©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Localization of the Insulin-like Growth Factor II Binding Site to Amino Acids 15081566 in Repeat 11 of the Mannose 6-Phosphate/Insulin-like Growth Factor II Receptor (*)

Bernhard Schmidt (§) , Christina Kiecke-Siemsen , Abdul Waheed (¶) , Thomas Braulke , Kurt von Figura

From the (1)Georg-August-Universität, Zentrum für Biochemie und Molekulare Zellbiologie, Abteilung Biochemie II, Gosslerstrasse 12d, D-37073 Göttingen, Federal Republic of Germany

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF-II receptor) binds insulin-like growth factor II (IGF-II) with high affinity. To localize the IGF-II binding site within the 15 repeating units that form the extracytoplasmic domain of the receptor, purified human M6P/IGF-II receptor was digested with thermolysin, and the fragments were analyzed for their ability to bind I-IGF-II in a cross-linking assay. Two IGF-II-binding receptor fragments of 23 and 37 kDa were purified. Sequence analysis revealed that the fragments consist of disulfide connected peptides comprising amino acids 1331-1566 and 1331-1697 of the receptor repeats 9-12. In a second approach we expressed truncated forms of the M6P/IGF-II receptor fused to the C terminus of the extracytoplasmic domain of the 46-kDa mannose 6-phosphate receptor. Fusion proteins containing M6P/IGF-II receptor repeats 10-15, 10-11, or 11-15 bound IGF-II, whereas a fusion protein containing the single repeat 10 failed to bind. This result indicates that repeat 11 (amino acids 1508-1650) is sufficient for binding of IGF-II. Residues 1508-1566, which are shared by the 23-kDa IGF-II-binding fragment and repeat 11, are proposed to form the IGF-II binding site of the M6P/IGF-II receptor.


INTRODUCTION

The mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF-II receptor)()is a 300-kDa transmembrane protein, which contains binding sites for two different types of ligands in its extracytoplasmic domain. On the one hand, lysosomal enzymes can bind via the mannose 6-phosphate residues of their oligosaccharide side chains to the receptor. This binding is critical for targeting of lysosomal enzymes from the Golgi or the plasma membrane to the lysosomes(1, 2, 3) . On the other hand, the receptor can bind the non-glycosylated insulin-like growth factor II (IGF-II)(4) . Binding at the plasma membrane is followed by internalization and degradation of IGF-II in the lysosomes(5) .

The M6P/IGF-II receptor is a type I transmembrane protein(6) . The extracytoplasmic domain is composed of 15 repeating units, each of 134-167 amino acids in length, which are homologous to each other and to the extracytoplasmic domain of the 46-kDa mannose 6-phosphate receptor (MPR46)(7) . Within the extracytoplasmic domain the M6P/IGF-II receptor binds 2 mol of mannose 6-phosphate (8) and 1 mol of IGF-II (9)/mol of receptor. The binding of IGF-II may be a characteristic of mammals, that has been demonstrated so far for the receptors from human (10-13), bovine(9, 14) , opossum(15) , and rat(14, 16) , while the receptors from chicken (17, 18) and frog (18) lack the binding of IGF-II.

IGF-II is a single-chain polypeptide of 67 amino acids, that shares a high degree of homology with IGF-I and insulin(19) . The majority of extracellular IGF-II is bound to IGF-binding proteins and acts as a mitogen for different cell types (20) and as a fetal growth factor (21). IGF-II binds to the M6P/IGF-II receptor with a K of 0.2-14.5 nM(9, 14, 15) at a single binding site, which is different from the two binding sites for mannose 6-phosphate(9, 11, 12, 14) , but the influence of the binding of the one ligand on the affinity for the other ligand remains still unclear. The exact locations of the binding sites for mannose 6-phosphate and IGF-II still need to be defined. Studies using truncated forms of the extracytoplasmic domain of the bovine M6P/IGF-II receptor indicate that for binding of mannose 6-phosphate repeats 1-3 and 7-9(22, 23) , and for binding of IGF-II repeats 5-11 (24) are sufficient.

In the present study, we characterized two IGF-II-binding fragments derived from human M6P/IGF-II receptor digested with thermolysin. The sequence analysis of the fragments revealed that each fragment consists of several peptides, cross-linked by disulfide bridges. The peptides of the smaller of the two fragments comprise the C terminus of repeat 9, the entire repeat 10, and the N terminus of repeat 11. In a complementary approach truncated forms of the human M6P/IGF-II receptor fused to the extracytoplasmic domain of the MPR46 were expressed and tested for their ability to bind IGF-II. Whereas the fusion proteins encoding repeat 10 alone did not bind IGF-II, fusion proteins containing repeats 10-15, 11-15, and 10-11 bound IGF-II. Taking the results of both approaches together, we conclude that the N terminus of repeat 11 comprising amino acids 1508-1566 contains the binding site for IGF-II.


MATERIALS AND METHODS

Reagents

Recombinant human IGF-II was obtained from Dr. W. Märki (Ciba-Geigy, Basel, Switzerland) and iodinated with NaI to a specific activity of about 10 mCi/mg with the aid of chloramine T as described(25) . Thermolysin, trypsin, subtilisin, and V8 protease were from Sigma, and disuccinimidylsuberate (DSS) from Pierce. Enzymes used for in vitro mutagenesis were from New England Biolabs. NaI and [S]methionine (>800 Ci/mmol) were purchased from Amersham Corp. The anti-MPR46 antisera and the monoclonal anti-MPR46 antibody 10C6 have been described previously(26, 27) . All other reagents were of analytical grade.

Purification and Digestion of the M6P/IGF-II Receptor

The M6P/IGF-II receptor was purified from human placenta as described previously(28) . The purified receptor was dialyzed overnight against 50 mM imidazole, pH 7.0, 0.05% Triton X-100 and precipitated with acetone (80% final concentration) at -20 °C overnight. After centrifugation at 30.000 g for 45 min at 4 °C the pellet was dried under vacuum and dissolved in 20 mM NaP, pH 7.5, 20 mM NaCl to a final concentration of 0.17 mg/ml. In an analytical scale, 3-µg aliquots of the receptor were incubated with 1, 2, 10, and 20% (w/w) thermolysin, trypsin, subtilisin, or V8 protease in 24 µl of 20 mM NaP, pH 7.5, 20 mM NaCl at 37 °C for 16 h. The digestion was stopped by the addition of 10 mM EDTA, and the receptor fragments were analyzed in a IGF-II cross-linking assay.

Purification of IGF-II-binding M6P/IGF-II Receptor Fragments

For purification of IGF-II-binding fragments, three aliquots of 2.0-2.3 mg M6P/IGF-II receptor were incubated with 2% (w/w) thermolysin at 37 °C for 16 h as described above. After the addition of EDTA, lyophilization, and dissolving in 600 µl of water, each aliquot was loaded on a Superdex HR200-size exclusion column (Pharmacia Biotech Inc.) equilibrated with 20 mM NaP, pH 7.5, 20 mM NaCl. The receptor fragments were eluted from the column with a flow rate of 0.5 ml/min and collected in fractions of 2 ml. One µl of each fraction was analyzed in the IGF-II cross-linking assay. Fractions 42-47 (pool I) and fractions 49-54 (pool II) of the three runs were combined, lyophilized, and dialyzed against 5 mM Tris/HCl, pH 7.5, 0.1% SDS overnight. The dialyzed samples were loaded on a preparative SDS-polyacrylamide gel (10% acrylamide for pool I and 12% for pool II). Following electrophoresis a small strip of the gel was cut off and stored. The fragments in the remaining gel were blotted on a polyvinylidene difluoride (PVDF) membrane, stained with 0.2% Coomassie Blue, 50% methanol, 10% acetic acid for 15 min, and washed three times with 45% methanol, 7% acetic acid for 5 min. The stained receptor fragments were subjected to an N-terminal sequence analysis. The gel strip was cut into small strips, which were homogenized in 200 µl of 5 mM Tris/HCl, pH 7.5, 0.1% SDS. The receptor fragments were extracted by incubation at 37 °C overnight. After centrifugation in an Eppendorf centrifuge, the supernatant was lyophilized and the receptor fragments precipitated by an incubation in 500 µl of 85% acetone, 5% trimethylamine, 5% acetic acid, 5% water for 1 h at 4 °C(29) . After centrifugation, the pellet was dissolved in 30 µl of 20 mM NaP, pH 7.5, 20 mM NaCl and analyzed in the IGF-II cross-linking assay.

Cross-linking of IGF-II to the M6P/IGF-II Receptor and Its Fragments

Cross-linking of IGF-II to the M6P/IGF-II receptor was carried out according to the procedure of Waheed et al. (12). One µg of the dialyzed and precipitated M6P/IGF-II receptor or 3 µg of the receptor fragment mixture or aliquots of purified fragments or dialyzed medium containing the fusion proteins were incubated with 1-4 10 dpm I-IGF-II in 30-60 µl of 20 mM NaP, pH 7.5, 20 mM NaCl, 0.05% Triton X-100 overnight at 4 °C. To estimate the half-maximal inhibition of cross-linking of I-IGF-II to the fusion proteins, binding and cross-linking were carried out in the presence of 10 to 10M unlabeled IGF-II. After binding cross-linking to I-IGF-II was accomplished by adding 0.06 mM DSS and incubation for 15 min on ice. The reaction was stopped by the addition of 125 mM Tris/HCl, pH 6.8, 1% SDS, 10% glycerol and an incubation at 95 °C for 5 min. The samples were subjected to SDS-PAGE under non-reducing conditions according to the protocol of Laemmli et al.(30) . Cross-linked I-IGF-II was detected by autoradiography and quantified by densitometry of the x-ray film.

Amino Acid Sequencing

In the area of the Coomassie Blue-stained bands, the PVDF membrane was cut off, washed with 20% methanol, and subjected to N-terminal sequencing as described previously(31) . The determined amino acid sequences were compared with the amino acid sequences of the M6P/IGF-II receptor and thermolysin by means of the computer program GPMAW (Lighthouse Data, Odense, Denmark).

Generation of MPR46-M6P/IGF-II Receptor Constructs

Four constructs were generated coding for different M6P/IGF-II receptor repeats fused to the extracytoplasmic domain of human MPR46. All constructs contain the MPR46 cDNA coding for the extracytoplasmic domain of MPR46 (nucleotides 1-715, amino acids 1-190, Ref. 32) inserted in the pBEH expression vector(33) . To generate this MPR46/pBEH plasmid, a BamHI site was inserted into the MPR46 cDNA at position 710 and the EcoRI/BamHI fragment encoding nucleotides 1-715 was cut out and subcloned into the pBEH plasmid.

Fused to the 3` site of the MPR46 cDNA, the constructs contain the indicated truncated human M6P/IGF-II receptor cDNAs(6) . Construct 10-15 contains nucleotides 4246-7028 with a stop codon in position 7027, coding for amino acids 1367-2293 (repeat 10-15); construct 10 contains nucleotides 4246-7028 with a stop codon in position 4693, coding for amino acids 1367-1515 (repeat 10); construct 10-11 contains nucleotides 4246-7028 with a stop codon in position 5098, coding for amino acids 1367-1650 (repeat 10-11); and construct 11-15 contains nucleotides 4669-7028 with a stop codon in position 7027, coding for amino acids 1508-2293 (repeat 11-15). These truncated M6P/IGF-II receptor cDNAs were generated using a KpnI/KpnI fragment comprising nucleotides 3242-7851 of the M6P/IGF-II receptor cDNA in M13mp18 plasmid as template for site-directed mutagenesis according to the protocol of Nakamaye and Eckstein(34) . BglII sites were created at the 5` end of repeat 10 or 11 and at the 3` end of the stop codons listed above. The BglII/BglII fragments were cloned into the MPR46/pBEH plasmid opened by BamHI. In each construct the fusion was represented by amino acid 190 of MPR46 and the first amino acid of either repeat 10 (threonine 1367) or repeat 11 (methionine 1508) of M6P/IGF-II receptor. For each construct the presence of the mutations was confirmed by fluorescent dye terminator cycle sequencing and analysis on an automated DNA sequencer (Applied Biosystems). Oligonucleotide primer for mutagenesis and sequencing were synthesized using an 381A solid-phase synthesizer (Applied Biosystems) and purified on a MonoQ anion exchange column (Pharmacia).

Tissue Culture and DNA Transfection

BHK cells (clone 21) were transfected with 20 µg of expression vector as described previously(35) . Stable colonies were transferred to a 24-well tissue culture plate. When cells were grown to confluence, the rate of expression of the fusion proteins into the medium was determined by an enzyme-linked immunoassay using anti-human MPR46 antiserum as described previously(26) . Cell colonies expressing the fusion proteins were transferred to 35-mm cell culture dishes for metabolic labeling and immunoprecipitation. For Western blot and IGF-II binding analysis, cells were transferred to 75-cm cell culture flasks. After cells had grown to confluence they were starved for 24 h in 10 ml of serum-free medium. Medium was collected, dialyzed against 20 mM NaP, pH 7.5, 20 mM NaCl, and concentrated to 200 µl by ultrafiltration. Equal amounts of media were used for Western blot and IGF-II binding analysis.

Metabolic Labeling, Immunoprecipitation, and PMP Affinity Chromatography

Confluent cells in 35-mm cell culture dishes were starved for 1 h in methionine-free medium and labeled with 0.8 ml of methionine-free medium containing 2.5% dialyzed fetal calf serum and 40 µCi of [S]methionine for 24 h. Medium was collected and dialyzed against 50 mM Tris/HCl, pH 7.5, 150 mM NaCl at 4 °C and subjected to immunoprecipitation as described previously (36) using anti-human MPR46 antiserum(26) . Binding of S-labeled secretions containing fusion proteins to PMP-Sepharose was carried out as described previously(37) .

Western Blot Analysis

Aliquots of 5-100 µl of dialyzed and concentrated cell medium containing the fusion proteins were subjected to Western blot analysis with ECL detection as described previously (38) using the monoclonal anti-human MPR46 antibody 10C6 (27).


RESULTS

IGF-II Binding to the M6P/IGF-II Receptor and Its Proteolytic Fragments

To examine the binding of IGF-II to the M6P/IGF-II receptor purified from human placenta, a cross-linking assay was performed. After incubation of the receptor with I-IGF-II, the mixture was subjected to cross-linking with DSS, SDS-PAGE and autoradiography. A broad signal in the range of 250-300 kDa was detected (Fig. 1, lane2), which resolves upon shorter exposure into distinct bands that coincide with bands observed when the gel was stained by silver or a polyclonal receptor antibody (data not shown). When I-IGF-II was subjected to cross-linking in the absence of the receptor (Fig. 1, lane1), or when the receptor was incubated with I-IGF-II in the presence of an excess of unlabeled IGF-II (lane3), the products of 250-300 kDa disappeared, thus identifying the latter as M6P/IGF-II receptor-I-IGF-II cross-link products.


Figure 1: Cross-linking of I-IGF-II to M6P/IGF-II receptor and its fragments. M6P/IGF-II receptor (1 µg) purified from human placenta was cross-linked to I-IGF-II via the cross-linker DSS and analyzed by SDS-PAGE (10% acrylamide) under nonreducing conditions and autoradiography as described under ``Materials and Methods'' (lane2). As controls cross-linking was carried out in the presence of 10M unlabeled IGF-II (lane3) or in the absence of receptor (lane1). Fragments derived from an incubation of 3 µg of M6P/IGF-II receptor for 16 h with 2% thermolysin were cross-linked to I-IGF-II in the absence (lane4) or presence (lane5) of 10M unlabeled IGF-II or in the absence of receptor (lane6). The apparent molecular masses of the cross-link products are indicated on the left.



In order to localize the IGF-II binding site within the receptor, M6P/IGF-II receptor was incubated with different proteases, including trypsin, thermolysin, subtilisin, and V8 protease. The proteolytic fragments were analyzed for IGF-II binding. Thermolysin was found to produce a series of products that retain IGF-II binding activity. Fig. 1(lane4) shows the pattern of IGF-II-binding fragments, obtained after incubation of M6P/IGF-II receptor for 16 h with 2% (w/w) thermolysin. A major 37-kDa and two minor 33- and 23-kDa I-IGF-II cross-linked products were detectable. When the digest was incubated in the presence of unlabeled IGF-II, the binding of I-IGF-II was inhibited completely (Fig. 1, lane5). Thermolysin or its autocatalytic fragments did not bind I-IGF-II (Fig. 1, lane6). The cross-linked products of 23-37 kDa are likely to contain receptor fragments of 1-2 repeats (the average mass of one repeat is about 16 kDa).

Purification of IGF-II-binding M6P/IGF-II Receptor Fragments

In order to identify the IGF-II-binding fragments by direct amino acid sequencing, 6.5 mg of M6P/IGF-II receptor were digested with thermolysin and the fragments purified by a combination of size exclusion chromatography and preparative SDS-PAGE. The elution profile of the size exclusion chromatography (Fig. 2A) indicates that the receptor was preferentially degraded to small fragments with molecular masses of less than 15 kDa. The fractions were analyzed for binding of IGF-II (Fig. 2B). The IGF-II-binding fragments were detected in fractions 40-54, which correspond to molecular masses of 30-50 kDa.


Figure 2: Purification of IGF-II-binding M6P/IGF-II receptor fragments by size exclusion chromatography. 6.5 mg of M6P/IGF-II receptor were digested with 2% thermolysin in three aliquots of 2.0, 2.2, and 2.3 mg and separated on a Superdex HR200-size exclusion column in three runs as described. A, the elution profile of the separation of 2.0 mg of receptor fragments is shown. Elution (arrows) and molecular masses in kDa of standard proteins are indicated. B, 1 µl of each fraction was subjected to cross-linking to I-IGF-II, SDS-PAGE (10% acrylamide), and autoradiography. The apparent molecular masses of the cross-linked products (on the left) and the number of fractions analyzed (below) are indicated. Fractions 42-47 (pool I) and 49-54 (pool II) of the three runs were combined.



Fractions 42-47 (yielding the majority of cross-linked products of 33 and 37 kDa) and fractions 49-54 (yielding the 23-kDa cross-linked product) were pooled separately for further purification by SDS-PAGE. In preliminary experiments, we showed that the receptor and its fragments retain their ability to bind IGF-II after SDS-PAGE and extraction from the gel, if SDS is removed by an acid acetone precipitation. Following separation on a preparative SDS-polyacrylamide gel, a small strip of the gel was cut off for cross-linking analysis (see below). The remaining gel was blotted on a PVDF membrane, followed by Coomassie Blue staining. Fractions 42-47 (Pool I) contained four stainable fragments with the apparent masses of 37, 40, 45, and 63 kDa, and fractions 49-54 (Pool II) contained five stainable fragments with apparent masses of 23, 25, 27, 33, and 37 kDa (see Fig. 3A). To test the ability of these fragments to bind IGF-II, each gel strip was cut into eight small strips. After extraction with 0.1% SDS and removal of SDS by an acid acetone precipitation, binding of IGF-II was analyzed. In Pool I extracts containing the 37- and 40-kDa Coomassie Blue-stainable fragments yielded I-IGF-II cross-link products of 33-37 kDa (data not shown). In Pool II the extract containing the 23-kDa Coomassie Blue-stainable fragment yielded an I-IGF-II cross-link product of 23 kDa (Fig. 3B, lane4), and the extracts containing the 33- and 37-kDa stainable fragments cross-linked products of 33 and 37 kDa (Fig. 3B, lanes1 and 2). A weak signal observed in lane5 could not be assigned to a Coomassie Blue-stainable fragment. Altogether, three IGF-II-binding fragments with apparent masses of 23, 37, and 40 kDa could be isolated from the thermolysin digest of the receptor.


Figure 3: Cross-linking of I-IGF-II to purified M6P/IGF-II receptor fragments. IGF-II-binding M6P/IGF-II receptor fragments collected after size exclusion chromatography (see Fig. 2, pool II) were purified by preparative SDS-PAGE (12% acrylamide). Following electrophoresis one part of the gel was blotted on a PVDF membrane and stained with Coomassie Blue. The other part of the gel was cut into eight small strips, which were extracted with 0.1% SDS. After removal of SDS by an acid acetone precipitation, the extracted fragments were subjected to cross-linking to I-IGF-II. PartA, the PVDF membrane containing the Coomassie Blue-stained receptor fragments is shown. On the left the apparent molecular masses of the fragments are indicated in kDa. On the right the numbers and positions where the strips were cut from the gel are indicated. PartB, the I-IGF-II cross-linking analysis of the extracts from the eight gel strips is shown. The number of gel strips (above) and the apparent molecular masses of the cross-linked products (on the left) are indicated.



Sequencing of the IGF-II-binding Fragments

The PVDF membranes containing the 23-, 37-, or 40-kDa IGF-II-binding fragments were subjected to sequence analysis by automatic Edman degradation. The results of the sequence analysis of the 23- and 37-kDa fragment are shown in Fig. 4. Sequencing of the 40-kDa fragment failed to provide any signal. Sequence analysis of the 23-kDa fragment showed one to seven amino acid signals per cycle. The computer analysis of this data revealed, that the amino acids could be assigned to the N-terminal sequences of three M6P/IGF-II receptor peptides (sequence 23A: amino acids 1360-1378; 23B: 1331-1339; 23C: 1552-1561) (see Fig. 5) and the N terminus of thermolysin (sequence 23E). The remaining signals (X) could neither be assigned to M6P/IGF-II receptor nor to thermolysin.


Figure 4: Sequencing of the IGF-II-binding M6P/IGF-II receptor fragments. Sequencing of the IGF-II-binding M6P/IGF-II receptor fragments of 23 and 37 kDa yielded several amino acid signals per cycle. By computer analysis, the amino acids could be assigned to the N-terminal sequences of human M6P/IGF-II receptor peptides (23A-23C, 37A, 37B, and 37D) and to the N-terminal sequence of thermolysin (23E). Some weak amino acid signals could not be assigned (X). The sequencing yield (in pmol) is indicated below each amino acid (, an exact quantification is not possible; -, amino acid is not detectable). It should be noted that serine and threonine cannot be quantified exactly, and cysteine cannot be determined during sequencing by Edman degradation.




Figure 5: Alignment of the IGF-II-binding peptides with the sequence of the M6P/IGF-II receptor. The amino acid sequence of the human M6P/IGF-II receptor in the region of repeats 9-12 is shown. Sequences determined by sequence analysis are labeled by grayboxes and labeled at the top. Disulfide bridges following the model of Lobel et al. (7) are indicated. The number of cysteines within the complete receptor sequence are partly indicated.



Sequencing of the 37-kDa fragment resulted in one to five amino acid signals per cycle, which could be assigned to the N-terminal sequences of three peptides derived from the M6P/IGF-II receptor (sequence 37A: amino acid 1360-1378; 37B: 1331-1340; 37D: 1692-1697) (see Fig. 5). The remaining signals (X) could not be assigned.

Peptides 23B, 23C, 37B, and 37D were sequenced up to their C termini. The C termini of peptide 23A and 37A, which share common N-terminal sequences, could not be identified. However, they extend beyond the cycle 19 given in Fig. 4, as indicated by signals in cycle 22 (leucine) and 23 (serine).

Peptide 23B terminates in front of a thermolysin cleavage site (N-terminally of valine 1341). For peptide 37B we assume that it consists of 11 rather than 10 residues (as indicated by the sequence analysis) and that the C-terminal residue (proline 1340) was washed out, as often observed in automatic Edman degradation. With this assumption peptide 37B is identical to peptide 23B. Additionally, peptide 37D and, with the assumption of a washout of the C-terminal glycine 1562, peptide 23C terminate in front of a thermolysin cleavage site (leucine 1698 and valine 1563).

For the alignment of the peptides, it should be noted that nonreducing conditions were used in all experimental steps. Receptor peptides comigrating in SDS-PAGE are therefore assumed to be linked by disulfide bonds. According to the model of Lobel et al.(7) , for disulfide bonding of the receptor, peptide 23B is expected to be connected with peptide 23A by a disulfide bridge between cysteines 1333 and 1361 (see Fig. 5). To allow disulfide binding of peptide 23A with a following peptide of the 23-kDa fragment, we have to postulate that peptide 23A extends at least up to cysteine 1516, to allow its disulfide binding to cysteine 1553 of peptide 23C. Peptide 23C contains a second cysteine residue (cysteine 1559), which is proposed to be linked in the receptor to cysteine 1566. The sequence data are compatible with a linkage of cysteine 1566 to the dipeptide alanine 1565-cysteine 1566, which can be cut out by thermolysin, while more extended sequences for cysteine 1566 containing peptides are excluded by the sequence analysis. The molecular mass of the 23-kDa IGF-II-binding fragment calculated from the sequenced peptides is 20.3-23.9 kDa, if the C terminus of peptide 23A is assumed to be at one of the thermolysin cleavage sites between cysteine 1516 and peptide 23C. This value fits well to the determined molecular mass of 23 kDa. Altogether the 23-kDa fragment consists of three peptides (plus the unidentified dipeptide alanine-cysteine, see above) connected to each other by disulfide bridges. The peptides extend from amino acid 1331 to 1562 (or 1566), which correspond to the C terminus of repeat 9, the entire repeat 10, and about 40% of the N terminus of repeat 11.

The peptides recovered from the 37-kDa fragment can be interpreted in an analogous way (see Fig. 5). Peptide 37B, which is identical to peptide 23B, and peptide 37A are connected by the disulfide bridge between cysteine 1333 and 1361. Peptide 37A is predicted to be disulfide bounded to peptide 37D via cysteine 1652 and cysteine 1695. The calculated molecular mass of the 37-kDa fragment is 34.9 kDa, if the C terminus of peptide 37A is assumed to be at the first thermolysin cleavage site following cysteine 1652 (N-terminally of valine 1654). The calculated value of 34.9 kDa and the determined molecular mass of 37 kDa of the purified fragment agree well. Thus, the 37-kDa fragment consists of three disulfide-connected peptides extending from amino acid 1331 to 1697, which corresponds to the C terminus of repeat 9, the entire repeats 10 and 11, and the N-terminal third of repeat 12.

Expression and IGF-II Binding of Truncated Forms of the M6P/IGF-II Receptor

The IGF-II-binding M6P/IGF-II receptor fragments of 23 and 37 kDa contain amino acid sequences, which are located in repeats 9-12. To analyze, which of these receptor repeats are involved in the binding of IGF-II, we expressed different truncated forms of the M6P/IGF-II receptor in BHK cells (Fig. 6). To increase the probability of a correct folding and disulfide connection of the truncated receptor forms, all recombinants were designed to terminate at the C-terminal amino acids of the receptor repeats as defined by Lobel et al.(7) . The receptor repeats were expressed in eucaryotic cells as fusion proteins connected to the C terminus of the extracytoplasmic domain of the MPR46. This strategy was chosen for the following reasons. 1) We speculated that folding of a single M6P/IGF-II receptor repeat would possibly be facilitated by expressing it as a fusion protein with a second receptor repeat. As a repeat, which does not bind IGF-II and yet may assist folding we chose the extracytoplasmic domain of the MPR46. The extracytoplasmic domain of the MPR46 shows a sequence homology of 14-28% to each of the 15 repeats, which form the extracytoplasmic domain of the M6P/IGF-II receptor(7) . This includes the conservation of six cysteines proposed to form disulfide bonds within each repeat. 2) The extracytoplasmic domain of the MPR46 provides a N-terminal signal peptide, which guarantees the translocation of the fusion proteins into the lumen of the endoplasmic reticulum. Since the constructs lack the transmembrane domains of the MPR46 and the M6P/IGF-II receptor, the fusion proteins should behave as secretory proteins. 3) The extracytoplasmic domain of the MPR46 serves as a tag for immunological detection of the fusion proteins and for binding to a PMP affinity column due to the mannose 6-phosphate binding site in the MPR46 domain(39) .


Figure 6: MPR46-M6P/IGF-II receptor fusion proteins. The four fusion proteins consist of the extracytoplasmic domain of the human MPR46 (graybox) fused to the N terminus of variable human M6P/IGF-II receptor repeats (whiteboxes). The numbers of the N- and C-terminal M6P/IGF-II receptor amino acids are indicated.



The DNA constructs were cloned into the expression vector pBEH and transfected into BHK cells. To isolate the fusion proteins, cells were incubated for 24 h in serum-free medium. After concentration and dialysis, the fusion proteins were analyzed by Western blot with an antibody against MPR46 and by chromatography on a PMP-Sepharose column in a mannose 6-phosphate-dependent manner (data not shown). These results demonstrated that the MPR46 domain of the fusion proteins folded correctly. Binding of IGF-II was tested in the cross-linking assay with I-IGF-II.

M6P/IGF-II Receptor Repeats 10-15 Bind IGF-II

Four fusion proteins containing different repeats of the M6P/IGF-II receptor were expressed (Fig. 6). In our first approach, we expressed repeats 10-15 (amino acids 1367-2293 of the M6P/IGF-II receptor) fused to the extracytoplasmic MPR46 domain. Western analysis of secretions of cells expressing the fusion protein 10-15 revealed a 135-kDa polypeptide (Fig. 7A, lane2). Its mass fits well to the theoretical mass of 138 kDa (105 kDa for repeats 10-15 and 33 kDa for the MPR46 extracytoplasmic domain). This calculation does not regard N-linked oligosaccharides possibly connected to one or more of the six potential N-glycosylation sites of repeats 10-15. When binding of IGF-II to the fusion protein 10-15 was analyzed, a cross-link product of 145 kDa was detected (Fig. 7B, lane2). Its mass corresponds to the calculated mass of the protein cross-linked to IGF-II (138 + 7.5 kDa). Half-maximal inhibition of I-IGF-II cross-linking was observed at about 10 nM unlabeled IGF-II (). The results demonstrate that repeats 10-15 are sufficient for binding of IGF-II.


Figure 7: Western blot and I-IGF-II cross-linking of the MPR46-M6P/IGF-II receptor fusion proteins. PartA, concentrated media containing the fusion proteins were subjected to Western analysis using a monoclonal anti-human MPR46 antibody for detection of the four fusion proteins (lanes 2-5). The amount of media was adopted to yield signals of similar intensity for MPR46, assuming that the equal MPR46 signals reflect equal molarities of fusion proteins. PartB, the same amount of concentrated media as in A was subjected to I-IGF-II cross-linking (lanes 2-5). As a control for IGF-II binding, 1 µg of M6P/IGF-II receptor was analyzed (lane1). In the experiment shown in B, the signal for fusion protein 10-15 cross-linked to I-IGF-II (lane2) was lower than in other experiments. The apparent molecular masses of proteins and cross-linked products are indicated. In A and B, SDS-PAGE was performed using 10% acrylamide.



The Single M6P/IGF-II Receptor Repeat 10 Fails to Bind IGF-II

Repeat 10 was the only repeat that was spanned in its full length by the peptides forming the 23-kDa IGF-II-binding proteolytic fragment. To examine whether repeat 10 is sufficient to bind IGF-II, fusion protein 10 was expressed. It consists of the M6P/IGF-II receptor repeat 10 (amino acids 1367-1515) fused to the extracytoplasmic domain of the MPR46 (see Fig. 6). By Western analysis, fusion protein 10 has a mass of 51 kDa (Fig. 7A, lane5), which fits well to the theoretical mass of 50 kDa. Analyzed in the IGF-II cross-linking assay, no cross-linked product could be detected (Fig. 7B, lane5), even after prolonged exposure of the x-ray film (data not shown).

M6P/IGF-II Receptor Repeats 10-11 Bind and Cross-link IGF-II

The failure of fusion protein 10 to bind IGF-II can be explained in two ways; the IGF-II-binding repeat may be located carboxyl-terminally of repeat 10, or repeat 10 binds and cross-links IGF-II only in the presence of the C-terminal repeats. To determine whether the presence of repeat 11 is required for the binding of IGF-II, we expressed the fusion protein 10-11, containing repeats 10-11 (amino acids 1367-1650, see Fig. 6). In Western analysis we detected a polypeptide with the mass of 65 kDa (Fig. 7A, lane4), which corresponds to the theoretical mass. Cross-linked to IGF-II a product of 65 kDa was detected (Fig. 7B, lane4), which deviates from the calculated mass of 72.5 kDa. Half-maximal inhibition of I-IGF-II cross-linking was estimated at about 200 nM unlabeled IGF-II (). These results indicate that the presence of repeat 11 is essential for binding or cross-linking of IGF-II.

M6P/IGF-II Receptor Repeat 11 Binds and Cross-links IGF-II Independently of Repeat 10

To analyze, if repeat 11 binds and cross-links IGF-II independent of repeat 10, we expressed the fusion protein 11-15, which contains repeats 11-15 (amino acids 1508-2293 of the M6P/IGF-II receptor, see Fig. 6). The molecular mass of 125 kDa in the Western analysis fits well to the theoretical mass of 122 kDa (Fig. 7A, lane3). Subjected to the IGF-II binding assay, a cross-linked product of 130 kDa was detected, which corresponds to the calculated mass (Fig. 7B, lane3). Half-maximal inhibition of cross-linking was estimated at about 40 nM unlabeled IGF-II (). Additional cross-linked products in the range of 10-45 kDa bind IGF-II with similar affinity, indicating that they consist of fragments of fusion protein 11-15. Since this fragments failed to bind anti-MPR46 antibody in Western analysis (Fig. 7A, lane3), we suggest that they lack the MPR46 domain. These results clearly demonstrate that repeat 11 is sufficient for binding of IGF-II.


DISCUSSION

We selected two different strategies to localize the binding site for IGF-II at the M6P/IGF-II receptor. In our first approach we purified two proteolytic receptor fragments of 23 and 37 kDa, which retained their ability to bind IGF-II. The amino acid sequences of this fragments overlap in the region of the C terminus of receptor repeat 9, the entire repeat 10, and the N-terminal 40% of repeat 11, indicating that this sequence of M6P/IGF-II receptor (amino acids 1331-1566) is sufficient for IGF-II binding.

Garmroudi and MacDonald (40) recently published a study on the localization of the IGF-II cross-linking site of the rat and bovine M6P/IGF-II receptor. In contrast to the approach used in this study, binding and cross-linking to IGF-II was carried out before the receptor was proteolytically fragmented. Several I-IGF-II cross-linked peptides with molecular masses of 14-18 kDa were isolated. N-terminal sequencing revealed that the peptides extend from the C-terminal fourth of repeat 10 to the N-terminal half of repeat 11 (corresponding to amino acids 1479-1581 of the human M6P/IGF-II receptor). Since lysine residues are missing in the C-terminal fourth of repeat 10, the authors concluded that IGF-II was cross-linked to one or several of the four lysine residues in the N-terminal half of repeat 11. Due to the experimental approach, it could not be determined whether the isolated fragments contain the IGF-II binding site, since the sites for binding and cross-linking might reside in different receptor repeats. In view of the results presented here, it is apparent that the cross-linking sites in repeat 11 are close to the IGF-II binding site and that only lysine residues 1509 and 1545 remain as candidate residues for cross-linking.

In our second approach we expressed truncated forms of the M6P/IGF-II receptor in cells and analyzed their ability to bind IGF-II. We could show that three fusion proteins containing repeats 10-15, 10-11, and 11-15 could be cross-linked to IGF-II, demonstrating that repeat 11 (amino acids 1508-1650) is sufficient for binding of IGF-II. The value of 10 nM unlabeled IGF-II leading to half-maximal inhibition of I-IGF-II cross-linking to fusion protein 10-15 corresponds to the value of 15 nM determined for the entire M6P/IGF-II receptor. The values of 40 and 200 nM determined for the fusion proteins 11-15 and 10-11 indicate that the strength of binding IGF-II is influenced by the presence of repeats 10 and 12-15.

In a related study Dahms et al.(24) analyzed truncated forms of the bovine M6P/IGF-II receptor transiently expressed in COS cells. They found that a construct encoding repeats 5-11 could be cross-linked to IGF-II, whereas a construct encoding repeats 5-10 failed to be cross-linked. These results showed that the presence of repeat 11 is necessary for the cross-linking to IGF-II. It remained unknown, however, whether repeat 11 is sufficient for IGF-II binding or whether one or several of repeats 5-10 are required to form the IGF-II binding site.

Taking the results of our two approaches, we can conclude that the N-terminal 40% of repeat 11, comprising amino acids 1508-1566, contains the binding site for IGF-II. Since IGF-II also binds to the IGF-I receptor, the insulin receptor(41) , and IGF-binding proteins (42), we searched for sequence homology between the IGF-II binding site of the M6P/IGF-II receptor residues 1508-1566 and the other IGF-II-binding polypeptides. We did not find an obvious homology with the IGF-I and the insulin receptor. This is not unexpected, as the amino acid residues of IGF-II mediating the binding to the M6P/IGF-II receptor and the other two receptors are located on different sites of the IGF-II molecule(43, 44) . The amino acid residues critical for binding of IGF-II to IGF-binding proteins form a patch that partly overlaps with the patch formed by the amino acid residues essential for binding to the M6P/IGF-II receptor(43, 44) . In spite of the overlapping binding sites at the ligand, obvious sequence homologies between the IGF-II binding sites at the M6P/IGF-II receptor and the IGF-binding proteins were not detectable.

In summary our data suggest that the N-terminal 40% of M6P/IGF-II receptor repeat 11 is sufficient to form the binding site for IGF-II. All M6P/IGF-II receptor constructs were expressed N-terminally fused to the extracytoplasmic domain of the MPR46. It should therefore be tested whether repeat 11 or a fragment of it would form a IGF-II binding site when expressed as such.

  
Table: Characterization of the binding of IFG-II to M6P/IGF-II receptor and the fusion proteins

Cross-linking of M6P/IGF-II receptor and fusion proteins to I-IGF-II was carried out in the absence and presence of 10 to 10M unlabeled IGF-II as indicated. After SDS-PAGE and autoradiography, cross-linked I-IGF-II was quantified by densitometry of the x-ray film and expressed as percentage of I-IGF-II bound in the absence of unlabeled IGF-II. All values represent the mean and standard deviation derived from 14-20 independent determinations.



FOOTNOTES

*
This work was supported by Grant Schm830/1-2 from the Deutsche Forschungsgemeinschaft and a grant from the Fonds der Chemischen Industrie. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 49-551-395951; Fax: 49-551-395979; E-mail: Gschmidt@pizza.uni-bc2.gwdg.de.

Present address: Saint Louis University, School of Medicine, Dept. of Biochemistry and Molecular Biology, 1402 S. Grand Blvd., Saint Louis, MO 63104.

The abbreviations used are: M6P/IGF-II receptor, mannose 6-phosphate/insulin-like growth factor II receptor; MPR46, 46-kDa mannose 6-phosphate receptor; IGF, insulin-like growth factor; BHK, baby hamster kidney; DSS, disuccinimidylsuberate; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride; ECL, enhanced chemiluminiscence; PMP, pentamannosye phosphate.


ACKNOWLEDGEMENTS

We thank Dr. W. S. Sly for providing the cDNA of the human M6P/IGF-II receptor, Kirsten Theiling and Wolfgang Lehnert for their help in preparing the DNA constructs, and Dr. R. Pohlmann for preparing the MPR46/pBEH plasmid. We thank Klaus Neifer for assistance during amino acid sequencing.


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