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The mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R) has diverse ligand-binding properties contributing to its roles in lysosome biogenesis and growth suppression. Optimal receptor binding and internalization of mannose 6-phosphate (Man-6-P)-bearing ligands requires a dimeric structure leading to bivalent high-affinity binding, presumably mediated by cooperation between sites on both subunits. Insulin-like growth factor II (IGF-II) binds to a single site on each monomer. It is hypothesized that IGF-II binding to cognate sites on each monomer occurs independently, but bivalent Man-6-P ligand binding requires cooperative contributions from sites on both monomers. To test this hypothesis, we co-immunoprecipitated differentially epitope-tagged soluble mini-receptors and assessed ligand binding. Pairing of wild-type and point-mutated IGF-II binding sites between two dimerized mini-receptors had no effect on the function of the contralateral binding site, indicating IGF-II binding to each side of the dimer is independent and manifests no intersubunit effects. As expected, heterodimeric receptors composed of a wild-type monomer and a mutant bearing two Man-6-P-binding knockout mutations form functional IGF-II binding sites. In contrast to prediction, such heterodimeric receptors also bind Man-6-P–based ligands with high affinity, and the amount of binding can be attributed entirely to the immunoprecipitated wild-type receptors. Anchoring of both C-terminal ends of the heterodimer produces optimal binding of both IGF-II and Man-6-P ligands. Thus, IGF-II binds independently to both subunits of the dimeric M6P/IGF2R. Although wild-type/mutant heterooligomers from readily when mixed, it appears that multivalent Man-6-P ligands bind preferentially to wild-type sites, possibly by cross-bridging receptors within clusters of immobilized receptors.
The mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R) is a 300-kDa transmembrane glycoprotein that has diverse ligand-binding properties that contribute to several important cellular functions [1, 2]. Insulin-like growth factor II (IGF-II) binding to the M6P/IGF2R leads to uptake into the cell and degradation of the growth factor in lysosomes [3–6]. This activity reduces IGF-II availability in the pericellular milieu, thereby decreasing its binding to mitogenic IGF-I receptors, which contributes substantially to the function of the M6P/IGF2R as a growth or tumor suppressor. Binding of lysosomal enzymes by the receptor is mediated by mannose 6-phosphate (Man-6-P) groups on N-linked oligosaccharides, and this mechanism is critical to lysosome biogenesis . There are also a number of glycoproteins other than the lysosomal enzymes that bind to the receptor by a Man-6-P-dependent mechanism, including thyroglobulin, proliferin, granzyme B, and latent transforming growth factor-β [1, 2, 8]. Several ligands, such as retinoic acid, urokinase-type plasminogen activator receptor (uPAR), and plasminogen, have also been reported to interact with the M6P/IGF2R via novel binding sites [9–13].
The human M6P/IGF2R consists of a large extracytoplasmic domain (ectodomain) of 2265 amino acid residues, a 23-residue transmembrane domain, and a short, 164-residue cytoplasmic domain [14, 15]. The ectodomain comprises fifteen repeats having 14–28% sequence identity. Each of the repeats is formed by a disulfide-bonded, crossed antiparallel β-sheet sandwich that resembles a flattened β-barrel . Ligand binding experiments have mapped the Man-6-P binding domains mainly to repeats 3 and 9, wherein mutation of critical residues can reduce ligand affinity  and in agreement with the structure of repeat 3 deduced from x-ray crystallography . The main amino acid residues involved in IGF-II binding are located within repeat 11, but residues within repeat 13 cooperate with repeat 11 to enhance ligand binding affinity [19–22].
Until recently, the M6P/IGF2R was considered to be monomeric in structure, and this view was supported by studies on the physicochemical properties of the solubilized receptor . However, recent work by York et al.  demonstrated that phosphomannosyl ligands with multiple Man-6-P moieties, such as β-glucuronidase, induced the cell-surface M6P/IGF2R to form dimers, which enhanced the rate of receptor internalization. These studies provided the first evidence of ligand-mediated cross-bridging of receptor monomers into a dimeric structure that interacted with apparently increased efficiency with the endocytic apparatus. IGF-II binding failed to produce such an increase in receptor internalization, which supported the hypothesis that IGF-II binds to its sites on the individual monomeric receptors . Further insight into the mechanism of dimerization was provided by Byrd et al. , who showed that dimer formation could occur independently of ligand binding, presumably mediated by direct interactions between the ectodomains of each monomer. Kreiling et al.  found that there is not a specific M6P/IGF2R dimerization domain, but rather there are interactions that exist between dimer partners all along the ectodomain of the receptor. Collectively, these studies led to the hypothesis that production of high-affinity ligand binding arises from cooperation between Man-6-P binding sites on each monomeric partner [1, 27]. The dimer-based model for high-affinity Man-6-P binding has recently received support from structural analysis of repeats 1–3 of the receptor's ectodomain by Olson et al. . Although binding of IGF-II by the M6P/IGF2R does not induce receptor dimerization  and it is known that IGF-II binds the receptor with one-to-one stoichiometry , it is unknown whether dimerization of the receptor has any effect on IGF-II binding. That is, would a defective IGF-II binding site on one monomer interfere with IGF-II binding on the other monomer?
The present work was designed to test the hypothesis that IGF-II binds independently to its binding sites on each receptor monomer, but that Man-6-P ligand binding is bivalent, requiring cooperative interaction of cognate sites on both monomers of the dimeric receptor. To test this hypothesis, we have measured ligand binding by dimers formed from cDNA constructs encoding repeats 1 to 15 of the M6P/IGF2R ectodomain. Our co-immunoprecipitation data indicate that oligomer formation does occur between receptors bearing different C-terminal epitope tags. Heterodimeric receptors composed of a wild-type monomer and a mutant bearing an IGF-II binding knockout mutation can form fully functional phosphomannosyl binding sites. In contrast, such receptor dimers are capable of binding IGF-II to the wild-type side, but not to the mutant side of the dimer. Overall, analysis of IGF-II binding in such receptor dimers suggests that each half of the dimer is capable of binding IGF-II independently of ligand occupancy of the contralateral site. A heterodimeric receptor composed of a wild-type monomer and a mutant bearing two Man-6-P binding knockout mutations can form functional IGF-II binding sites. However, such receptors are also capable of high-affinity Man-6-P binding, with the amount of ligand binding being directly proportional to the amount of the wild-type receptor present. These results can be explained either by a sterically improbable intramolecular binding mechanism or if binding of a multivalent ligand forces receptors to realign within the immunoprecipitated complexes to promote preferential cross-bridging between wild-type receptors.
The M6P/IGF2R ectodomain is critical for receptor ligand binding and dimerization [19, 25, 29, 30]. Therefore, receptor constructs for testing these functions were designed to encode all 15 repeats of the ectodomain of the M6P/IGF2R followed by either an 8-residue FLAG epitope tag or a 12-residue Myc tag (Fig. 1A). Distinct epitope tags were used to allow detection of heterologous interactions between mini-receptors. Two forms of the FLAG and Myc epitope-tagged mini-receptors, 1–15 wild-type and 1–15 I1572T (I/T) were transiently expressed alone or co-expressed in HEK 293T human embryonic kidney cells. Cell extracts were prepared using Triton X-100 and analyzed for relative expression levels of the mini-receptors by immunoblotting with M2 α-FLAG or 9E10 α-Myc antibodies (data not shown).
The two differentially tagged mini-receptors were constructed to assess the possibility of intersubunit effects between receptors. Several groups have determined that the I1572T mutation residing in the heart of the IGF-II binding domain in repeat 11 disrupts IGF-II binding to the receptor [22, 31–33]. The ligand blotting data in Figure 1C confirm that the wild-type mini-receptors used in this study could bind IGF-II, whereas the I/T mutant mini-receptors could not. In contrast, both wild-type and mutant mini-receptors bound the phosphomannosylated pseudoglycoprotein PMP-BSA (Fig. 1B). The presence of the different epitope tags on the mini-receptors had no apparent effect on ligand binding in this assay.
Previous work from our laboratory suggested the possibility of negative cooperativity of ligand binding by the oligomeric M6P/IGF2R . Thus, prior to examination of FLAG- and Myc-tagged mini-receptors, we assessed the ligand binding characteristics of a mixture of wild-type and mutant FLAG-tagged mini-receptors. To accomplish this, cells were transfected with a mixture of cDNAs in which the proportion of mutant cDNA to wild-type cDNA was increased while the total amount of cDNA was held constant. The effects of the I/T mutation on both IGF-II and PMP-BSA binding were analyzed using FLAG-tagged mini-receptors in a mixed immunoprecipitation, which ensured that both C-terminal ends were anchored to the resin in the same way (Fig. 2). Binding of 125I-PMP-BSA to mixed mini-receptors, which was measured to establish a baseline of ligand binding function, was not affected by the proportion of wild-type to mutant mini-receptor, suggesting that the I/T mutation did not interfere with functional phosphomannosyl ligand binding (Fig. 2A). Binding of 125I-IGF-II to immunoprecipitated mini-receptors was measured to assess if IGF-II binds the wild-type mini-receptor in the presence of the I/T mutant mini-receptors (Fig 2B). It was predicted that the presence of the I/T mutant mini-receptors would not interfere with IGF-II binding to the wild-type receptors because IGF-II is a monovalent ligand that should bind independently to each available receptor . The data in Figure 2B support this idea, as the total amount of IGF-II binding tended to follow the line displayed on the bar graph, which was calculated based on the percentage of wild-type vs. mutant receptor cDNAs input into the original transfection (Fig. 2B).
To determine if co-transfected mini-receptors interact in a possible oligomeric complex, 293T cell lysates containing co-expressed FLAG and Myc epitope-tagged mini-receptors were analyzed by an immunoprecipitation assay using M2 α-FLAG affinity resin. The resin pellets were washed to remove unbound proteins, separated by reducing SDS-PAGE, and analyzed by immunoblotting with the α-FLAG or α-Myc antibodies to determine if the Myc epitope-tagged mini-receptors interacted with the FLAG epitope-tagged mini-receptors (Fig. 3A–3D). Cells were transfected either with 30 μg of cDNA encoding the FLAG- and Myc-tagged mini-receptors alone or with a combination of 15 μg of each differentially tagged mini-receptor cDNA. PhosphorImager analysis of the blots revealed that essentially all of the expressed FLAG-tagged mini-receptors precipitated by incubation with the M2 affinity resin (Fig. 3A vs. 3B). If the level of expression of mutant vs. wild-type receptors reflects the proportion of their respective cDNAs in the transfection, and based on random association to form dimers, it was projected that co-immunoprecipitation of the FLAG-tagged mini-receptors from a 1:1 transfection pool would yield a 1:2:1 distribution of wild-type homodimers, wild-type/mutant heterodimers, and mutant homodimers, respectively. The data in Figure 3, panels C and D, indicate that ~50% of the co-expressed Myc-tagged mini-receptors were co-immunoprecipitated with the FLAG-tagged mini-receptors (Fig. 3C vs. 3D), suggesting that about half the Myc-tagged mini-receptors existed as homodimers (which do not precipitate in this assay) and the other half formed heterodimers with the FLAG-tagged mini-receptors. The data in Figure 3C and 3D indicate that the Myc-tagged mini-receptor did not immunoprecipitate in the absence of a FLAG-tagged partner. In addition, it is noteworthy that the presence of the I/T mutation had no apparent effect on the interaction leading to co-immunoprecipitation.
These data indicate that differentially epitope-tagged M6P/IGF2R mini-receptors were capable of association as asymmetric oligomers, but they do not indicate whether these structures are functional in ligand binding. To test this property, co-immunoprecipitated mini-receptors were subjected to direct binding analysis using radiolabeled ligands (Fig. 3E and 3F). For this purpose, differentially tagged mini-receptors were co-immunoprecipitated using a FLAG-based antibody from lysates of cells transfected with a 1:1 ratio of receptor cDNAs. We would expect that ~25% of the Myc-tagged mini-receptors would be present as homodimers, and thus would not precipitate in this assay. Thus, it was projected that PMP-BSA binding to the co-immunoprecipitated mini-receptors would yield approximately 75% of the binding observed with individually immunoprecipitated FLAG-tagged mini-receptors (Fig. 3E). Binding of 125I-PMP-BSA to immunoprecipitated mini-receptors was not affected by the proportion of wild-type vs. I/T mutant mini-receptors in the mixture, suggesting that the I/T mutation did not interfere with the formation of oligomers that are functional in phosphomannosyl ligand binding and establishing a baseline of ligand binding function (Fig. 3E).
Binding of 125I-IGF-II to immunoprecipitated mini-receptors was measured to assess if IGF-II binds independently to the asymmetric heterodimers (Fig. 3F). In this assay, we expected that 25% of the dimers formed would be Myc-tagged symmetric homodimers and therefore would not be immunoprecipitated by the M2 resin, 25% would be FLAG-tagged symmetric homodimers, and the remaining 50% of the dimers formed would be FLAG- and Myc-tagged asymmetric heterodimers. The calculations projected that the percentage of binding would follow the line displayed on the bar graph. However, the data show that when a mutant FLAG-tagged mini-receptor served as the “bait” for immunoprecipitation, binding of IGF-II to the asymmetric heterodimers was suppressed or not detected as readily as expected. One possibility to explain this functional deficit may be interference from the pairing of two different C-terminal epitope tags.
To test whether the FLAG or Myc epitope tags interfere with the formation of fully functional receptors in asymmetric heterodimers by having only half of the complex tethered to the resin, a reciprocal immunoprecipitation was performed. Cell lysates containing co-expressed FLAG and Myc epitope-tagged mini-receptors were analyzed by an immunoprecipitation assay using protein-G Sepharose and 9E10 α-Myc antibody. Immunoblots revealed that essentially all of the input Myc-tagged mini-receptors precipitated in the assay (Fig. 4A vs. 4B). The data in Figure 4, panels C and D, indicate that ~50% of the co-expressed FLAG-tagged mini-receptors were co-immunoprecipitated with the Myc-tagged mini-receptors (Fig. 4C vs. 4D). As was observed in α-FLAG-based immunoprecipitations (Fig 3), the presence of the I/T mutation had no effect on the interaction leading to co-immunoprecipitation.
To assess the ligand binding function of these asymmetric heterodimers, co-immunoprecipitated mini-receptors were subjected to direct binding analysis using radiolabeled ligands (Fig. 4E–4F). Based on the premise that only 75% of the dimers formed during this assay would be precipitable using the Myc-based immunoprecipitation, the amount of 125I-PMP-BSA binding was calculated and represented according to the line on the bar graph (Fig. 4E). The data in Figure 4E for the co-immunoprecipitated mini-receptors were consistent with this expectation and the results seen with complementary α-FLAG immunoprecipitation (Fig. 3E).
Binding of 125I-IGF-II to immunoprecipitated mini-receptors was measured to determine if IGF-II binds independently to both sides of the asymmetric hetero-oligomers (Fig. 4F). It was projected that the percentage of binding would follow the line displayed on the bar graph; however, when the I/T mutant Myc-tagged mini-receptor served as the bait for immunoprecipitation by the resin, binding of IGF-II to the asymmetric hetero-oligomers was interfered with or not detected as readily as expected. These results are consistent with the results observed with α-FLAG immunoprecipitation from the same panel of mini-receptor transfections (Fig. 3F). It appeared that no matter which epitope tag of the hetero-oligomer was anchored to the immunoprecipitating resin, IGF-II binding was suppressed when the tethering partner (bait) was the I/T mutant. To test the possibility that properties of the Myc epitope tag might somehow be responsible for this phenomenon, the effects of a different epitope tag, hemagglutinin (HA), were examined in pairing with FLAG-tagged receptors. However, these results (data not shown) were consistent with the results obtained with FLAG- and Myc-tagged partners (Fig. 3F) even though a different epitope tag (HA instead of Myc) was combined with the FLAG epitope tag.
Two forms of the FLAG-tagged mini-receptors, 1–15 wild-type and 1–15 R426A/R1325A (R2A), were constructed to assess the possibility of intersubunit effects for the phosphomannosyl ligand binding sites of the mini-receptors (Fig. 5A). These mini-receptors were transiently expressed alone or co-expressed in 293T cells. Cell extracts were analyzed for relative expression levels of the mini-receptors by immunoblotting with M2 α-FLAG antibody (data not shown).
Binding of 125I-IGF-II by immunoprecipitated wild-type versus R2A mutant mini-receptors was independent of the proportion of wild-type to mutant mini-receptors in the mixture, suggesting that the R2A mutation did not disrupt IGF-II binding (Fig. 5B). These data supported the hypothesis that IGF-II binding to the co-immunoprecipitated mini-receptors would be nearly the same as that observed with the mini-receptors expressed individually. Binding of 125I-PMP-BSA to immunoprecipitated mini-receptors was measured to assess whether the presence of the mutant mini-receptors affected PMP-BSA binding to the wild-type mini-receptors (Fig. 5C). In these experiments, the null hypothesis states that there is no cross-talk between binding sites within the mixture and thus binding should simply reflect the proportions of wild-type and mutant receptors in the transfection panel. Thus, we projected that the percentage of binding based on contributions of the mutant (no binding activity) and wild-type (100% binding activity) mini-receptors in the mixture would follow the line displayed on the bar graph and suggests that the wild-type binding site on one receptor is not affected by the presence of a mutant mini-receptor that is not capable of binding PMP-BSA.
Two forms of the Myc epitope-tagged mini-receptors, 1–15 wild-type and 1–15 R426A/R1325A (R2A) (Fig. 6A), were constructed to assess whether these co-transfected differentially tagged mini-receptors can be immunoprecipitated as oligomeric complexes. These mini-receptors were transiently expressed alone or co-expressed in 293T cells, and analyzed for relative expression levels of the mini-receptors by immunoblotting with α-FLAG and α-Myc antibodies (data not shown).
The cell lysates with volumes normalized for expression of the FLAG-tagged mini-receptor were analyzed by an immunoprecipitation assay using M2 α-FLAG affinity resin. PhosphorImager analysis of the immunoblots confirmed that essentially all of the expressed FLAG-tagged mini-receptors precipitated by incubation with the M2 affinity resin (Fig. 6B vs. 6C). The data in Figure 6, panels D and E, indicate that ~50% of the co-expressed Myc-tagged mini-receptors were co-immunoprecipitated with the FLAG-tagged mini-receptors (Fig. 6D vs. 6E), suggesting a balanced distribution between Myc-tagged mini-receptor homo-oligomers and hetero-oligomers with the FLAG-tagged mini-receptors. The presence of the R2A mutation had no detectable effect on the interaction leading to co-immunoprecipitation.
Binding of 125I-IGF-II to immunoprecipitated wild-type vs. R2A mutant mini-receptors was equivalent, suggesting that the R2A mutation had no discernible effect on the formation of oligomers that are functional in IGF-II binding (Fig. 6F). These data were consistent with the prediction that, since 25% of the oligomers formed should be homo-oligomers of Myc-tagged mini-receptors, IGF-II binding by the co-immunoprecipitated mini-receptors should have yielded approximately 75% of the binding observed with individually immunoprecipitated, FLAG-tagged mini-receptors.
Binding of 125I-PMP-BSA to these asymmetric hetero-oligomers was measured to determine whether PMP-BSA binding would show interactive effects between the wild-type and mutant partners (Fig. 6G). As was seen before when a mutant FLAG-tagged mini-receptor acted as the bait molecule in the immunoprecipitation, binding of PMP-BSA to the asymmetric hetero-oligomers was suppressed. These results are consistent with the results observed in experiments with the I/T mutation, suggesting that the ligand binding functions of the receptor's ectodomain can operate independently of one other within each receptor and relative to receptor partners, suggesting again that this effect depends on tethering the C-terminal ends of the extracytoplasmic domains.
The rationale for this work was to improve understanding of how the subunits of the multimeric M6P/IGF2R participated in binding the two main classes of ligands, IGF-II and phosphomannosylated glycoproteins. In mammals, binding of IGF-II by the M6P/IGF2R is thought to contribute to growth homeostasis. Previous work has shown that the receptor operates optimally as a dimer and we wanted to determine what effect the dimeric structure may have on IGF-II binding. It has been suggested that IGF-II binding to the M6P/IGF2R requires contributions of repeats 11 and 13, but only within a single polypeptide chain [20, 22, 33]. It is well established that the receptor binds Man-6-P ligands in a multivalent fashion [1, 18, 35]. However, because the receptor has two Man-6-P binding domains within a single polypeptide chain, it remains uncertain whether this bivalent binding activity is a property of a single monomeric receptor or the result of cooperative interaction between the two subunits of a dimeric receptor. There is strong evidence that, in the cell, the preferred mode of binding is through a dimeric structure as shown by York et al., who found that a multivalent phosphomannosylated ligand cross-bridged the dimeric receptor to promote optimal internalization . This conclusion was reinforced by the work done by Byrd et al. , who analyzed mutant receptors bearing a substitution of Arg for Ala at position 1325 that knocks out Man-6-P ligand binding to the repeat 9 site. Scatchard plot analysis showed that these mutant receptors were still able to bind bivalent Man-6-P ligands with high-affinity, leading to the conclusion that high-affinity binding in that case must be due to alignment of two repeat 3 Man-6-P binding domains on paired monomers. Furthermore, Olson et al.  demonstrated via x-ray projection models that the closest distance between the two Man-6-P binding sites of one monomeric receptor is 45–70 Å, indicating that a single diphosphorylated oligosaccharide, with a maximum distance of ~30 Å , could not bind to the Man-6-P binding domains of both repeats 3 and 9 simultaneously . The present study was designed to test if ligand binding by the dimeric receptor is cooperative, based on the hypothesis that IGF-II binds independently to cognate sites on both monomers of the dimeric receptor, but that Man-6-P ligand binding would require cooperation of both monomers. For this purpose we developed a quantitative assay for heterodimer formation that was based on immunoprecipitation of differentially epitope-tagged receptors. The availability of the I1572T mutant allowed us to address the initial question whether a non-functional IGF-II binding site would interfere with the function of a wild-type binding site when paired in a single heterodimeric structure.
Association assays indicated that immunoprecipitation between differentially epitope-tagged mini-receptors was feasible. These assays also indicated that immunoprecipitation was not preferential to the epitope used to tag the receptor, as there was no discernable difference between the Myc- and HA-tagged receptors in co-immunoprecipitation with the FLAG-tagged receptor. As expected, we observed that essentially all of the FLAG-tagged mini-receptors were precipitated by incubation with M2 resin. In experiments with the FLAG-tagged receptor as the bait, ~50% of the Myc-tagged mini-receptors were co-precipitated from a cell lysate prepared from cells co-transfected with equal amounts of the tagged mini-receptor cDNA. This strongly suggests, but does not prove, that the mini-receptors associated in a 1:2:1 relationship: 25% FLAG homodimers, 50% FLAG-Myc heterodimers, and 25% Myc homodimers (which would not precipitate in this assay). The simplest interpretation of these data relative to the structure of the receptor is that the mini-receptors were in the form of dimers. Byrd et al.  showed via mutational analysis that receptors with only one functional Man-6-P binding site exhibited high-affinity binding of Man-6-P-containing ligands. Given that high-affinity binding of a bivalent ligand is due to cooperative interaction with two or more receptor binding sites , these data suggested that oligomerization of the receptor contributes to high-affinity binding. In addition, native gel electrophoresis demonstrated that the receptor could be separated into monomeric and dimeric forms in the presence or absence of Man-6-P-containing ligands . York et al  demonstrated by sucrose gradient sedimentation and gel filtration that the receptor bound to a multivalent Man-6-P-containing ligand, β-glucuronidase, exhibited a sedimentation coefficient and Stokes radius that were consistent with a complex of two receptor molecules plus one molecule of ligand. However, when these experiments were performed with IGF-II, the receptor appeared to exist as a monomer. Internalization experiments performed with 125I-IGF-II in the presence of β-glucuronidase revealed that β-glucuronidase accelerated the rate of IGF-II uptake, suggesting that intermolecular cross-linking of receptors enhanced receptor endocytosis . Thus, all the available data are consistent with the conclusion that the M6P/IGF2R functions as a dimer in high-affinity binding of Man-6-P-bearing ligands, but possibly not for IGF-II. Our work with soluble forms of the receptor ([25, 26, 34] and the present study) indicates that the ectodomain of the receptor is capable of dimer formation in the absence of ligand.
Affinity cross-linking of 125I-IGF-II to a mutant repeat 11 mini-receptor revealed that the I1572T mutation completely abolished IGF-II binding . This finding was confirmed by Linnell et al.  by utilizing surface plasmon resonance of a truncated receptor containing extracytoplasmic repeats 10–13, which contained the I1572T mutation in repeat 11. However, the mechanism by which this mutation abrogates IGF-II binding is still not clear. Structural analysis of repeat 11 identified the presumptive IGF-II binding site in a hydrophobic pocket at the end of a β-barrel structure . Ile1572 was found to lie near, but not directly within, this putative IGF-II binding site. This mutation involves substituting a polar residue, Thr, for a bulky, nonpolar residue, Ile, which might have altered the IGF-II binding pocket by inducing a conformational change that reduces binding energy or makes the site less hydrophobic. In any case, this type of effect should be regional and have minimal influence on the wild-type IGF-II binding site on an adjacent mini-receptor within a dimer. Our experiments support this prediction, showing that the pairing of wild-type and 11572T mutant IGF-II binding sites between two dimerized mini-receptors had no effect on the function of the contralateral binding site. The mutant site does not prevent the wild-type site from binding IGF-II and pairing with a wild-type subunit does not repair the defect in the mutant site inducing it to bind IGF-II. This indicates that IGF-II binding to each side of the dimer is independent. Symmetric heterodimers (having identical epitope tags, both of which are tethered to the resin bead) achieve the predicted amount of binding as described above. Tethering of both sides of the dimer likely mimics the structure obtained when anchored in the membrane, in accordance with the notion that this structure is the receptor's normal functional state.
The most interesting and unexpected finding of the present study is that asymmetric heterodimers (having different epitope tags, of which only one is tethered to the resin bead) demonstrate complex binding behavior. Dimers of this type exhibit the predicted amount of binding only if the heterodimer is tethered by the wild-type partner. In contrast, we found that if the heterodimer is tethered to the resin by the mutant partner, the amount of binding observed is substantially less than expected. This complex binding behavior seen with IGF-II binding must only be a local effect, as binding of PMP-BSA, which binds to other sites in the ectodomain of these heterodimers, resulted in the predicted amount of ligand binding. This loss of binding function observed with asymmetric heterodimers may be due to deformation of the dimeric structure. One possible explanation for failure to form a dimer of correct structure could be steric hindrance between the Myc tag and the M2 resin bead. This is envisioned to cause the Myc-tagged receptor partner to be bent outward away from the tethered FLAG-tagged partner, potentially resulting in distortion of the IGF-II binding pocket and consequent reduced ability to bind IGF-II. This effect is likely not due to reduced contact between repeats 11 and 13, as repeat 11 is capable of binding IGF-II even in the absence of repeat 13 [20, 22]. These data suggest that the failure to tether the tail of the extracytoplasmic domain results in the inability to form appropriate contacts between dimeric partners. Follow-up experiments using structural approaches will be needed to address this possibility.
Localization of the two Man-6-P binding domains was previously reported by Westlund et al. , who subjected the M6P/IGF2R to partial proteolytic digestion using subtilisin. They determined that repeats 1–3 and 7–10 can independently bind Man-6-P-containing ligands. Dahms et al.  further defined the location of Man-6-P binding sites by using mutational analysis to establish the importance of specific Arg residues in the function of both Man-6-P binding sites. They determined that Arg426 and Arg1325 in repeats 3 and 9, respectively, are essential components of the receptor's high-affinity Man-6-P binding sites. The structure of repeats 1–3 of the bovine M6P/IGF2R in the presence of Man-6-P was solved by Olson et al. . This work revealed key amino acid residues in the binding site of repeat 3 that were important for Man-6-P binding. In particular it was found that the guanidinium group of Arg435 (corresponding to Arg111 of the CD-MPR and Arg426 of the human M6P/IGF2R) forms one or two critical hydrogen bonds with the 2'-hydroxyl group of the mannose ring but these represent only two out of more than a dozen non-covalent interactions between ligand and receptor within the binding pocket. This leads to the question as to whether the R→A mutation actually causes a loss of binding energy sufficient to abrogate Man-6-P binding or whether the mutated binding pocket becomes distorted, preventing Man-6-P binding. Glucose 6-phosphate (Glc-6-P) is unable to bind to the two Man-6-P binding sites of the receptor . The only difference between Glc-6-P and Man-6-P is the position of the 2'-hydroxyl group. The equatorial 2-hydroxyl group of Glc-6-P is not in the correct position to make the necessary hydrogen bonds with the critical Arg; however, the axial 2-hydroxyl of Man-6-P is. Therefore it seems that the binding energy of the site must be decreased with the R→A mutation preventing Man-6-P from binding to the Man-6-P binding sites.
Based on this analysis and the need for cooperative interaction between two binding sties, we expected the wild-type/R2A heterodimers to show equivalent Bmax with reduced affinity; however, ligand binding analysis (data not shown) revealed a 50% decrease in Bmax while the affinity remained unchanged. This surprising finding led us to hypothesize that the large multivalent Man-6-P-based ligands such as PMP-BSA can bind bivalently to wild-type subunits of heterodimeric receptors juxtaposed on the resin. Whether these represent nearby heterodimers or receptors interacting in large oligomeric clusters is not yet clear. It is known from earlier work that the M6P/IGF2R is concentrated in coated pits on the plasma membrane and that there is a greater than 60-fold enrichment of receptor in clathrin-coated pits when compared to microsomes [37, 38]. During receptor-mediated endocytosis when there is a high accumulation of receptors into one location, it is possible they can interact in higher-order oligomeric structures. This would explain why we do not see a decrease in affinity of phosphomannosyl ligands between wild type and R2A mutant receptors. This accumulation of receptors may be mimicked on the resin bead during an immunoprecipitation such that the multivalent ligand binding selects binding-competent partners, promoting preferential cross-bridging between wild-type receptors and forming higher oligomeric structures on the resin bead. As was seen with the IGF-II binding mutant, asymmetric heterodimers that are tethered by mutant receptors show an unexpected decrease in PMP-BSA binding. It thus appears that tethering of the C-terminal end of the ectodomain is important for both IGF-II and PMP-BSA binding.
In summary, the major findings of this work are consistent with a dimer model for M6P/IGF2R oligomerization as all co-immunoprecipitations resulted in a predicted outcome of pull-down or ligand binding irrespective of the tag or mutant. IGF-II was found to bind independently to sites on each monomeric partner while high-affinity binding of multivalent Man-6-P ligands was proportional to the number of wild-type binding sites available in a mixture of mutant/wild-type receptors. Tethering to the resin of asymmetric heterodimers suggests the importance of anchoring the tail of the ectodomain for both IGF-II and Man-6-P ligand binding. Receptor binding on resin resembles a patchwork model where a multivalent ligand can cross-bridge between nearby wild-type sites to achieve high affinity binding. Additionally, further studies are needed to obtain more information on the structure of the M6P/IGF2R ectodomain particularly toward the C-terminal repeat 15 and its interaction with the membrane to assess if there are important associated proteins or receptor-lipid interactions that cannot be mimicked using epitope-tagged portions of the ectodomain. Finally, experiments in whole cells must address the possibility that the receptor can associate in patches during endocytosis.
D-Man-6-P disodium salt, the α(anti)-FLAG M2 antibody, the α-FLAG M2-agarose affinity gel, and the protein G-Sepharose reagents were obtained from Sigma (St. Louis, MO). The α-Myc 9E10 antibody was purchased from Upstate Biotechnology, Inc. (Hercules, CA) or the University of Nebraska Medical Center Monoclonal Antibody Facility (Omaha, NE). The anti-hemagglutinin (α-HA) antibody was purchased from Roche Diagnostics Corporation (Indianapolis, IN). Rabbit α-mouse IgG was from Dako Corp. (Carpinteria, CA), carrier-free Na125I and 125I-protein A was from PerkinElmer Life Sciences (Boston, MA). Recombinant human IGF-II was a gift of Lilly Research Laboratories (Indianapolis, IN). The pseudoglycoprotein pentamannosyl 6-phosphate-bovine serum albumin (PMP-BSA) was prepared as described previously . Radiolabeled PMP-BSA was prepared by iodination using precoated IODOGEN tubes from Pierce (Rockford, IL) according to the specifications of the manufacturer to a specific activity of 25–70 Ci/g. IGF-II was radioiodinated using Enzymobeads from Bio-Rad (Hercules, CA) to a specific activity of 33–114 Ci/g. The pCMV5 vector was provided by Dr. David W. Russell (University of Texas Southwestern Medical Center, Dallas, TX) . The 8.6-kilobase pair human M6P/IGF2R cDNA was a gift of Dr. William S. Sly (St. Louis University Medical Center, St. Louis, MO) . Oligonucleotides were synthesized by Integrated DNA Technologies (Coralville, IA). Other reagents and supplies were obtained from sources as indicated.
A soluble receptor containing all 15 extracytoplasmic repeats of the M6P/IGF2R followed by a FLAG epitope tag (DYKDDDDK), called 1–15F, was engineered from the hIGF2R cDNA as described previously . Two similar receptors, 1–15Myc and 1–15HA, were also generated using the same strategy with a COOH-terminal Myc (MEQKLISEEDLN) or HA (YPYDVPDYA) epitope tag engineered in place of the FLAG tag . Site-directed mutagenesis was carried out using the QuikChange™ mutagenesis kit from Stratagene (La Jolla, CA). Pfu-directed thermal cycling was conducted with a fragment of the human M6P/IGF2R cDNA encompassing two PflMI sites (nt 3847–6315) in pCRII (Invitrogen, Carlsbad, CA). Complementary primer pairs corresponding to nt 4840–4874 substituting T to C at nt 4862 creating the Ile1572 → Thr (I1572T) mutation. The resultant product was digested with PflMI and subcloned into a shuttle vector containing the M6P/IGF2R cDNA. The presence of the mutation was confirmed by sequence analysis. Finally, the mutant construct was digested with EagI, and the 5.2-kilobase fragment was subcloned into the pCMV5 vector containing the FLAG-tagged (1–15F), Myc-tagged (1–15Myc), or HA-tagged (1–15HA) human receptor constructs creating the 1–15F I/T, 1–15Myc I/T, and 1–15HA I/T mutant receptors, respectively.
Soluble, FLAG- or Myc-tagged mini-receptors that were mutant for residues in the repeats 3 and 9 Man-6-P binding domains (R426A and R1325A, respectively) were prepared as follows: nucleotides (nt) 100–3242 of the M6P/IGF2R cDNA served as a template for amplification using a 5'-primer containing an EcoR I restriction site preceding the sequence corresponding to nt 1112–1129 of the receptor cDNA and a 3'-primer with sequence complementary to nt 2474–2487 of M6P/IGF2R cDNA, followed by an Xho I site. The products from these amplifications were digested with EcoR I and Xho I and subcloned into pBluescript SK II+ (pBSKII+) (Stratagene). This construct was subjected to two rounds of amplification with primers designed to incorporate the R426A mutation responsible for altering the ectodomain repeat 3 Man-6-P binding site using the Megaprimer approach . This first round of amplification involved producing the mutation, by amplifying from that site (nt 1425) to the 3' end of the mini-receptor (nt 2487). This “megaprimer” was then used in a second round of amplification with the 5'-primer used previously. The repeat 3 mutant amplification product was digested with EcoR I and Xho I and subcloned back into pBSKII+. An ~1 kb Bsm I fragment (sites at M6P/IGF2R nt 1408 and 2449) containing the mutation was removed from the megaprimer and subcloned into the corresponding positions of pBSKII+/Kpn, which contained a 2.2 kb Kpn I fragment derived from M6P/IGF2R nt 100–3242, creating a pBSKII+/Kpn-R426A mini-receptor with the 3rd repeat Man-6-P-binding mutation. The Kpn I fragment from this construct was then subcloned into pCMV5/R1325A that encoded either a FLAG- or Myc-tagged soluble receptor containing the 9th repeat Man-6-P-binding mutation synthesized as previously described , which had also been digested with Kpn I and Xmn I, creating a soluble M6P/IGF2R mini-receptor bearing Man-6-P binding-site mutations at both repeats 3 and 9 (1–15F R2A and 1–15Myc R2A).
Transient expression of soluble receptors was performed in 293T human embryonic kidney cells cultured in Dulbecco's modified Eagle medium supplemented with 5% fetal bovine serum and 5 μg/μl gentamycin at 37°C in a humidified atmosphere of 5% CO2/95% air. Transfection was carried out by a modification of the calcium phosphate method previously described . Cells were harvested three days after transfection and lysates were prepared by solubilization with 50 mM HEPES, pH 7.4, 1% Triton X-100, and 1 mM MgCl2, as previously described . After lysates were prepared, 25 μl aliquots were electrophoresed on a 6% SDS-PAGE gel under reducing conditions and transferred to BA85 nitrocellulose paper (Schleicher & Schuell). The blots were blocked with 4% nonfat milk in 50 mM HEPES, pH 7.4, 150 mM NaCl, 0.1% Tween 20 and probed with either the α-FLAG M2 antibody (1:2000), the α-Myc 9E10 antibody (1:500), or the α-HA antibody (1:250) followed by a secondary rabbit α-mouse IgG. The resulting antibody complex was developed with 125I-protein A and detected by means of autoradiography followed by PhosphorImager (Molecular Dynamics, Sunnyvale, CA) to quantify relative expression of the soluble receptors.
Aliquots of 293T cell lysates, containing equimolar amounts of expressed soluble receptors, were electrophoresed on a 6% SDS-PAGE gel under non-reducing conditions and transferred to BA85 nitrocellulose paper. The ligand blots were processed according to the procedure of Hossenlopp et al. , probed using 125I-IGF-II (~2 × 106 cpm/8 ml) or 125I-PMP- BSA (~2 × 106 cpm/8 ml) for 16 h at 4°C, and then developed by autoradiography.
Aliquots of 293T cell lysates, containing equimolar amounts of expressed FLAG-tagged soluble receptors, were incubated with 8 μl of packed M2 affinity resin in HEPES-buffered saline (HBS; 50 mM HEPES, pH 7.4, 0.15 M NaCl) plus 1% bovine serum albumin (BSA) and 5 mM Man-6-P for 3 h at 4°C on an end-over-end mixer. The resin was collected by centrifugation at 13,000 × g for 20 s. The resulting resin pellets were washed three times with 1 ml HBS containing 0.05% Triton X-100 (HBST). The resin pellets were heated to 100°C for 5 min and electrophoresed on a 6% SDS-PAGE gel under reducing conditions and transferred to BA85 nitrocellulose paper. The blots were probed with either α-FLAG M2, α-Myc 9E10, or α-HA antibodies, developed with 125I-protein A, followed by autoradiography and PhosphorImager analysis.
Aliquots of 293T cell lysates, containing equimolar amounts of expressed Myc-tagged soluble receptors, were incubated with 1 μl (1:100) of α-Myc 9E10 antibody in HBST for 16 h at 4°C. Protein G-Sepharose, preblocked by washing in HBS + 1% BSA, in the amount of 6 μl packed resin was introduced into the lysate-antibody mixture and allowed to incubate for 3 h at 4°C on an end-over-end mixer. The resin was collected by centrifugation at 13,000 × g for 20 s. The resulting resin pellets were washed three times with HBST. The pellets were heated to 100°C for 5 min in sample buffer and electrophoresed on a 6% SDS-PAGE gel under reducing conditions and transferred to BA85 nitrocellulose paper. The blots were probed with either α-FLAG M2 or α-Myc 9E10 antibodies, developed with 125I-protein A, followed by autoradiography and PhosphorImager analysis.
Aliquots of 293T cell lysates containing equimolar amounts of the expressed FLAG-tagged receptors were incubated with 8 μl of packed M2 affinity resin in HBS + 1% BSA for 3 h at 4°C. The addition of 5 mM Man-6-P at this point prevented the co-precipitation of endogenous phosphomannosylated ligands. The resin was collected by centrifugation at 13,000 × g for 20 sec. The resin pellets were washed three times with 1.0 ml of HBST. The ability of the immunoprecipitated receptors to bind 125I-IGF-II was measured by incubating the resin pellets with 2 nM 125I-IGF-II plus 100 nM unlabeled IGF-I in HBST 16 h at 4°C. The addition of IGF-I to the binding reaction prevented interference from IGF-binding proteins that exist in the cell lysates. The resin pellets were washed twice with 1.0 ml of HBST to remove unbound ligand, collected by centrifugation, and counted in a γ-counter. Specific 125I-IGF-II binding was determined by subtracting counts/minute ligand bound in replicate reactions carried out in the presence of 1 μM IGF-II. Binding of 125I-PMP-BSA was measured under similar conditions using 1 nM 125I-PMP-BSA in the presence or absence of 5 mM Man-6-P to compensate for nonspecific binding. The data were graphed and analyzed using GraphPad Prism™ software. Ligand binding parameters were measured by competitive binding analysis. Equal amounts of the receptor constructs were immunoadsorbed to M2 resin and incubated with 1 nM 125I-PMP-BSA in the presence of increasing concentrations of unlabeled PMP-BSA (from 0 to 500 nM) at 4°C for 3 h. The resin pellets were then washed and counted as described above. The data were fit to a model for one-site and two-site competitive binding using GraphPad Prism™ software.
We thank Dr. William S. Sly for providing the human M6P/IGF2R cDNA, Dr. David W. Russell for providing pCMV5, and The Rockefeller University for permission to use the 293T cells. We are grateful to Margaret H. Niedenthal of Lilly Research Laboratories for providing the IGF-II. We thank Barbara Switzer and the University of Nebraska Medical Center Monoclonal Antibody Facility for the production of the 9E10 antibody. We also thank Christine D. Dreis, Rosslyn Grosely, and Christopher M. Connelly for their input and technical support. This work is supported in part by National Institutes of Health Grant 5RO1CA91885 to R.G.M. J.L. Kreiling was the recipient of pre-doctoral stipend support provided by Graduate Studies; Bukey, McDonald, Emley, and Widaman fellowships; the Dr. Fred W. Upson Grant-in-aid award through the University of Nebraska Medical Center, and the NASA Space Grant Fellowship. M.A. Hartman was the recipient of pre-doctoral stipend support provided by Graduate Studies and Skala Fellowships through the University of Nebraska Medical Center and the GAANN Fellowship.