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INTRODUCTION |
The 46-kDa mannose 6-phosphate receptor
(MPR46)1 mediates the
transport of newly synthesized soluble lysosomal enzymes from the
trans-Golgi network (TGN) to the endosomal-prelysosomal
compartment. Following the pH-induced dissociation of the complexes,
the MPR either return to the TGN or undergo cycling via the plasma
membrane (1). Targeting information contained within the 67-residue cytoplasmic domain of this type I integral membrane glycoprotein is
responsible for the directed intracellular transport of MPR46 between
TGN, endosomes, and the plasma membrane as well as for retention from
lysosomal delivery. Mutational analyses have identified several signals
including a tyrosine (Tyr45-Arg-Gly-Val48) and
dileucine-based motif (Leu64-Leu65), and two
aromatic residues (Phe13 and Phe18)
required for efficient sorting in the TGN or rapid internalization at
the plasma membrane (2-4). Additionally, the di-aromatic
Phe18-Trp19 motif and the phosphorylation of
Ser57 have been reported to be important for endosomal
sorting (5, 6). Finally, Cys34 has been shown to be a
reversible palmitoylated residue that might anchor the tail to the
lipid layer and form a cytoplasmic loop (7). Both the intact
palmitoylation site and the correct length of the loop are critical to
prevent targeting of MPR46 to degrading compartments (7, 8).
The signal structures in the cytoplasmic tail of the MPR46 form
selective recognition sites for cytosolic proteins facilitating the
incorporation of the receptors into transport vesicles. The best
studied class of these cytosolic proteins comprises the family of
adaptor proteins (AP; Ref. 9). AP-1 and AP-2 are heterotetramers comprising two large ~100-kDa chains (
1 and
-adaptin for AP-1;
2 and
-adaptin for AP-2), a medium subunit of ~50 kDa (µ1 and µ2) and a small polypeptide of ~20 kDa (
1 and
2). AP-1 is
involved in protein sorting at the TGN, whereas AP-2 mediates
endocytosis from the plasma membrane. Recently, heterotetrameric AP-3
and AP-4 complexes have been identified (10, 11), which are proposed to
be involved in sorting at the TGN and endosomal membranes. The AP-3
complex containing a µ3A isoform is ubiquitously expressed, whereas
µ3B is a component of the neuronal-specific AP-3 variant. Biosensor
analysis has demonstrated the presence of several distinct binding
sites for AP-1 and AP-2 in the cytoplasmic tail of the MPR46, which do
not depend on dileucine signals (12). Using the yeast two-hybrid
system, µ1, µ2, and µ3 chains have been demonstrated to interact
with triple repeat sequences of tyrosine-containing sorting signals of
several integral membrane proteins (13). However, the composition of
residues surrounding the tyrosine residue and the position of the
tyrosine motif within the tail relative to the membrane appear to be
important in determining µ chain specificity (13, 14). The subunit of
the adaptor complexes responsible for recognition of the dileucine
motifs is a matter of debate (13, 15, 16).
In the present study we have examined the interaction of the entire
cytoplasmic tail of the MPR46 (MPR46-CT) and various tail mutants with
µ2 and µ3A using the yeast two-hybrid system. The results have
revealed that both µ2 and µ3A bind to the MPR46-CT. The
interactions are specific and depend on the presence of a complex
configuration of different signals comprising not only the tyrosine and
dileucine motif but also acidic amino acid residues localized between
these motifs.
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MATERIALS AND METHODS |
[35S]Methionine and the prestained Rainbow protein
marker were from Amersham Pharmacia Biotech Europe (Freiburg, Germany).
Oligonucleotide primers for PCR were synthesized by NAPS
(Göttingen, Germany). The following reagents were obtained
commercially as indicated: restriction enzymes and T4 DNA ligase (New
England Biolabs, Schwalbach, Germany); glutathione-agarose (Sigma);
Pfu and Taq DNA polymerase and the
Quick®Change site-directed mutagenesis kit (Stratagene
Cloning System, La Jolla, CA); TNT® coupled reticulocyte
lysate system (Promega Corp., Madison, WI).
Vectors--
The Gal4-DNA binding domain vector pAS2 and the
Gal4-activation domain vector pGAD424 were purchased from
CLONTECH Laboratories (Heidelberg, Germany). The
bacterial expression vector pGEX-4T-1 came from Amersham Pharmacia
Biotech Europe and pSPUTK was obtained from Stratagene.
Yeast and Bacterial Strains--
Yeast strains for two-hybrid
system (MATCHMAKER) were purchased from CLONTECH.
The yeast strain HF7c was used for cotransformation of the Gal4
constructs and SFY526 was used for
-galactosidase assays. The
Escherichia coli strains of DH5
and BL-21 were obtained from CLONTECH and Amersham Pharmacia Biotech
Europe, respectively.
Plasmids--
The mouse and rat medium chain-2 (µ2) and -3A
(µ3A) were kindly provided by Dr. Peter Schu (University of
Göttingen, Göttingen, Germany) and Dr. Margret Robinson
(University of Cambridge, Cambridge, United Kingdom), respectively.
Two-hybrid Plasmid Constructions--
The cDNAs of wild type
cytoplasmic tails of human MPR46 (MPR46-CT; Ref. 17) and human MPR300
(MPR300-CT; Ref. 18) were ligated in frame with the Gal4BD into the
BamHI/PstI-sites of pAS2. Truncations of the
MPR46-CT were generated by PCR introducing TGA stop codons at the
respective sites using the following oligonucleotide primers:
MPR46-CT-StopH63, ST63-F (5'-CGGGATCCCGCAGCGACTGGTAGTGGGA-3') and
ST63-R (5'-CGGGATCCCGTCAATGGTCATCCCTTTCTT-3'); MPR46- CTStopV48, ST63-F and ST48-R (5'-CGGGATCCCGTCACACACCACGATATGCTGC-3'); MPR46-CT StopA44, ST63-F and ST44-R (5'-CGGGATCCCGTCATGCTGCAGGCACATTTCG-3'); MPR46-CT
1-16, ST1-16-F (5'-CGGGATCCCGGCCTTCTGGCAGGATCTT-3') and
ST1-16-R (5'-CGGGATCCCGCTACATTGGTAATAAATG-3'). The polymerase chain
products were cloned in frame with the Gal4-DBD into the BamHI site of pAS2. Point mutations were generated by means
of the QuickChangeTM site-directed mutagenesis kit (Stratagene)
according to the manufacturer's protocol with the following
oligonucleotide probes: MPR46-CT-E55A/E56A, E55-F
(5'-GATGACCAGCTGGGGGCGGCGTCAGAAGAAAGGGAT-3') and E55-R
(5'-CATCCCTTTCTTCTGACGCCGCCCCCAGCTGGTCATC-3'). The following mutations,
MPR46-CT-E55A/E56A/E58A/E59A and
MPR46-CT-E55A/E56A/E58A/E59A/D61A/D62A, were introduced successively
using the mutagenized MPR46-CT-cDNA as a template with
primers: MPR46-CT-E55A/E56A/E58A/E59A, E58-F (5'-GGGGAGGAGTCAGCAGCAAGGGATGACCAT-3') and E58-R
(5'-ATGGTCATCCCTTGCTGCTGACTCCTCCCC-3'); MPR46-CT-E55A/E56A/E58A/E59A/D61A/D62A, D61-F
(5'-TCAGCAGCAAGGGCTGCCCATTTATTACCA-3') and D61-R
(5'-TGGTAATAAATGGGCAGCCCTTGCTGCTGA-3'). The mutation MPR46-CT-Y45A/V48A
was introduced using the wild type MPR46-CT cDNA as template with
primers Y45-F (5'-GTGCCTGCAGCAGCTCGTGGTGCGGGGGATGACCAG-3') and Y45-R
(5'-CTGGTCATCCCCCGCACCACGAGCTGCTGCAGGCAC-3'). For the MPR46-CT-StopH63-Y45A/V48A mutation, the MPR46-CT-StopH63 cDNA was
used as template with primers Y45-F and Y45-R. All mutations were
verified by sequencing the final products. The full coding regions of
µ2 and µ3A were amplified by PCR using the following oligonucleotide primers: µ2-F (5'-GAAGATCTTCATGATCGGAGGCTTATTCAT-3') and µ2-R (5'
GAAGATCTTCCTAGCAGCGGGTTTCATAAA-3'), µ3A-F
(5'
GGAATTCATGATCCACAGTCTATTTC-3') and µ3A-R
(5'
CGGGATCCCGTCATGTCCTCACTTGGAA-3'). The µ2 and µ3A PCR
products were digested with BglII and
EcoRI/BamHI, respectively, and cloned in frame
with the Gal4AD into the corresponding restriction sites of the GAL4
activation domain vector pGAD424.
Two-hybrid Analysis--
The yeast reporter strain HF7c was
cotransformed with wild type and mutant pAS2-MPR46-CT or
pAS2-MPR300-CT-constructs and the pGAD424-µ2 and -µ3A constructs,
respectively, using a lithium acetate-base method. To examine
specificity of the interaction, HF7c cells were cotransformed with the
plasmids pAS2 (Gal4DBD alone) and pAS2 lamin and the pGAD424-µ2 and
-µ3A constructs. The double transformants were grown on SD agar
medium lacking Trp, Leu, and His (+ 5 mM
3-amino-1,2,4-triazole, Sigma) for 5 days at 30 °C before positive
colonies were picked, restreaked onto triple minus plates, and assayed
for the lacZ phenotype.
Production and Purification of GST Fusion Proteins--
GST
fusion constructs were made by ligation of wild type MPR46-CT-cDNA
into the EcoRI- and SalI- restriction sites of
the vector pGEX-4T-1 (Amersham Pharmacia Biotech Europe). The
MPR300-CT-cDNA (amino acids 1-164) was amplified by polymerase
chain reaction using PfuTurboTM DNA polymerase
(Stratagene) with primers MPR300CT-F (5'-GGAATTCCGCAAGAAGAAGAGGAGGGAAACAGTG-3') and MPR300CT-R
(5'-GGAATTCCTCAGATGTGTAAGAGGTC-3'). The polymerase chain reaction
product was ligated into the EcoRI site of pGEX-4T-1. For
GST-MPR46-CT fusion protein expression, E. coli BL-21 cells
were transformed with the pGEX-4T-1-MPR46-CT construct and grown to an
A600 of 0.6 followed by induction of GST fusion
protein expression with isopropyl-1-thio-
-D-galactoside at a final concentration of 0.1 mM. After 4 h of
additional growth at 37 °C, E. coli BL-21 cells were
pelleted and lysed by sonification in 10 mM
phosphate-buffered saline, pH 7.4 (PBS), containing 1 mg/ml lysozyme,
25% (w/v) sucrose, 1 mM EDTA, 1% Triton X-100, and 1 mM phenylmethylsulfonyl fluoride. After centrifugation for 10 min at 10,000 × g, the supernatant was mixed with 1 ml (50% v/v) glutathione-agarose (Sigma), equilibrated in PBS,
incubated for 1 h at 4 °C, and washed three times with 20 bead
volumes of PBS containing 1% Triton X-100 and 1 mM
phenylmethylsulfonyl fluoride. The fusion protein was eluted with 50 mM Tris-HCl, pH 8.0, containing 15 mM reduced
glutathione. Eluted proteins were dialyzed overnight against PBS and
stored in aliquots at
70 °C. For MPR300-CT fusion protein
expression, E. coli BL21 cells were grown to an
A600 of 0.1 and induced with 0.2 mM
isopropyl-1-thio-
-D-galactopyranoside for 3 h at
30 °C (19). Lysis of E. coli BL21 cells, affinity purification, and elution of MPR300-CT fusion proteins were carried out
as described above.
In Vitro Translation of µ2 and µ3A--
The µ2 and µ3A
cDNA was subcloned into Bg/II restriction site of the
pSPUTK vector. The pSP64-luciferase construct was obtained from
Promega. In vitro translation was carried out by using the TNT® coupled reticulocyte lysate system in the presence of
40 µCi of [35S]methionine in a final volume of 50 µl
according to the manufacturer's instructions. 35S-Labeled,
in vitro translated products were centrifuged at
100,000 × g for 5 min to remove insoluble materials.
In Vitro Binding Assay--
The GST-MPR46-CT and GST-MPR300-CT
fusion proteins (25 µg) were incubated with the
35S-labeled µ2, µ3A, or luciferase translation product
(5 µl) at 4 °C for 2 h in 0.5 ml of binding buffer (50 mM HEPES, pH 7.5, containing 150 mM KCl, 10 mM MgCl2, 10% (v/v) glycerol, 1% (v/v) Triton
X-100, and 0.2% bovine serum albumin) followed by the addition of 50 µl (50% v/v) glutathione-agarose in binding buffer for 2 h at
4 °C. The beads were washed three times with binding buffer without
bovine serum albumin. The bound proteins were extracted by boiling in
SDS-sample buffer, separated by SDS-polyacrylamide gel electrophoresis
(12.5% acrylamide), and analyzed by phosphorimaging (Fujix BAS 1000, Fuji Photo Co., Japan).
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RESULTS |
Analysis of the Interaction of Wild Type MPR46 Cytoplasmic Tail
with µ2 and µ3A Subunits--
Reporter yeast cells were first
transformed with a plasmid encoding a fusion protein between the wild
type MPR46-CT or MPR300-CT and the Gal4 DNA binding domain
(pAS2-MPR46/300-CT). The strain was subsequently transformed with a
plasmid encoding the µ2 or µ3A adaptor subunit fused to the
transcriptional activation domain of Gal4 (pGAD424-µ2 or -µ3A).
After an incubation period of 5 days in the presence of
3-amino-1,2,4-triazole (5 mM), the cells expressing the
MPR46-CT fusion protein with either µ2 or µ3A exhibited a strong
growth in a medium lacking histidine, and positive galactosidase activity (Fig. 1). The MPR300-CT failed
to interact both with µ2 and µ3A.

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Fig. 1.
Interaction of MPR-CT with
µ2 and µ3A. Wild type
cytoplasmic tail of MPR46 or MPR300 fused to the Gal4 DNA
binding domain (pAS2) were coexpressed in HF7c cells with µ2 or µ3A
fused to the complementary Gal4 activation domain (pGAD424). The cells
were grown for 5 days at 30 °C in the presence of 5 mM
3-AT on plates with (+His) or without histidine
( His) and tested for expression of -galactosidase
activity (blue color).
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The MPR46-CT Binds Directly to the µ2 and µ3A Subunit in
Vitro--
To confirm the results obtained with the two-hybrid system,
the interaction of in vitro translated
[35S]methionine-labeled µ2 and µ3A with MPR46-CT and
MPR300-CT expressed and purified as GST fusion protein was examined.
Densitometric evaluation of the coprecipitated µ2 subunit and
correction by unspecific binding to GST alone revealed that about 4.3%
of total µ2 was specifically recovered on glutathione-agarose beads
(Fig. 2A). About 17% of the
total µ3A was coprecipitated with the MPR46-CT fusion protein,
whereas the in vitro translated
[35S]methionine labeled luciferase used as a control was
not. In coprecipitation experiments with the MPR300-CT fusion protein, 4.2% of total µ2 but almost no µ3A (1%) was recovered (Fig.
2B).

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Fig. 2.
Binding of in vitro
translated µ2 and
µ3A to wild type MPR46-CT and MPR300-CT.
A, equimolar amounts (0.7 nmol) of GST or wild type
GST-MPR46-CT fusion protein were incubated either with
35S-labeled µ2 (lanes 2 and
3), luciferase (lane 5), or µ3A
(lanes 7 and 8) for 2 h at
4 °C following absorption to glutathione-agarose beads for 1 h.
After washing the beads, the bound radioactive proteins were detected
by SDS-polyacrylamide gel electrophoresis and autoradiography. One
fifth of the total amount of the in vitro-translated µ2
(lane 1), luciferase (lane
4), and µ3A (lane 6) were applied on
the gel for comparison. B, GST or wild type GST-MPR300-CT
fusion protein (25 µg) were incubated with 35S-labeled
µ2 (lanes 10 and 11) or µ3A
(lanes 13 and 14) as described above.
Ten percent of the total amount of in vitro translated µ2
(lane 9) and µ3A (lane
12) was applied on the gel for comparison. Bound µ2
(lanes 3 and 11) or µ3A
(lanes 8 and 14) was quantified using
a phosphorimager and expressed as percentage of total µ2 or µ3A,
respectively. Binding was corrected by the unspecific binding of
35S-labeled µ2 (lanes 2 and
10) or µ3A (lanes 7 and
13) to GST, respectively. Autoradiograms of one
representative experiment out of three are shown.
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MPR46-CT Signal Structures Required for µ2 Interaction--
To
define the specificity and the site responsible for the interaction
with µ2 in more detail, several plasmids encoding mutant forms of
MPR46-CT (Fig. 3) and control constructs
were cotransformed with the pGAD424-µ2-plasmid. The N-terminal
deleted MPR46-CT
1-16 and the C-terminal truncated
MPR46-CT-StopH63, which lacks the dileucine motif, caused growth on
histidine-free medium and activation of the lacZ reporter
gene similar to the wild type MPR46-CT (Fig. 3; Table
I). Further C-terminal truncation
resulting in cytoplasmic domains still containing the tyrosine-based
signal (MPR46-CT-StopV48) or lacking both the dileucine and tyrosine
motives (MPR46-CT-StopA44) reduced the interaction to 25 and 4%,
respectively, of wild type MPR46-CT. The replacement of
Tyr45 and Val48 by alanine in the entire
cytoplasmic tail (MPR46-CT-Y45A/V48A) decreased
-galactosidase
activity to 75% of the wild type tail. When these two residues were
replaced in addition to the deletion of the dileucine motif
(MPR46-CT-StopH63-Y45A/V48A), the
-galactosidase activity was
reduced to 60% of the wild type tail. The inability of cells
cotransformed with Gal4-binding domain alone, lamin C, and µ2, to
grow in the absence of histidine (Fig. 3) or to activate
-galactosidase (Table I) confirmed the specificity in the
interaction between MPR46-CT and µ2. The lack of
-galactosidase
activity in cells coexpressing the wild type MPR300-CT fusion protein
and µ2 corresponded to the failure to grow on histidine-free medium (Fig. 1).

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Fig. 3.
Interaction of various mutant MPR46-CT
with µ2. A, schematic
illustration of wild type MPR46 cytoplasmic tail. The Gal4-DBD
(black) is N-terminally fused to the MPR46-CT construct
shown as single-letter code amino acid sequence.
The numbering starts with Q following the transmembrane domain
according to Johnson and Kornfeld (3). The tyrosine-based and the
dileucine motifs (gray box) as well as the
cluster of acidic amino acid residues (underlined) are
indicated. B, yeast cells were cotransformed with the
indicated pAS2 plasmids and plasmids expressing the pGAD424 fused to
µ2. The ability to grow on plates lacking histidine
( His) for 5 days was tested.
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Table I
Interaction between pGAD424-µ2 and various pAS2-Gal4 DBD fusions
determined by a liquid quantitative -galactosidase assay
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Interaction of Mutant MPR46-CT with µ3A--
When the various
MPR46-CT mutants were coexpressed with µ3A in SFY 526 cells,
quantification of the interaction measured by lacZ reporter
gene activity revealed that the deletion of the dileucine motif
(MPR46-CT StopH63) reduced
-galactosidase activity by 42% (Fig.
4). The MPR46-CT StopV48 and StopA44
mutants as well as the alanine substitution (Y45A/V48A) in the entire
or in the truncated receptor tail (StopH63-Y45A/V48A) interacted weakly with µ3A (10-27% of
-galactosidase activity of the wild type MPR46-CT). The N-terminal deleted mutant MPR46-CT
1-16 activated
-galactosidase similarly to the wild type tail. In control cells coexpressing lamin C, no interaction with µ3A was observed (Fig. 4).

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Fig. 4.
Specificity of the interaction between
µ3A with wild type and mutant MPR46-CT.
A, SFY 526 cells were transformed with the pAS2 plasmids
encoding the indicated Gal4-DBD fusions and with the pGAD424-µ3A
plasmid. The ability to grow on plates with (+His) or
without histidine ( His) is shown. B, after an
incubation for 3 days, -galactosidase activity was tested in a
filter assay. C, subsequently, five independent clones were
selected and grown to A600 values of 0.8 and
-galactosidase activity was measured using chlorophenol
red- -D-galactopyranoside as a substrate.
-Galactosidase activities in relative units are presented as mean
values ± S.D. (bars).
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The Cluster of Acidic Amino Acid Residues in MPR46-CT Contributes
to µ2 and µ3A Interaction--
To determine whether the cluster of
acidic amino acids (residues 50-62) is responsible for the reduced
binding of mutant MPR46-CT StopV48 to µ2 and µ3A in comparison with
the StopH63 mutant as well as for residual adaptor subunit binding in
the StopH63-Y45A/V48A mutant, substitutions of acidic residues in the
context of the entire tail were tested (Fig.
5). The coexpression of the mutant MPR46-CT containing double substitution of Glu55,
Glu56, and the quadruple substitution of Glu55,
Glu56, Glu58, and Glu59 by alanine
with µ2 reduced
-galactosidase activity by 22 and 31%,
respectively (Fig. 5C). These mutations impaired the
interaction with µ3A more strongly (20 and 18% of
-galactosidase
activity of the wild type MPR46-CT). The substitution of
Glu55, Glu56, Glu58,
Glu59, Asp61, and Asp62 by alanine
reduced the interaction both with µ2 and µ3A to 26 and 5%,
respectively, of wild type MPR46-CT.

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Fig. 5.
Medium adaptor subunit binding to MPR46-CT
mutated in its acidic amino acid cluster. A, schematic
representation of the C terminus of the Gal4-DBD MPR46-CT construct
comprising the acidic amino acid cluster (underlined). The
name of each mutant is listed on the left indicating which
amino acid has been changed to alanine (boldface).
B, two-hybrid analysis of yeast cells expressing Gal4-DBD
fusion proteins containing MPR46-CT mutants and the Gal4 activation
domain fusion proteins containing µ2 (left half of the
plate) or µ3A (right half of the plate) adaptor subunits.
The cells were grown for 5 days at 30 °C on plates with
(+His) or without histidine ( His).
C, -galactosidase activities were measured in six
selected independent clones grown to A600 values
of 0.8. -Galactosidase activities in relative units are presented as
mean values ± S.D. (bars).
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 |
DISCUSSION |
Studies using the yeast two-hybrid system have established that
tyrosine-based sorting signals in cytoplasmic domains of membrane receptors are important for the interaction with µ1, µ2, and µ3 subunits of adaptor complexes AP-1, -2, and-3, respectively (10, 13,
14). In the present study, we have examined the full-length cytoplasmic
tail of the MPR46 containing several independent signal structures
including tyrosine- and dileucine-based motifs (residues 45-48 and
residues 64 and 65, respectively), aromatic residues (residues 13, 18, and 19), and clusters of acidic amino acids (residues 50-62) for its
capability to interact with medium chains of AP-2 and AP-3 in the yeast
two-hybrid system. Here we report that the tyrosine motif and a distal
cluster of acidic amino acid residues but not the dileucine signal
within the MPR46 tail are sufficient for effective binding to µ2. In
contrast, the interaction of the receptor tail with µ3A depends on a
complex C-terminal structure comprising tyrosine and dileucine motifs
as well as a strong hydrophilic sequence.
The phenylalanine residues 13 and 18 as well as the tyrosine-based
motif 45YRGV48 have been shown to mediate the
internalization of the MPR46 from the cell surface (3). Whereas the
former signal overlaps with the specific endosomal sorting signal
18FW19, the tyrosine motif has the
characteristics of the consensus motif YXX
(where Y is
tyrosine, X is any amino acid, and
is a bulky
hydrophobic amino acid) which can be found within the cytoplasmic
domains of many endocytic transmembrane proteins recognizing µ2 (13,
20). Recently, crystallization studies of µ2 complexed with
tyrosine-containing internalization signal peptides have revealed that
the peptides are bound to an extended conformation with separate
pockets for both Y and
residues (21). Consistent with the
prediction that the dileucine signals, which are characteristically surrounded by polar and/or charged residues, will not be able to bind
to the hydrophobic pocket structure of µ2 (21), the dileucine signals
of CD 3
have been proposed to interact with
2 adaptor subunits
(16). However, surface plasmon resonance data have demonstrated the
binding of dileucine-based sorting signals within the cytoplasmic tail
of the major histocompatibility complex class II invariant chain to the
µ2 chain of AP-2 (18). Here we show that the dileucine motif of the
MPR46 tail is not critical for the interaction with µ2 in agreement
with studies testing the binding of AP-2 to MPR46 tail peptides (15).
Furthermore, the present data with C-terminal truncated MPR46 tails or
with substitution of Tyr45 and Val48 by alanine
in the context of the entire tail showed that, in addition to the
tyrosine motif, a second signal in the stretch of amino acids 49-63 is
required for efficient µ2 binding. Indeed, this study demonstrates
that the substitution of six acidic residues within the compact cluster
of acidic amino acids in this region (50DDQLGEESEERDD62)
prevents the interaction with µ2. The positive electrostatic surface
potential near the YXX
binding site of µ2 (21) may support this interaction. Additionally, the acidic residues may also be
important in vivo for the binding of the MPR46-CT to µ2 because these amino acids are part of a casein kinase-2 phosphorylation site at Ser57 (23). Since the nonphosphorylated MPR46 are
not cycled via the plasma membrane (6), these receptors should fail to
interact with AP2. Both a tyrosine-based motif and a cluster of acidic residues have also been reported to function as independent signals within the cytoplasmic tail of furin to mediate TGN localization and
internalization from the cell surface (24, 25). Residues 2-16 of the
MPR46 tail, which have been reported to affect AP-2 binding (12), are
not important for MPR46 tail binding to the µ2 chain, suggesting that
these juxtamembrane residues may be involved in the binding of other
AP-2 subunits. In contrast to the coprecipitation of µ2 with the
MPR300-CT, this interaction could not be demonstrated in the two-hybrid
assay. The reason for these discrepancies is unclear.
AP-3 has been shown to be involved in a clathrin-independent transport
of membrane proteins to lysosomes in mammalian cells or to the vacuole
in yeast, and to lysosome-related storage granules arising from the
endocytic pathway like melanosomes in melanocytes, synaptic vesicles in
neurons, as well as synaptic-like microvesicles in neuroendocrine cells
(26-30). Using the yeast two-hybrid system, tyrosine-based sorting
motifs YXX
of LAMP I and CD63 have been shown to interact
with µ3A and µ3B chains (10, 27) whereas in the cytoplasmic tails
of LIMP II, tyrosinase, and vacuolar alkaline phosphatase or Vam3p,
dileucine residues and acidic clusters were found to be important for
AP-3 binding (26, 28, 31). Interestingly, we show for the first time
the requirement for multiple C-terminal motifs within the cytoplasmic
tail of the MPR46 comprising tyrosine- and dileucine-based signals, and
clustered acidic amino acid residues to interact efficiently with
µ3A. Quantification of µ3A interactions with various MPR46 tails by
measurement of lacZ reporter gene activities revealed that
the different signals were not additive but appeared to function in a
cooperative manner. In addition, the data showed that the acidic
residues Glu55, Glu56, Glu58, and
Glu59 are more critical for efficient binding to µ3A than
to µ2. At least two signals located in the cytoplasmic tail of LIMP
II, tyrosinase and synaptotagmin, have also been reported to be
critical for correct intracellular sorting (28, 30, 32). It is likely that the structural distance between the tyrosine- and dileucine-based motifs and the acidic cluster in the cytoplasmic tail of MPR300 as well
as their position with respect to the membrane (33, 34) compared with
the MPR46 tail may be responsible for the failure of the MPR300 tail to
bind to µ3A.
Our present data are in contradiction to studies in which the MPR46
tail peptide immobilized on a sensor surface fails to bind AP-3
complexes (28). However, similar discrepancies have been reported for
the µ3A/AP-3 binding to the LAMP I tail (27, 28). The different
results obtained with our two-hybrid approach and with the plasmon
resonance technique using the MPR46 tail (28) might be explained by the
usage of µ3A compared with purified AP-3 complexes from brain cytosol
containing µ3B. Another possibility might be the formation of
receptor tail dimers in the two-hybrid system resembling prevalent
dimeric MPR46 forms present in membranes (35), which might facilitate
the binding of µ3A dimers. The physiological significance of MPR46
binding to µ3A, which preferentially interacts with lysosomal
membrane proteins, is unclear. However, substitution of
Cys34 or deletion of amino acid residues 20-23 or 24-29
as well as of the C-terminal end of the cytoplasmic tail interfere with
endosomal sorting processes (7, 8, 22) and might direct the MPR46 to
organelles for degradation in an AP-3-dependent manner.