Interaction of the Cation-dependent Mannose 6-Phosphate Receptor with GGA Proteins*

The GGAs (Golgi-localizing, (cid:1) -adaptin ear homology domain, ARF-binding) are a multidomain family of pro- teins implicated in protein trafficking between the Golgi and endosomes. Recent evidence has established that the cation-independent (CI) and cation-dependent (CD) mannose 6-phosphate receptors (MPRs) bind spe-cifically to the VHS domains of the GGAs through acidic cluster-dileucine motifs at the carboxyl ends of their cytoplasmic tails. However, the CD-MPR binds the VHS domains more weakly than the CI-MPR. Alignment of the C-terminal residues of the two receptors revealed a number of non-conservative differences in the acidic cluster-dileucine motifs and the flanking residues. Mutation of these residues in the CD-MPR cytoplasmic tail to the corresponding residues in the CI-MPR conferred either full binding (H63D mutant), intermediate binding (R60S), or unchanged binding (E56F/S57H) to the GGAs as determined by in vitro glutathione S -transferase pull-down assays. Furthermore, the C-terminal methionine of the CD-MPR, but not the C-terminal valine of the CI-MPR, inhibited GGA binding. Addition of four alanines to the C-terminal valine of the CI-MPR also se-verely reduced GGA binding, demonstrating the importance of the spacing of the acidic cluster-dileucine

In mammalian cells, newly synthesized lysosomal enzymes are modified posttranslationally to acquire the mannose 6-phosphate recognition marker (1). These enzymes bind to the lumenal domains of sorting receptors through their mannose 6-phosphate recognition markers at the trans-Golgi network (TGN) 1 and are targeted to acidified endosomes and lysosomes.
The 46-kDa cation-dependent mannose 6-phosphate receptor (CD-MPR) and the 275-kDa cation-independent mannose 6-phosphate receptor (CI-MPR/insulin-like growth factor-II receptor) are type I membrane-spanning glycoproteins known to mediate the processes of recognition and sorting of these enzymes (2). Recently a family of multidomain proteins named the Golgi-localized, ␥-ear-containing, ARF-binding proteins (or GGAs) was discovered in mammals and in yeast (3)(4)(5)(6)(7). These proteins have been shown to facilitate protein trafficking from the TGN to lysosomes in mammalian cells and to the vacuole in yeast (4,5,8,9). The GGAs were found to bind to both the CI-MPR and CD-MPR in yeast two-hybrid systems and in GST pull-down assays (10 -13). The binding was demonstrated to be a specific interaction between acidic cluster-dileucine signals found near the C termini of the MPRs and the N-terminal VHS domains of the GGAs. The GGAs also bind to the ADP-ribosylation factor-GTP complex (ARF-GTP) that is localized to the TGN (6,9,14) and to clathrin (11,14). These observations are consistent with a model for lysosomal enzyme trafficking involving the recruitment of the MPRs and their cargo by the GGAs to the site of transport vesicle formation.
Results from yeast two-hybrid experiments suggest a significant difference in the affinity of binding between the CI-MPR and CD-MPR and their GGA-binding partners. In these studies, the C-terminal tail of the CI-MPR bound to the VHS domains of the GGAs with higher affinity than did the corresponding region of the CD-MPR (10,13). There also was a difference in the relative affinity of the CD-MPR cytoplasmic tail for the VHS domains of the different GGAs. Puertollano et al. (10) showed that the CD-MPR bound well to the VHS domain of GGA1, but only poorly to that of GGA3, and not at all to GGA2. In contrast, they found that the cytoplasmic tail of the CI-MPR bound to the VHS domains of all three GGAs with about equal affinity. Takatsu et al. (13) on the other hand found that the cytoplasmic tail of the CI-MPR but not the CD-MPR interacted with the VHS domain of GGA1.
In the present study we have examined the basis for the differences in binding of the two MPRs to the GGAs. GST fusion peptides were generated with various single and double amino acid substitutions of the CD-MPR C-terminal region to more closely mimic the sequence of the CI-MPR C-terminal peptide. The ability of these GST peptides to bind to GGAs1-3 was measured in GST pull-down assays. These experiments have identified several residues that significantly increase binding to the VHS domains of the GGAs. Cell lines stably transfected with the CD-MPR mutants that have improved GGA binding exhibit increased efficiency of cathepsin D sort-ing. This is consistent with the idea that lysosomal sorting at the TGN requires the direct participation of GGAs.

EXPERIMENTAL PROCEDURES
Antibodies-The anti-Myc monoclonal antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-HA monoclonal was from Covance (Berkeley, CA). Anti-bovine CD-MPR polyclonal antibody and anti-human cathepsin D polyclonal antibody were generated in this laboratory.
Plasmid Construction-The construct for the GST fusion encoding the wild-type bovine CI-MPR 163-amino acid cytoplasmic tail was made by PCR and ligation into the vector pGEX6P-1 (Amersham Biosciences) as described (11). Residues 1-145 of the CI-MPR tail were subsequently deleted out by designing primers and using the QuickChange TM system (Stratagene, La Jolla, CA) to generate a GST CI-MPR peptide fusion incorporating only the C-terminal 18 amino acids of the cytoplasmic tail. The GST CD-MPR peptide construct was generated by annealing sense and antisense oligonucleotides corresponding to the C-terminal 18 amino acids of the bovine CD-MPR cDNA and ligating the doublestranded products into EcoRI/XhoI-digested pGEX-5X-3 (Amersham Biosciences). Mutagenesis of the CI-and CD-MPR cytoplasmic tails was performed using primers incorporating the desired mutations with the QuickChange system. myc-GGA1pCR3.1 and GGA3pCR3.1 were generous gifts from Juan Bonifacino (National Institutes of Health). GGA2-HApRK5 was kindly provided by Veli-Pekka Lehto (University of Oulu). To construct myc-GGA1 and GGA2-HA in the pFB1 vector (Invitrogen, Carlsbad, CA) for expression in SF9 insect cells, myc-GGA1pCR3.1 and GGA2-HApRK5 were double-digested with BamHI/NotI, and the cDNA inserts were ligated with BamHI/NotI-digested pFB1 to generate myc-GGA1pFB1 and GGA2-HApFB1. myc-GGA3pFB1 was constructed in two steps as follows. A SalI restriction site was engineered into myc-GGA1pFB1 by substituting the codons for Leu-10 and Glu-11 of the GGA1 cDNA by the QuickChange method. Similarly, a SalI site was engineered into GGA3pCR3.1 by substituting the codons for Glu-7 and Ser-8 of the GGA3 cDNA. myc-GGA3pFB1 was subsequently constructed by substituting the SalI/NotI fragment of myc-GGA1pFB1 encoding the GGA1 cDNA with the corresponding SalI/NotI fragment encoding the GGA3 cDNA. The various truncation mutants were generated using primers incorporating a stop codon at the desired position with the Quick-Change system.
Mutations in full-length CD-MPR were introduced into the cDNA subcloned in the vector pBillNeo (15) by the QuickChange method using oligonucleotide pairs incorporating the desired mutations. All constructs and mutations were confirmed to be correct by dideoxynucleotide sequencing.
Protein Expression-The various GST CI-and CD-MPR fusion proteins were expressed in the Escherichia coli strain BL21(RIL) (Stratagene) essentially as described (16). Cells from 1 liter of culture were lysed into 20 ml of CellLytic B reagent (Sigma), sonicated briefly, and centrifuged at 27,000 ϫ g at 4°C for 15 min to remove insoluble material. The clarified lysate was then mixed by tumbling at 4°C for 4 h with glutathione-Sepharose 4B (Amersham Biosciences) pre-equilibrated with 20 mM Tris⅐Cl, pH 7.5, containing 0.1% Triton X-100. Following 4 washes with the 20 mM Tris, 0.1% Triton X-100 buffer and a single wash with detergent-free 50 mM Tris⅐Cl, pH 8.0, the GST fusion proteins were competitively eluted with 10 mM reduced glutathione in 50 mM Tris⅐Cl, pH 8.0. Proteins eluted with reduced glutathione were dialyzed overnight against phosphate-buffered saline before use in pulldown experiments.
myc-GGA1pFB1, GGA2-HApFB1, and myc-GGA3pFB1 were transformed into E. coli DH10Bac competent cells to generate recombinant bacmids as per the manufacturer's protocol (Invitrogen). Bacmid DNAs were transfected into SF9 insect cells to produce recombinant baculoviruses that were amplified and used to express the various GGAs in the insect cells. Insect cells expressing the GGA proteins were routinely harvested 48 h post-infection, lysed into cold buffer A (25 mM Hepes-KOH, pH 7.2, 125 mM potassium acetate, 2.5 mM magnesium acetate, 1 mM dithiothreitol, and 0.4% Triton X-100) containing protease inhibitor mixture (Roche Diagnostics, Indianapolis, IN) by sonication, and centrifuged at 20,000 ϫ g for 10 min. The supernatant containing the GGA protein was stored at Ϫ80°C for use in the binding assays.
Binding Assays-The binding of the various GST fusions with the GGA proteins was assayed in buffer B (buffer A with 0.1% Triton X-100) in a final volume of 300 l in 1.5-ml pre-siliconized microcentrifuge tubes (Midwest Scientific, Valley Park, MO). Routinely, the GST fusion proteins were first immobilized at room temperature on 30 l of packed glutathione-Sepharose to concentrations of 3-6 mg/ml. The beads with bound proteins were pelleted by centrifugation at 750 ϫ g for 1 min and the beads washed once with cold buffer B, and 300 l of the SF9 cell lysate in buffer A at a final concentration of 2-5 mg/ml was added to the washed beads. To determine binding to AP-1, 300 l of bovine adrenal cytosol (8 mg/ml) in buffer B was added to the washed beads. The binding reactions were allowed to proceed for 3 h at 4°C with tumbling, following which the samples were subjected to centrifugation at 750 ϫ g for 1 min. An aliquot of the supernatant was saved, and the pellets were washed four times each by resuspension in 1 ml of cold buffer B by centrifugation at 750 ϫ g. The washed pellets were resuspended in SDS sample buffer and heated at 100°C for 5 min. Unless indicated otherwise, 1% of each pellet and supernatant fraction was loaded on SDS gels.
Electrophoresis and Immunoblotting-Proteins were resolved on 10% SDS-polyacrylamide gels and transferred to nitrocellulose. Blots were blocked with TBST (10 mM Tris⅐Cl, pH 7.5, 150 mM NaCl, 0.1% Tween 20) containing 5% non-fat milk for 1 h at room temperature. The blots were then probed with primary antibodies as indicated in the individual figure legends, followed by horseradish peroxidase-conjugated anti-mouse IgG. The immunoreactive bands were visualized on x-ray films using enhanced chemiluminescence (Amersham Biosciences).
Stable Cell Transfectants-Stable cell lines expressing the various CD-MPR mutants were generated by transfection of the pBillNeo constructs into CI-MPR negative mouse L cells (D-9) and the subsequent selection of clones propagated in G418-containing media. Transgene protein expression was determined by Western blotting. The CD-MPR protein levels of the various cell lines were compared with the nontransfected D-9 cells by loading equal quantities of total protein from cell lysates on SDS gels, transferring to nitrocellulose, and quantifying the immunoreactive bands by ECL using the Kodak Digital Imaging System (Eastman Kodak Co.).
Pulse-Chase and Cathepsin D Sorting Assays-Metabolic labeling and sorting of cathepsin D by the various CD-MPR mutant cell lines were performed essentially as described (17). Briefly, mouse L cells were plated onto 6-well plates 2 days prior to labeling. Cell monolayers at ϳ90% confluency were washed twice with cysteine/methionine-free ␣-minimum Eagle's medium and labeled for 1 h with 1 ml of 1 mCi/ml Tran 35 S-label (ICN Radiochemicals, Irvine, CA) in the presence of 20 mM Hepes and 5 mM mannose 6-phosphate. Excess unlabeled methionine (10 mM final concentration) was added to initiate a 4-h chase. Cells were then placed on ice and processed as described (18). The amount of cathepsin D sorted was determined by immunoprecipitating equivalent aliquots of the lysed cell and media high speed supernatants overnight at 4°C with rabbit anti-human cathepsin D antiserum and protein A-Sepharose beads (Repligen, Cambridge, MA). After four washes, immunoprecipitates were subjected to 10% SDS-PAGE under non-reducing conditions. Gels were treated with EN 3 HANCE (PerkinElmer Life Sciences), dried, and exposed to Kodak X-Omat AR film at Ϫ70°C. The resultant autoradiogram was used as a template to excise from the gel the regions corresponding to the processed and secreted forms of cathepsin D. The gel slices were then processed, and the associated radioactivity was determined as described (17). The percentage of cathepsin D sorted was calculated as the ratio of radioactivity in the processed form to the sum of the processed and secreted forms.

Effect of Mutations in the CD-MPR Tail on GGA Binding-
Although both the CD-MPR and the CI-MPR contain acidic cluster-dileucine sequences near the C terminus of their cytoplasmic tails, inspection of the amino acid sequences of the bovine species shows several differences in this region as well as in the flanking residues (Fig. 1). Setting the first leucine of the dileucine motif to 0, there are differences at positions Ϫ1, Ϫ4, Ϫ7, and Ϫ8 and at ϩ2 and ϩ3. To evaluate the importance of these differences in binding to the various GGAs, GST fusion peptides incorporating the C-terminal 18 amino acids of the receptors were generated with single or double substitutions of the CI-MPR residues into the equivalent position of the CD-MPR peptide backbone. The ability of these various mutant GST peptides to bind GGAs in pull-down assays was then compared with binding by the wild-type CI-MPR and CD-MPR peptides. In preliminary experiments, we found that the GST peptides with the wild-type MPR sequences bound GGAs to the same extent as the GST full-length cytoplasmic tails. Fig. 2A shows that GGA2 binds much better to the wild-type CI-MPR peptide than to the wild-type CD-MPR peptide, confirming the previous report (10). Thus, analysis of 1% of the GST CI-MPR peptide pellet gave a signal equivalent to that seen with 25% of the GST CD-MPR pellet. Mutation of the His residue at position Ϫ1 of the CD-MPR sequence to Asp resulted in a striking increase in GGA2 binding, equivalent to that seen with the CI-MPR peptide. Mutation of the Arg residue to a Ser at position Ϫ4 of the CD-MPR sequence greatly enhanced GGA2 binding as well, although not quite to the same extent as the His to Asp substitution. When the Glu and Ser flanking residues at positions Ϫ8 and 7 were changed to Phe and His, respectively, there was no significant alteration in GGA2 binding, indicating that these residues do not have a role in the interaction with GGA2. In contrast, the residues downstream of the dileucines strongly affect GGA2 binding. Thus substitution of the Pro and Met residues at positions ϩ2 and 3 with alanines resulted in GGA2 binding equal to that observed with the CI-MPR peptide. This result is similar to that reported by Puertollano et al. (10) with GGA3 using the yeast two-hybrid assay. The same results were obtained when the binding of GGA1 and GGA3 to the various peptides was tested (Fig. 2, B-D). In both cases the CI-MPR peptide bound the GGAs much better than the CD-MPR peptide, and the His 3 Asp, Arg 3 Ser, and Pro 3 Ala/Met 3 Ala substitutions enhanced binding, whereas the Glu 3 Phe/Ser 3 His mutation had no detectable effect on binding.
The C-terminal Met Residue Inhibits GGA Binding-The finding that the Pro 3 Ala/Met 3 Ala substitution enhanced GGA binding indicated that one or both of these residues was inhibitory. To pursue this point, GST peptides were prepared in which the Pro and Met residues were individually mutated to alanines. When these were tested for binding to the GGA1 VHS

FIG. 2. Effect of mutations in the CD-MPR tail on GGA binding.
A-D, specific mutations of the C-terminal amino acid residues of the CD-MPR cytoplasmic tail to the corresponding residues in the CI-MPR tail greatly enhance binding to all three GGA VHS domains in GST pull-down experiments. 1% of the pellet (P) and supernatant (S) were resolved on 10% SDS gels, transferred to nitrocellulose, and probed with either the anti-Myc or anti-HA antibody as described under "Experimental Procedures." *, highlights the fact that the GST CD-MPR peptide does bind to the VHS domain albeit very weakly relative to the GST CI-MPR peptide, 25% of pellet and 5% of supernatant were loaded in this case.

FIG. 3. C-terminal methionine of CD-MPR is inhibitory to GGA binding.
Substitution of the C-terminal valine of the CI-MPR to methionine greatly impairs binding of the GST CI-MPR peptide to the VHS domain of GGA1 and to a somewhat lesser extent to GGA2. Proteins resolved on SDS gels were tranferred to nitrocellulose, and immunoblotting was performed as described under "Experimental Procedures."

FIG. 4. Binding of MPRs to the VHS domains requires a precise spacing between the dileucines and the free C terminus.
An immunoblot of the binding reaction shows that extension of the terminal carboxyl moiety by an additional four alanines from the native C terminus drastically reduces binding of both the GST-CI MPR pep (peptide) and the GST CD-MPR PM 3 AA pep (peptide) to all three GGA VHS domains. domain, the peptide with the Met 3 Ala mutation exhibited a striking increase in GGA1 binding, whereas the peptide with the Pro 3 Ala substitution behaved similar to the wild-type CD-MPR peptide (Fig. 2D). This demonstrates that the Cterminal Met completely accounts for the inhibitory effect. Because the C-terminal residue in the bovine CI-MPR is a valine, we asked whether substitution of this bulky hydrophobic residue with a methionine would influence GGA binding. As shown in Fig. 3, a GST CI-MPR peptide with the Val to Met substitution bound GGA1 and GGA2 poorly when compared with the wild-type peptide, supporting the interpretation that a Met at the ϩ3 position inhibits binding to the GGAs.
Addition of Alanines to the C Termini of the MPRs Inhibits GGA Binding-The acidic cluster/dileucine motifs of both MPRs are located two residues from the C terminus of the cytoplasmic tails (Fig. 1). In sortilin, another membrane receptor known to interact with the GGAs, the acidic cluster cluster/ dileucine motif is one residue from the carboxyl end (12). This prompted us to ask whether the positioning of the acidic cluster-dileucine motif relative to the C terminus of the protein influences binding to the GGAs. To explore this issue we constructed GST CI-MPR and CD-MPR peptides with four alanine residues extending beyond the usual C-terminal residue. The CD-MPR peptide also contained the Pro 3 Ala/Met 3 Ala substitution so that GGA binding could be more easily detected. As shown in Fig. 4, the addition of the four alanines strikingly inhibited binding to all three GGAs. These findings indicate that the position of the acidic cluster-dileucine motif relative to the C terminus of the cytoplasmic tail has a major effect on GGA binding. (17) published before the discovery of the GGAs, we reported that CI-MPR-negative mouse L cells expressing CD-MPR with the Pro 3 Ala/Met 3 Ala mutation sorted the acid hydrolase cathepsin D more efficiently than cells expressing greater amounts of wild-type CD-MPR. The current findings suggest that the improved sorting efficiency may have been the result of enhanced binding to the GGAs. To determine whether this correlation holds for the other mutations that increase GGA binding, CI-MPR-negative L cells (clone D-9) were stably transfected with plasmids encoding bovine CD-MPRs with either Arg 3 Ser, His 3 Asp, or Glu 3 Phe/Ser 3 His substitutions. The ability of these cells to sort cathepsin D in pulse/chase experiments was measured and compared with cells expressing wild-type CD-MPR or CD-MPR with the Pro 3 Ala/Met 3 Ala mutation. The results are summarized in Table I. The non-transfected D-9 cells sorted 44 Ϯ 3% of the cathepsin D due to the endogenous CD-MPR. The sorting efficiency increased to 69 Ϯ 4% in cells expressing 12-fold more wild-type CD-MPR but was essentially unchanged in a cell line expressing only 4-fold more wild-type CD-MPR.

MPR Binding to the GGAs Correlates with Cathepsin D Sorting in Cells-In a previous study
Cells expressing CD-MPR with the Glu 3 Phe/Ser 3 His mutation sorted 53 Ϯ 2% of cathepsin D, which is not significantly different from the non-transfected D-9 cells. This mutation does not enhance GGA binding. In contrast, cells expressing CD-MPR with either the Arg 3 Ser or the His 3 Asp mutations exhibited a significant increase in cathepsin D sorting (57 Ϯ 2 and 70 Ϯ 4%, respectively) even though the receptor expression level was only two times the basal level. Both of these mutations increase GGA binding with the His 3 Asp substitution having a greater effect than the Arg 3 Ser substitution. The cells expressing receptor with the Pro 3 Ala/Met 3 Ala mutation sorted cathepsin D very efficiently (84 Ϯ 1%), confirming our previous findings. The expression level of this mutant receptor was 4.5-fold the basal level, which may explain why it is more effective than the His 3 Asp and Arg 3 Ser mutants. Taken as a whole, these data reveal a strong correlation between GGA binding and the efficiency of cathepsin D sorting in cells.
Another potential explanation for the improved ability of the mutant receptors to sort cathepsin D is that the amino acid substitutions enhance binding to the AP-1 adaptor protein complex that is also implicated in packaging the MPRs into transport vesicles in the TGN (19). The various GST fusion peptides were therefore tested for their ability to bind AP-1 in pull-down assays. As shown in Fig. 5, the full-length CD-MPR cytoplasmic tail bound only a trace amount of AP-1 in this assay compared with strong binding by the CI-MPR full-length cytoplasmic tail. None of the GST peptides having wild-type or mutant acidic cluster-dileucine sequences bound detectable levels of AP-1. This indicates that the AP-1 binding to the full-length CI-MPR cytoplasmic tail observed in this assay is not mediated by the acidic cluster-dileucine motif, in agreement with our previous study (11). Furthermore, it suggests that the improved efficiency of cathepsin D sorting exhibited by To determine whether the increased sorting efficiency exhibited by the CD-MPR mutants was due to improved binding to AP-1, GST CI/CD-MPR tail FL (full-length cytoplasmic tail) and GST CI/CD-MPR pep (peptide) wild-type and mutants were tested for their ability to bind AP-1 in in vitro pull-down assays. 10% of pellet (P) and 3% of supernatant (S) were loaded on the SDS gel. As seen from the immunoblot, neither the wild-type GST CD-MPR peptide nor the mutants that showed markedly enhanced GGA interaction display any binding to AP-1.  three of the CD-MPR mutants is not a consequence of enhanced binding to AP-1.

DISCUSSION
The results presented in this study provide a number of new insights concerning the interaction of the GGAs with acidic cluster-dileucine motifs. First, our data show that the weak binding of the CD-MPR to the GGAs relative to that observed with the CI-MPR is due to two amino acid differences in the acidic clusters of these receptors along with a difference in the C-terminal residue located two residues downstream of the dileucines. Within the acidic clusters, we found that an Asp residue at the Ϫ1 position greatly enhanced GGA binding compared with a His at this position. This is consistent with our previous study (11) showing that multiple residues in the acidic cluster of the CI-MPR influence binding to GGA2. A Ser at position Ϫ4 also improved GGA binding relative to an Arg at that position. In both instances the favorable residue is present in the acidic cluster of the CI-MPR, whereas the unfavorable residue is found in the CD-MPR acidic cluster.
The other key observation is that a Met at the C-terminal position, as occurs in the CD-MPR, inhibits GGA binding, whereas a Val at that position, as found in the bovine CI-MPR, allows strong binding. This finding is significant because it indicates that the GGAs recognize the C-terminal residue as well as the acidic cluster-dileucine motif. Furthermore, because the addition of four alanines to the C-terminal residue strongly inhibited GGA binding, it appears that the location of the acidic cluster-dileucine motif two residues upstream from the C terminus is critical for optimal binding to the GGAs. The effects of these residues on binding was found with all three GGAs, demonstrating that the different GGAs recognize common elements in the acidic cluster-dileucine motifs.
While this manuscript was in preparation, two groups (20,21) reported the crystal structure of the VHS domains of human GGA1 and GGA3 complexed with the acidic clusterdileucine motifs of the MPRs. The VHS domain is a righthanded superhelix of eight helices. Helices 6 and 8 form a surface that makes extensive contacts with amino acid residues Ϫ5 to ϩ3 of the CI-MPR cytoplasmic tail and residues Ϫ4 to ϩ3 of the CD-MPR cytoplasmic tail. Among these residues, the dileucines and the aspartate at position Ϫ3 have the most extensive interactions with the VHS-binding site, whereas the other residues appear to contribute to a lesser extent to the binding. These authors also implicated the C-terminal flanking residues of the acidic cluster-dileucine motifs in the binding to the VHS domain. They noted that the C-terminal side-chain protrudes into a hydrophobic pocket. Furthermore, Misra et al. (20) found that MPR sequences with two residues distal to the dileucines gave optimal binding, whereas those with one or three bound more weakly, and those with zero or four did not bind. Peptides containing amidated C-terminal residues also bound very weakly. Thus binding to the VHS domains requires a precise spacing between the dileucines and the free C terminus. Finally, Misra et al. (20) reported that the CI-MPR peptide bound 4 -6-fold better to the VHS domains of GGA1 and GGA3 than the CD-MPR peptide as determined by isothermal titration calorimetry. These findings are in good agreement with our results. Our data extend the work of Misra et al. (20) and Shiba et al. (21) by identifying the residues that account for the differences in CI-MPR and CD-MPR binding to the VHS domains.
The second important finding in our study is the strong correlation between the apparent binding affinities of the various acidic cluster-dileucine motifs to GGAs1-3 in the in vitro pull-down assays and the efficiency of cathepsin D sorting in mouse L cells expressing CD-MPRs with the different muta-tions. These data provide evidence that GGAs play a key role in packaging CD-MPRs with bound acid hydrolases into transport vesicles that are delivered to the endosome/lysosome system. A similar correlation emerged from studies of CI-MPRs that have a variety of mutations in their acidic cluster-dileucine motifs (11,18,(22)(23)(24). None of the mutations in the CD-MPR acidic cluster-dileucine motif that enhanced GGA binding resulted in AP-1 binding as measured by the pull-down assays. In fact, the full-length CD-MPR cytoplasmic tail bound AP-1 very poorly in this assay in contrast to the findings with the CI-MPR cytoplasmic tail. This probably reflects the insensitivity of the assay because Honing and co-workers (25) have reported that the cytoplasmic tail of the CD-MPR binds AP-1 with high affinity. These investigators used surface plasmon resonance to identify two independent sequences (amino acids 34 -43 and 49 -67) that bind AP-1 with K d values of 14 nM. Whereas the 49 -67 sequence includes the acidic cluster-dileucine motif, they showed that mutation of the dileucines to alanines had no effect on AP-1 binding. This is significant because mutation of the dileucines abolishes GGA binding, and cells expressing CD-MPRs with this mutation exhibit extremely poor cathepsin D sorting (17,26). Thus although AP-1 may have a role in packaging the CD-MPR into clathrin-coated vesicles at the TGN, either it handles only a small fraction of the cargo relative to the GGAs or it relies on the GGAs to present the MPRs for incorporation into AP-1 transport vesicles. Our data do not distinguish between these possibilities.
Finally, it is curious that the CD-MPR, in contrast to the CI-MPR, would evolve with an acidic cluster-dileucine motif that fails to provide optimal interaction with the GGAs. The CD-MPR does function to sort acid hydrolases in cells, so perhaps it works sufficiently well so that pressure has not been exerted to evolve into a more efficient receptor. Alternatively, the CD-MPR may be subject to some type of regulation that modulates GGA interaction. The cytoplasmic tail of the CD-MPR contains a casein kinase II phosphorylation site just upstream of the acidic cluster-dileucine motif (the Ser is at position Ϫ7, see Fig. 1). The importance of phosphorylation of Ser-57 is not clear at this time. Mauxion et al. (19) have suggested that phosphorylation of this serine facilitates AP-1 recruitment to the TGN. On the other hand, substitution of Ser-57 with an alanine or aspartate did not influence acid hydrolase sorting in mouse cells (17,27). It may turn out that phosphorylation of Ser-57 regulates binding to the GGAs under some circumstances.