Multiple C-terminal Motifs of the 46-kDa Mannose 6-Phosphate Receptor Tail Contribute to Efficient Binding of Medium Chains of AP-2 and AP-3*

Stephan Storch and Thomas BraulkeDagger

From Department of Biochemistry, Children's Hospital, University of Hamburg, D-20246 Hamburg, Germany

Received for publication, June 23, 2000, and in revised form, November 8, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The interaction of adaptor protein (AP) complexes with signal structures in the cytoplasmic domains of membrane proteins is required for intracellular sorting. Tyrosine- or dileucine-based motifs have been reported to bind to medium chain subunits (µ) of AP-1, AP-2, or AP-3. In the present study, we have examined the interaction of the entire 67-amino acid cytoplasmic domain of the 46-kDa mannose 6-phosphate receptor (MPR46-CT) containing tyrosine- as well as dileucine-based motifs with µ2 and µ3A chains using the yeast two-hybrid system. Both µ2 and µ3A bind specifically to the MPR46-CT. In contrast, µ3A fails to bind to the cytoplasmic domain of the 300-kDa mannose 6-phosphate receptor. Mutational analysis of the MPR46-CT revealed that the tyrosine-based motif and distal sequences rich in acidic amino acid residues are sufficient for effective binding to µ2. However, the dileucine motif was found to be one part of a consecutive complex C-terminal structure comprising tyrosine and dileucine motifs as well as clusters of acidic residues necessary for efficient binding of µ3A. Alanine substitution of 2 or 4 acidic amino acid residues of this cluster reduces the binding to µ3A much more than to µ2. The data suggest that the MPR46 is capable of interacting with different AP complexes using multiple partially overlapping sorting signals, which might depend on posttranslational modifications or subcellular localization of the receptor.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 (beta 1 and gamma -adaptin for AP-1; beta 2 and alpha -adaptin for AP-2), a medium subunit of ~50 kDa (µ1 and µ2) and a small polypeptide of ~20 kDa (sigma 1 and sigma 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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

[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 beta -galactosidase assays. The Escherichia coli strains of DH5alpha 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 Delta 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-beta -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-beta -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).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -galactosidase activity (blue color).

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.

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 Delta 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 beta -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 beta -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 beta -galactosidase (Table I) confirmed the specificity in the interaction between MPR46-CT and µ2. The lack of beta -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 beta -galactosidase assay

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 beta -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 beta -galactosidase activity of the wild type MPR46-CT). The N-terminal deleted mutant MPR46-CT Delta 1-16 activated beta -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, beta -galactosidase activity was tested in a filter assay. C, subsequently, five independent clones were selected and grown to A600 values of 0.8 and beta -galactosidase activity was measured using chlorophenol red-beta -D-galactopyranoside as a substrate. beta -Galactosidase activities in relative units are presented as mean values ± S.D. (bars).

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 beta -galactosidase activity by 22 and 31%, respectively (Fig. 5C). These mutations impaired the interaction with µ3A more strongly (20 and 18% of beta -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, beta -galactosidase activities were measured in six selected independent clones grown to A600 values of 0.8. beta -Galactosidase activities in relative units are presented as mean values ± S.D. (bars).



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 YXXphi (where Y is tyrosine, X is any amino acid, and phi  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 phi  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 3gamma have been proposed to interact with beta 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 YXXphi 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 YXXphi 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.


    ACKNOWLEDGEMENTS

We are grateful to Peter Schu and Margret S. Robinson for generously providing the µ2 and µ3A cDNA, respectively.


    FOOTNOTES

* This work was supported by Deutsche Forschungsgemeinschaft Grant BR990/8-2 and by the Fonds der Chemischen Industrie.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.

Dagger To whom correspondence should be addressed: Dept. of Biochemistry, Children's Hospital, University of Hamburg, Martinistr. 52, D-20246 Hamburg, Germany. Tel.: 49-40-42803-4493; Fax: 49-40-42803-8504; E-mail: braulke@uke.uni-hamburg.de.

Published, JBC Papers in Press, November 8, 2000, DOI 10.1074/jbc.M005548200


    ABBREVIATIONS

The abbreviations used are: MPR46, mannose 6-phosphate receptor with molecular mass of 46 kDa; MPR, mannose 6-phosphate receptor; MPR300, mannose 6-phosphate receptor with molecular mass of 300 kDa; CT, cytoplasmic tail; DBD, DNA binding domain; AD, activation domain; GST, glutathione S-transferase; TGN, trans-Golgi network; AP, adaptor protein complex; µ2, medium chain of AP-2; µ3A, medium chain of AP-3; PBS, phosphate-buffered saline.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES


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