
asked where in the cell these chimeras reside. One possibility was that
mutants which failed to be targeted to the LE/Lys would enter a
recycling pathway between the plasma membrane and an early endocytic
compartment. This would be in agreement with previous experiments
suggesting that ssHRPPselectin
C1 co-localizes with the
classic marker for a recycling itinerary, the transferrin receptor,
when expressed in PC12 cells (26). One prediction for such behavior was
that a significant proportion of the mutant chimeras should appear on
the plasma membrane during recycling. The level on the surface should
be intermediate between a protein that is efficiently targeted to
lysosomes, e.g. ssHRPP-selectin and a protein
that fails to be internalized, e.g.
ssHRPP-selectin763. To determine whether this is the case,
we employed an 125I-Fab anti-HRP binding assay to
detect the chimeras on the cell surface. As seen in Fig.
7,
ssHRPP-selectin763 showed the highest level at
the plasma membrane, twice that of ssHRPP-selectin.
Analysis of the other chimeras revealed that
ssHRPP-selectin
C1, ssHRPP-selectinKCPL, and
ssHRPP-selectinP767A displayed an intermediate level
between ssHRPP-selectin and
ssHRPP-selectin763, whereas
ssHRPP-selectinNPHS and
ssHRPP-selectinKDDG were present at the same level as
ssHRPP-selectin. Since substitution of KCPL, deletion of
the C1 domain, or substitution for proline 767 did not affect or had
only a little effect on internalization rates
(24),2 the appearance of
these chimeras at the plasma membrane strongly suggests an increased
pool of recycling protein.
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ssHRPP-selectinC1 and
ssHRPP-selectinKCPL Are Localized in Trn-positive
Endosomes--
The recycling pathway of H.Ep.2 cells has previously
been characterized in detail, and Trn has been found to be the best
marker of this itinerary (31, 32, 41). To determine whether
ssHRPP-selectin
C1, ssHRPP-selectinKCPL, and
Trn are present within the same endosomes, cells expressing ssHRPP-selectin, ssHRPP-selectin
C1, or
ssHRPP-selectinKCPL were loaded with 125I-Trn,
treated with MES buffer to remove the plasma membrane-bound ligand, and
then the co-localization of HRP activity and iodinated ligand was
examined by exploiting the DAB-induced density shift procedure. PNS
obtained from these cells was split in two equal parts. One of them was
incubated with DAB and H2O2, and the another one with HB alone. Samples were layered on 1-16% Ficoll gradients and
centrifuged to equilibrium as described under "Experimental Procedures." In control cells expressing ssHRPP-selectin,
the DAB reaction shifted 24% of the total 125I-Trn
radioactivity, whereas in cells expressing
ssHRPP-selectin
C1 or
ssHRPP-selectinKCPL, significantly larger amounts
of ligand were shifted: 54% and 52%, respectively. Based on these
data, we assume that ssHRPP-selectin moves transiently
through the Trn-positive endosomes en route to the lysosome.
Importantly, both ssHRPP-selectin
C1 and
ssHRPP-selectinKCPL reveal significant co-localization with
Trn, strongly suggesting a recycling itinerary for these chimeras.
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DISCUSSION |
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In the present work, we have identified a novel LTS in the cytoplasmic tail of P-selectin. Green et al. (21) had found previously that a putative LTS of P-selectin is located within the membrane-proximal C1 domain of the cytoplasmic portion of this protein, the amino acid sequence of which is shown in Fig. 1. Using site-directed mutagenesis, we have engineered a series of tetrapeptide substitutions within HRP-P-selectin chimeras to further characterize lysosomal targeting information within the C1 domain. The assays introduced in the present study have allowed us to quantify even minor differences in endosomal/lysosomal sorting. Summarizing the results of both lysosomal targeting and HRP proteolysis experiments, we have uncovered a novel determinant within the C1 domain of P-selectin: KCPL. This LTS appears to be the sole element of the cytoplasmic tail necessary for lysosomal targeting since neither the disruption of the two other tetrapeptides within the C1 domain, KDDG and NPHS, nor the removal of the C2 domain affected lysosomal targeting. Whether this sequence is also sufficient for lysosomal targeting has not yet been directly established. Carrying out such experiments may not provide simple answers, since by placing it into another context, KCPL may well operate differently, as has been observed elsewhere. For example, the transfer of the Leu-Ile signal of LIMP2 to CD36 and CD8 plasma membrane proteins did not induce diversion of these proteins to lysosomes (8). In addition, the spacing of an LTS relative to the transmembrane domain in proteins possessing a short cytoplasmic tail has been found to be critical for proper functioning of the signal (45).
The HRP proteolysis data are more complex than those obtained by subcellular fractionation, since we observed some difference between ssHRPP-selectin and mutants in which KCPL has not been inactivated. We suspect that this may reflect some differential targeting between subcompartments of the late endocytic pathway. It is striking, however, that mutation of KCPL to tetra-alanine resulted in levels of HRP proteolysis similar to those seen for tailless ssHRPP-selectin763, strongly suggesting that KCPL contains all the information needed to accomplish the lysosomal delivery of P-selectin.
The effect of tetra-alanine substitution of KCPL was very marked:
lysosomal targeting and HRP clipping were reduced by 7- and
15-fold, respectively, of the levels found for
ssHRPP-selectin (Fig. 3). By contrast,
inactivation of the LTSs of other transmembrane proteins and receptors
caused no more than a 3-fold reduction of targeting compared with that
of the intact protein, suggesting that additional sequences may be
involved in those cases (9, 27, 29, 43). The existence of multiple
lysosomal sorting signals has indeed been demonstrated for and
chains of CD3 (48), for the invariant chain (Ii) of class II major
histocompatibility complex (49) and EGFR (27-29). In principal, by
acting in concert, multiple LTSs could provide for a targeting activity
equivalent to that of KCPL within P-selectin.
Many LTSs also operate to mediate internalization at the plasma
membrane, as is the case for the interleukin-6 signal transducer gp130
(50), LAMP1 (45, 51, 52), Ii (49), lysosomal acid phosphatase (53, 54),
LIMP2 (8), and CD 3 and
chains (48). These data indicate that
in most cases the LTS and internalization signal are co-linear (see
Table I). The relationship between internalization and lysosomal targeting is generally acknowledged, although its functional significance remains to be investigated. One of
the sources of confusion is that there is no simple correlation between
internalization and degradation rates for proteins targeting to
lysosomes via the plasma membrane (9, 27-29, 43, 45). Thus, whether
the multiple signals function to increase the overall efficiency of
targeting by acting together at one rate-limiting step or operate at
different stages is not clear.
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Does KCPL operate at more than one step in directing the protein to late endocytic compartments? A detailed search for internalization signals in the cytoplasmic tail of P-selectin did not succeed in identifying any potential motifs despite the presence of an obvious candidate sequence within the C2 domain (YGVFTNAAF) that provides a good match with known internalization signals. Instead, mutation of most amino acids throughout the cytoplasmic tail affects the efficiency of internalization (24). This finding, coupled to internalization experiments using HRP-P-selectin chimeras2 firmly supports the view that KCPL is a signal that mediates endosome-to-lysosome trafficking without affecting internalization.
As illustrated in Table I, the LTS of P-selectin, KCPL, has no obvious similarity to a large number of lysosomal targeting determinants, most of which also act as internalization signals in other proteins. Our data also indicate that the proline 767 provides a major contribution to the LTS. Interestingly, proline located within sorting signals that center on the NPXY motif was shown to be a key amino acid, thought to be involved in stabilizing the predicted "tight turn" (1, 5), whereas in other signals it does not seem to be important.
P-selectin has been shown previously to be efficiently endocytosed in many cell lines (20, 24, 25). If inactivation of KCPL has no effect on the internalization ability of this chimera but prevents degradation, then where in the cell is HRPP-selectinKCPL likely to accumulate?
In H.Ep.2 cells, recycling and lysosomally directed tracers have been found to be internalized into the same compartment (31, 33, 34) where their sorting occurs. These endosomes were morphologically identified as MVBs (30, 32-34). The MVBs undergo a gradual maturation process involving removal of recycling receptors such as Trn receptor (TrnR), and retention of lysosomally directed proteins such as EGFR. By contrast, subsequent transfer of endocytosed proteins from late or mature endosomes to lysosomes is believed to be a discontinuous process requiring heterotypic fusion between fully matured but LAMP1-negative MVB and pre-existing LAMP1-positive, acid hydrolase active lysosomes (32, 55).
Our findings suggest that KCPL is most likely to operate where the segregation of recycling and degradative pathways occurs. Electron microscopy revealed that, during the maturation of MVBs, EGFR are recruited from the outer membrane of MVB onto internal vesicles while TrnR remains on the perimeter membrane (34, 56). This recruitment onto inner vesicles is critical for the lysosomal degradation of the EGFR. Felder et al. (27) have documented that the tyrosine kinase of the EGFR controls sorting of internalized receptor through spatial segregation within the MVB. Thus, wild-type EGFR was shown to be recruited to internal vesicles of MVBs and subsequently degraded, whereas kinase-negative receptor underwent increased recycling and was localized to the outer membrane of the MVBs followed by removal to small tubulovesicles (27).
We speculate therefore that KCPL promotes the retention of P-selectin
within the maturing MVBs, possibly by promoting inclusion in inwardly
budding vesicles along with the EGFR. When KCPL-dependent retention within MVBs is abolished, then P-selectin normally destined for degradation is likely to be diverted to the recycling pathway which
may occur by default (4, 57). This scheme is supported by experiments
(Fig. 8) showing that the level of
ssHRPP-selectinKCPL on the plasma membrane is greater than
that of ssHRPP-selectin, as would be expected if the mutant
protein efficiently recycles. Moreover, DAB cytochemistry experiments
in the present work demonstrate that, although the wild-type
HRP-P-selectin chimera is found within 125I-EGF-containing
endosomes and lysosomes, the distribution of ssHRPP-selectinKCPL and ssHRPP-selectinC1
overlapped with 125I-TrnR-containing compartments. Other
studies have also revealed increased levels of recycling for an EGFR
lacking a degradation signal (29), as well as for a LAMP1 mutant in
which the LTS was displaced within the cytoplasmic tail (45).
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The data obtained in the present study coupled with published observations from other laboratories demonstrate an abundance of different sorting signals involved in lysosomal targeting. What are the reasons for such a heterogeneity? One possibility is the need to provide for the different physiological and signaling functions of proteins routed to lysosomes. For example, some receptors of hormones, growth factors, and cytokines utilize the degradative pathway to desensitize the cell during down-regulation. In these cases, rapid clearance of these proteins may require efficient internalization and degradation. P-selectin transiently appearing on the plasma membrane of activated endothelial cells during inflammatory response was also found to be quickly cleared (58). A prolonged appearance on the plasma membrane might lead to over-recruitment of leukocytes with a subsequent uncontrolled inflammation. A similar rapid trafficking has been observed in nonendothelial cells in which P-selectin is rapidly transported to the plasma membrane and then efficiently delivered to lysosomes for degradation (21-23). On the other hand, resident lysosomal membrane proteins, traveling from the plasma membrane to their final destination, may not have the same need for rapid clearance or degradation. Such different trafficking requirements would explain the variety of signals, but, clearly, further studies will be needed to investigate the basis for LTS diversity.
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ACKNOWLEDGEMENTS |
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We thank Prof. C. R. Hopkins, Dr. C. E. Futter, Dr. M. Marsh, Dr. S. Moss, Dr. K. Römisch, Dr. E. Hewitt, and Dr. M. Arribas for discussion and critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by a Wellcome Trust fellowship (to A. B.).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. Tel.: 44-171-380-7808;
Fax: 44-171-380-7805; E-mail: d.cutler{at}ucl.ac.uk.
1
The abbreviations used are: LTS, lysosomal
targeting signal; EGF, epidermal growth factor; EGFR, EGF receptor;
HRP, horseradish peroxidase; Trn, transferrin; TrnR, Trn receptor; PBS,
phosphate-buffered saline; MES, 4-morpholineethanesulfonic acid; PNS,
postnuclear supernatant; Lys, lysosome; NAGA,
N-acetyl--D-glucosaminidase; LTI, lysosomal
targeting index; LE, late endosome; DAB, 3,3
-diaminoBenzidine; MVB,
multivesicular bodies.
2 A. D. Blagoveshchenskaya and D. F. Cutler, unpublished observations.
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REFERENCES |
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