High-affinity ligand binding by wild-type/mutant heteromeric complexes of the mannose 6-phosphate/insulin-like growth factor II receptor Michelle A. Hartman 1 , Jodi L. Kreiling 2 , James C. Byrd 1 and Richard G. MacDonald 1 1 Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, USA 2 Department of Chemistry, University of Nebraska, Omaha, NE, USA The mannose 6-phosphate ⁄ insulin-like growth factor II receptor (M6P ⁄ IGF2R) is a 300 kDa transmembrane glycoprotein that has diverse ligand-binding properties contributing 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 deg- radation of the growth factor in lysosomes [3–6]. This activity reduces IGF-II availability in the pericellular Keywords insulin-like growth factor II; ligand binding; mannose 6-phosphate; mannose 6-phosphate ⁄ insulin-like growth factor II receptor; oligomerization Correspondence R. G. MacDonald, Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, 985870 Nebraska MED CTR, Omaha, NE 68198 5870, USA Fax: +1 402 559 6650 Tel:+1 402 559 7824 E-mail: rgmacdon@unmc.edu (Received 17 October 2008, revised 19 December 2008, accepted 21 January 2009) doi:10.1111/j.1742-4658.2009.06917.x The mannose 6-phosphate ⁄ insulin-like growth factor II receptor 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 struc- ture 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-immunopre- cipitated 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. By 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 indepen- dently to both subunits of the dimeric mannose 6-phosphate ⁄ insulin-like growth factor II receptor. Although wild-type ⁄ mutant hetero-oligomers form 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. Abbreviations Glc-6-P, glucose 6-phosphate; HA, hemagglutinin; HBS, Hepes-buffered saline; HBST, HBS containing 0.05% Triton X-100; IGF-II, insulin-like growth factor II; M6P ⁄ IGF2R, mannose 6-phosphate ⁄ insulin-like growth factor II receptor; Man-6-P, mannose 6-phosphate; pBSKII+, pBluescript SK II+; PMP-BSA, pentamannosyl 6-phosphate-BSA. FEBS Journal 276 (2009) 1915–1929 ª 2009 The Authors Journal compilation ª 2009 FEBS 1915 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 [7]. There are also a number of glycoproteins other than the lyso- somal enzymes that bind to the receptor by a Man-6- P-dependent mechanism, including thyroglobulin, proliferin, granzyme B and latent transforming growth factor-b [1,2,8]. Several ligands, such as retinoic acid, urokinase-type plasminogen activator receptor 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 extracy- toplasmic 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 b-sheet sandwich that resembles a flattened b-barrel [16]. 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 [17], and such mapping is in agreement with the structure of repeat 3 deduced from X-ray crystallography [18]. 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 sup- ported by studies on the physicochemical properties of the solubilized receptor [23]. However, a recent study by York et al. [24] demonstrated that phos- phomannosyl ligands with multiple Man-6-P moieties, such as b-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 struc- ture that interacted with apparently increased efficiency with the endocytic apparatus. IGF-II bind- ing 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 [24]. Further insight into the mechanism of dimerization was provided by Byrd et al. [25], who showed that dimer formation could occur indepen- dently of ligand binding, presumably mediated by direct interactions between the ectodomains of each monomer. Kreiling et al. [26] 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. [18]. Although binding of IGF-II by the M6P ⁄ IGF2R does not induce receptor dimerization [24] and it is known that IGF-II binds the receptor with one-to-one stoichiome- try [28], it remains 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 study aimed 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 measured ligand binding by dimers formed from cDNA constructs encoding repeats 1–15 of the M6P ⁄ IGF2R ectodo- main. Our co-immunoprecipitation data indicate that oligomer formation does occur between receptors bearing different C-terminal epitope tags. Hetero- dimeric receptors composed of a wild-type monomer and a mutant bearing an IGF-II binding knockout mutation can form fully functional phosphomannosyl binding sites. By 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, the analysis of IGF-II binding in such receptor dimers suggests that each half of the dimer is capable of binding IGF-II independently of the 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 by binding of a multivalent ligand forcing receptors to realign within the immunoprecipitated complexes, thus promoting preferential cross-bridging between wild-type receptors. Ligand binding by the dimeric M6P ⁄ IGF2R M. A. Hartman et al. 1916 FEBS Journal 276 (2009) 1915–1929 ª 2009 The Authors Journal compilation ª 2009 FEBS Results Transient expression and ligand binding properties of FLAG and Myc epitope-tagged M6P/IGF2R mini-receptors The M6P ⁄ IGF2R ectodomain is critical for receptor ligand binding and dimerization [19,25,29,30]. There- fore, receptor constructs for testing these functions were designed to encode all 15 repeats of the ectodo- main of the M6P⁄ IGF2R followed by either an eight- 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 anti-FLAG or 9E10 anti-Myc immunoglobulins (Igs) (data not shown). The two differentially tagged mini-receptors were constructed to assess the possibility of intersubunit effects between receptors. Several studies have deter- mined 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 shown in Fig. 1C confirm that the wild- type mini-receptors used in the present study could bind IGF-II, whereas the I ⁄ T mutant mini-receptors could not. By contrast, both wild-type and mutant mini-receptors bound the phosphomannosylated pseudoglycoprotein pentamannosyl 6-phosphate-BSA (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. Ligand binding by immunoprecipitated FLAG-tagged M6P/IGF2R mini-receptors Previous studies suggested the possibility of negative cooperativity of ligand binding by the oligomeric M6P ⁄ IGF2R [34]. 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, whereas 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 [ 125 I]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-recep- tor, suggesting that the I ⁄ T mutation did not interfere with functional phosphomannosyl ligand binding (Fig. 2A). Binding of [ 125 I]IGF-II to immunoprecipi- tated 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 pre- dicted that the presence of the I ⁄ T mutant mini-recep- tors 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 avail- Myc FLAG FLAG Myc COOH COOH COOH COOH H 2 N H 2 N H 2 N H 2 N * * Construct name: 1-15F 1-15F I/T 1-15Myc 1-15Myc I/T 9 113 51 - 1 F 1-15F I/T - 151 My c 1-15 M cIT y / IGF-II PMP-BSA Endogenous Endogenous A B C Fig. 1. Schematic diagram and ligand blot analysis of FLAG and Myc epitope-tagged M6P ⁄ IGF2R mini-receptors. (A) The receptor constructs are shown in linear format from the amino terminus to the carboxyl terminus, with repeats of the ectodomain shown as rectangles. The shaded rectangles indicate repeats 3 and 9, to which the main determinants of Man-6-P binding have been mapped. The stippled rectangles represent repeat 11 containing the principal residues responsible for IGF-II binding, and the aster- isk denotes the I>T mutation at residue 1572 (I ⁄ T), which abro- gates IGF-II binding. The black rectangles represent the FLAG or Myc epitope tags on the carboxyl terminus. (B, C) Equimolar amounts of the transfected cell lysates were electrophoresed on 6% nonreducing SDS ⁄ PAGE gels. The proteins were transferred to BA85 nitrocellulose, processed for ligand blotting and probed for binding of either [ 125 I]PMP-BSA (B) or [ 125 I]IGF-II (C) and developed by autoradiography. The autoradiograms of representative blots are shown. M. A. Hartman et al. Ligand binding by the dimeric M6P ⁄ IGF2R FEBS Journal 276 (2009) 1915–1929 ª 2009 The Authors Journal compilation ª 2009 FEBS 1917 able receptor [24]. The data shown in Fig. 2B support this idea because the total amount of IGF-II binding tended to follow the line displayed in the bar graph, which was calculated based on the percentage of wild- type versus mutant receptor cDNAs input into the original transfection (Fig. 2B). Ligand binding by co-immunoprecipitated FLAG- and Myc-tagged M6P/IGF2R mini-receptors To determine whether 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 anti-FLAG affinity resin. The resin pellets were washed to remove unbound proteins, separated by reducing SDS ⁄ PAGE, and analyzed by immunoblotting with anti-FLAG or anti-Myc Igs to determine whether the Myc epitope- tagged mini-receptors interacted with the FLAG epitope-tagged mini-receptors (Fig. 3A–D). Cells were transfected either with 30 lg of cDNA encoding the FLAG- and Myc-tagged mini-receptors alone or with a combination of 15 lg 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 incuba- tion with the M2 affinity resin (Fig. 3A versus B). If the level of expression of mutant versus wild-type receptors reflects the proportion of their respective cDNAs in the transfection, and based on random asso- ciation to form dimers, it was projected that co-immu- noprecipitation 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, respec- tively. Figure 3C,D indicates that approximately 50% of the co-expressed Myc-tagged mini-receptors were co-immunoprecipitated with the FLAG-tagged mini- receptors (Fig. 3C versus D), suggesting that approxi- mately 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. Figure 3C,D indicates that the Myc-tagged mini-receptor did not immuno- precipitate 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 associa- tion as asymmetric oligomers, but they do not indicate whether these structures are functional in ligand bind- ing. To test this property, co-immunoprecipitated mini-receptors were subjected to direct binding analysis using radiolabeled ligands (Fig. 3E,F). For this pur- pose, 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 approximately 25% of the Myc-tagged mini-receptors would be pres- ent as homodimers, and thus would not precipitate in this assay. Thus, it was projected that PMP-BSA bind- ing to the co-immunoprecipitated mini-receptors would yield approximately 75% of the binding observed with individually immunoprecipitated FLAG-tagged mini- 0 30 30 µg 1-15F cDNA µg 1-15I/T cDNA 0 0 25 50 75 100 125 150 125 I-PMP-BSA binding c.p.m. × 10 3 125 I-IGF-II binding c.p.m. × 10 3 0 10 20 30 40 A B Fig. 2. Analysis of ligand binding to soluble 1-15 and 1-15 I ⁄ T mutant FLAG epitope-tagged receptors immunoprecipitated with anti-FLAG resin. Cell lysates, containing equimolar amounts of expressed soluble receptors, were immunoprecipitated with M2 anti-FLAG affinity resin and assayed for binding of [ 125 I]PMP-BSA (A) or [ 125 I]IGF-II (B). The lines in each graph indicate the amount of binding predicted if the wild-type and mutant receptors are binding ligand independently. The triangles indicate a progressive shift in the ratio of wild-type to mutant receptor cDNA transfected into cells. Values represent the mean ± SD of three replicate measure- ments for each condition. These data represent the means of four independent experiments. [Correction added on 5 March 2009 after first online publication: in Fig. 2B ‘ 125 I-PMP-BSA binding’ was cor- rected to ‘ 125 I-IGF-II binding’.] Ligand binding by the dimeric M6P ⁄ IGF2R M. A. Hartman et al. 1918 FEBS Journal 276 (2009) 1915–1929 ª 2009 The Authors Journal compilation ª 2009 FEBS receptors (Fig. 3E). Binding of [ 125 I]PMP-BSA to immunoprecipitated mini-receptors was not affected by the proportion of wild-type versus I ⁄ T mutant mini- receptors in the mixture, suggesting that the I ⁄ T muta- tion 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 [ 125 I]IGF-II to immunoprecipitated mini- receptors was measured to assess whether 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 immuno- precipitated 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 in 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 sup- pressed or not detected as readily as expected. One possibility for explaining this functional deficit may comprise interference from the pairing of two different C-terminal epitope tags. Ligand binding by FLAG- and Myc-tagged M6P/IGF2R mini-receptors via reciprocal co-immunoprecipitation To test whether the FLAG or Myc epitope tags inter- fere with the formation of fully functional receptors in asymmetric heterodimers by having only half of the complex tethered to the resin, a reciprocal immunopre- cipitation was performed. Cell lysates containing co-expressed FLAG and Myc epitope-tagged mini- Transfected construct (µg) 1-15F 1-15F I/T 1-15Myc 1-15Myc I/T 30 0 0 0 15 15 0 0 15 15 0 0 0 0 15 15 15 15 0 0 0 0 0 30 30 0 0 0 IB: α α -FLAG IB: α -FLAG IB: α -Myc IB: α -Myc IP: α -FLAG IP: α -FLAG C D E F B A Transfected construct (µg) 1-15F 1-15F I/T 1-15Myc 1-15Myc I/T 30 0 0 0 15 15 0 0 15 15 0 0 0 0 15 15 15 15 0 0 0 0 0 30 30 0 0 0 0 25 50 75 100 125 150 125 I-PMP-BSA binding c.p.m. × 10 2 0 10 20 30 40 125 I-IGF-II binding c.p.m. × 10 3 Fig. 3. Co-immunoprecipitation and ligand binding by FLAG and Myc epitope-tagged asymmetric dimeric soluble receptors immuno- precipitated with M2 anti-FLAG resin. The ability of 1-15Myc to co-immunoprecipitate with 1-15F was measured by immunoprecipi- tating equimolar amounts of the 1-15F soluble receptor with M2 anti-FLAG affinity resin from 293T lysates of the M6P ⁄ IGF2R mini-receptors. After immunoprecipitation, the resin pellets were collected, washed, heated with sample buffer and analyzed by 6% reducing SDS ⁄ PAGE. The proteins were transferred to BA85 nitro- cellulose, immunoblotted with anti-FLAG M2 (A, B) or anti-Myc 9E10 (C, D) Igs and developed with [ 125 I]protein A. As a control, cell lysate in the amount that was used during the immunoprecipi- tation was directly loaded onto the gel (A, C). Cell lysates, contain- ing equimolar amounts of expressed FLAG-tagged soluble receptors, were immunoprecipitated with M2 anti-FLAG affinity resin and then incubated in the presence of 1 n M [ 125 I]PMP-BSA (E) or 2 n M [ 125 I]IGF-II (F) for 3 h at 4 °C. Bound ligand was determined by centrifuging the resin pellets, washing and counting the pellets in a c-counter. Radioactivity retained in the presence of either 5m M Man-6-P or 1 lM IGF-II was subtracted from each binding reaction to determine the specific binding for [ 125 I]PMP-BSA and [ 125 I]IGF-II, respectively. The lines in each graph (E, F) indicate the amount of binding predicted if the wild-type and mutant receptors are binding ligand independently. The tables indicate the amounts of the various cDNAs transfected into cells for each condition and apply to the data shown both above and below the table. Values represent the mean ± SD of three replicate measurements for each condition. These data represent the means of four indepen- dent experiments. [Correction added on 5 March 2009 after first online publication: in Fig. 3D ‘IP: a-Myc’ was corrected to ‘IP: a-FLAG’, and in Fig. 3F ‘ 125 I-PMP-BSA binding’ was corrected to ‘ 125 I-IGF-II binding’.] M. A. Hartman et al. Ligand binding by the dimeric M6P ⁄ IGF2R FEBS Journal 276 (2009) 1915–1929 ª 2009 The Authors Journal compilation ª 2009 FEBS 1919 receptors were analyzed by an immunoprecipitation assay using protein G-Sepharose and 9E10 anti-Myc Ig. Immunoblots revealed that essentially all of the input Myc-tagged mini-receptors precipitated in the assay (Fig. 4A versus B). Figure 4C,D indicates that approximately 50% of the co-expressed FLAG-tagged mini-receptors were co-immunoprecipitated with the Myc-tagged mini-receptors (Fig. 4C versus D). As was observed in anti-FLAG-based immunoprecipitations (Fig. 3), the presence of the I ⁄ T mutation had no effect on the interaction leading to co-immunoprecipi- tation. To assess the ligand binding function of these asym- metric heterodimers, co-immunoprecipitated mini- receptors were subjected to direct binding analysis using radiolabeled ligands (Fig. 4E,F). 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 [ 125 I]PMP-BSA binding was calculated and represented according to the line in the bar graph (Fig. 4E). The data shown in Fig. 4E for the co-immunoprecipitated mini-receptors were consistent with this expectation as well as the results observed with complementary anti-FLAG immunoprecipitation (Fig. 3E). Binding of [ 125 I]IGF-II to immunoprecipitated mini- receptors was measured to determine whether 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 in the bar graph; however, when the I⁄ T mutant Myc-tagged mini-receptor served as the bait for immu- noprecipitation 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 anti-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 A B C D 1-15F 1-15F I/T 1-15Myc 1-15Myc I/T 0 0 30 0 15 15 0 0 0 0 15 15 15 15 0 0 15 15 0 0 0 0 0 30 0 30 0 0 E F 1-15F 1-15F I/T 1-15Myc 1-15Myc I/T 30 0 0 0 15 15 0 0 15 15 0 0 0 0 15 15 15 15 0 0 0 0 0 30 30 0 0 0 0 10 20 30 40 50 0 10 20 30 40 50 60 70 80 90 Transfected construct (µg) Transfected construct (µg) IB: α -Myc IB: α -Myc IP: α -Myc IB: α -FLAG IB: α -FLAG IP: α -Myc 125 I-PMP-BSA binding c.p.m. × 10 2 125 I-IGF-II binding c.p.m. × 10 2 Fig. 4. Co-immunoprecipitation and ligand binding of FLAG and Myc epitope-tagged asymmetric dimeric soluble receptors immuno- precipitated with protein G-Sepharose. The ability of 1-15F to co-immunoprecipitate with 1-15Myc was measured by immunopre- cipitating equimolar amounts of the 1-15Myc soluble receptor in 293T lysates of the M6P ⁄ IGF2R mini-receptors with protein G-Sepharose previously incubated with anti-Myc 9E10 Ig. After immunoprecipitation, the resin pellets were collected, washed, heated with sample buffer and analyzed by 6% reducing SDS ⁄ PAGE. The proteins were transferred to BA85 nitrocellulose and immunoblotted with anti-Myc 9E10 (A, B) or anti-FLAG M2 (C, D) Igs. As a control, cell lysate in the amount that was used during the immunoprecipitation was directly loaded onto the gel (A, C). Cell lysates, containing equimolar amounts of expressed Myc-tagged soluble receptors, were immunoprecipitated with pro- tein G-Sepharose previously incubated with anti-Myc 9E10 Ig and assayed for binding of [ 125 I]PMP-BSA (E) or [ 125 I]IGF-II (F). The lines in each graph (E, F) indicate the amount of binding predicted if the wild-type and mutant receptors are binding ligand independently. The tables indicate the amounts of the various cDNAs transfected into cells for each condition and apply to the data shown both above and below the table. Values represent the mean ± SD of three representative measurements for each condition. These data represent the means of four independent experiments. [Correction added on 5 March 2009 after first online publication: in Fig. 4D ‘IP: a-FLAG’ was corrected to ‘IP: a-Myc’, and in Fig. 4F ‘ 125 I-PMP-BSA binding’ was corrected to ‘ 125 I-IGF-II binding’.] Ligand binding by the dimeric M6P ⁄ IGF2R M. A. Hartman et al. 1920 FEBS Journal 276 (2009) 1915–1929 ª 2009 The Authors Journal compilation ª 2009 FEBS 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 exam- ined 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. Ligand binding by double-mutant, FLAG-tagged M6P/IGF2R mini-receptors Two forms of the FLAG-tagged mini-receptors, 1-15 wild-type and 1-15 R426A ⁄ R1325A (R2A), were con- structed 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 anti-FLAG Ig (data not shown). Binding of [ 125 I]IGF-II by immunoprecipitated wild-type versus R2A mutant mini-receptors was inde- pendent 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 support the hypothesis that IGF-II binding to the co-immunoprecipitated mini-receptors would be almost the same as that observed with the mini-recep- tors expressed individually. Binding of [ 125 I]PMP-BSA to immunoprecipitated mini-receptors was measured to assess whether the presence of the mutant mini-recep- tors affected PMP-BSA binding to the wild-type mini- receptors (Fig. 5C). In these experiments, the null hypothesis proposes 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 in the bar graph, suggesting that the wild-type binding site on one receptor is not affected by the presence of a mutant mini-receptor that is incapable of binding PMP-BSA. Ligand binding by double-mutant FLAG and Myc-tagged co-immunoprecipitated M6P/IGF2R mini-receptors 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 Construct name: FLAG FLAG COOH COOH * 1-15F R2A H 2 N 1-15F H 2 N 9 113 A * 0 30 0 30 B C 0.0 2.5 5.0 7.5 10.0 12.5 15.0 0 10 20 30 40 125 I-IGF-II binding c.p.m. × 10 2 125 I-PMP-BSA binding c.p.m. × 10 3 µg 1-15F cDNA µg 1-15F R2A cDNA Fig. 5. Schematic diagram and ligand binding analysis of soluble 1-15 and 1-15R2A mutant FLAG epitope-tagged receptors immuno- precipitated with anti-FLAG resin. (A) The receptor constructs are shown in linear format from the amino terminus to the carboxyl terminus, with repeats of the ectodomain illustrated as rectangles. The stippled rectangles represent repeat 11 containing the principal residues responsible for IGF-II binding. The shaded rectangles indi- cated repeats 3 and 9, to which the main determinants of Man-6-P binding have been mapped and the asterisk denotes the RfiA mutations at residues 426 and 1325 (R2A), which abrogates Man- 6-P binding. The black rectangles represent the FLAG epitope tags on the carboxyl terminus. (B, C) Cell lysates, containing equimolar amounts of expressed soluble receptors, were immunoprecipitated with M2 anti-FLAG affinity resin and assayed for binding of [ 125 I]IGF-II (B) or [ 125 I]PMP-BSA (C). The lines in each graph indicate the amount of binding predicted if the wild-type and mutant recep- tors are binding ligand independently. The triangles indicate a progressive shift in the ratio of wild-type to mutant receptor cDNA transfected into cells. Values represent the mean ± SD of three replicate measurements for each condition. These data represent the means of four independent experiments. M. A. Hartman et al. Ligand binding by the dimeric M6P ⁄ IGF2R FEBS Journal 276 (2009) 1915–1929 ª 2009 The Authors Journal compilation ª 2009 FEBS 1921 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 immuno- blotting with anti-FLAG and anti-Myc Igs (data not shown). The cell lysates with volumes normalized for expres- sion of the FLAG-tagged mini-receptor were analyzed by an immunoprecipitation assay using M2 anti- 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 versus C). The data shown in Fig. 6D,E indicate that approx- imately 50% of the co-expressed Myc-tagged mini-receptors were co-immunoprecipitated with the FLAG-tagged mini-receptors (Fig. 6D versus E), 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 [ 125 I]IGF-II to immunoprecipitated wild- type versus R2A mutant mini-receptors was equivalent, suggesting that the R2A mutation had no discernible effect on the formation of oligomers that are func- tional in IGF-II binding (Fig. 6F). These data were consistent with the prediction that, because 25% of the oligomers formed should be homo-oligomers of Myc- tagged mini-receptors, IGF-II binding by the co-immu- noprecipitated mini-receptors should have yielded approximately 75% of the binding observed with 1-15F 1-15F R2A 1-15Myc 1-15Myc R2A 30 0 0 0 15 15 0 0 15 15 0 0 0 0 15 15 30 0 0 0 0 0 30 0 0 0 0 30 Myc Myc COOH COOH HN 2 HN 2 * 1-15Myc 1-15Myc R2A * A B C D E 1-15F 1-15F R2A 1-15Myc 1-15Myc R2A 30 0 0 0 15 15 0 0 15 15 0 0 0 0 15 15 30 0 0 0 0 0 30 0 0 0 0 30 F G 0 25 50 75 100 125 0 100 200 300 400 500 600 700 800 IB: α -FLAG IB: α -FLAG IP: α -FLAG Transfected construct (µg) Transfected construct (µg) IB: α -Myc IB: α -Myc IP: α -FLAG 125 I-IGF-II binding c.p.m. × 10 2 125 I-PMP-BSA binding c.p.m. × 10 3 Fig. 6. Schematic diagram of soluble 1-15 and 1-15 R2A mutant Myc epitope-tagged receptors, co-immunoprecipitation and ligand binding analysis of soluble 1-15 and 1-15R2A mutant FLAG and Myc epitope-tagged asymmetric soluble heterodimeric receptors. (A) The receptor constructs are shown in linear format from amino terminus to carboxyl terminus, with repeats of the ectodomain illus- trated as rectangles. The stippled rectangles represent repeat 11 containing the principal residues responsible for IGF-II binding. The shaded rectangles indicated repeats 3 and 9, to which the main determinants of Man-6-P binding have been mapped and the aster- isk denotes the RfiA mutations at residues 426 and 1325 (R2A), which abrogates Man-6-P binding. The black rectangles represent the Myc epitope tags on the carboxyl terminus. (B–E) The ability of 1-15Myc to co-immunoprecipitate with equimolar amounts of 1-15F soluble receptor with M2 anti-FLAG affinity resin from 293T cell lysates of M6P ⁄ IGF2R mini-receptors. After immunoprecipitation, the resin pellets were collected, washed and immunoblotted with anti-FLAG M2 (B, C) or anti-Myc 9E10 (C, D) Igs. As a control, cell lysate in the amount that was used during the immunoprecipitation was directly loaded on the gel (B, D). (F, G) Cell lysates, containing equimolar amounts of expressed FLAG-tagged soluble receptors, were immunoprecipitated with M2 anti-FLAG affinity resin and assayed for binding of 2 n M [ 125 I]IGF-II (F) or 1 nM [ 125 I]PMP-BSA (G) for 3 h at 4 °C. The lines in each graph (F, G) indicate the amount of binding predicted if the wild-type and mutant receptors are binding ligand independently. The tables indicate the amounts of the various cDNAs transfected into cells for each condition and apply to the data shown both above and below the table. Values represent the mean ± SD of three replicative measurements for each condition. These data represent the means of four indepen- dent experiments. [Correction added on 5 March 2009 after first online publication: in Fig. 6E ‘IP: a -Myc’ was corrected to ‘IP: a-FLAG’, and in Fig. 6F ‘ 125 I-PMP-BSA binding’ was corrected to ‘ 125 I-IGF-II binding’.] Ligand binding by the dimeric M6P ⁄ IGF2R M. A. Hartman et al. 1922 FEBS Journal 276 (2009) 1915–1929 ª 2009 The Authors Journal compilation ª 2009 FEBS individually immunoprecipitated, FLAG-tagged mini- receptors. Binding of [ 125 I]PMP-BSA to these asymmetric het- ero-oligomers was measured to determine whether PMP-BSA binding would show interactive effects between the wild-type and mutant partners (Fig. 6G). As observed previously when a mutant FLAG-tagged mini-receptor acted as the bait molecule in the immu- noprecipitation, binding of PMP-BSA to the asym- metric hetero-oligomers was suppressed. These results are consistent with the results obtained in experiments with the I ⁄ T mutation, suggesting that the ligand bind- ing functions of the receptor’s ectodomain can operate independently of one other within each receptor and relative to receptor partners, again indicating that this effect depends on tethering the C-terminal ends of the extracytoplasmic domains. Discussion The rationale for the present study 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 studies have shown that the receptor operates optimally as a dimer and, in the present study, we aimed 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 bind- ing domains within a single polypeptide chain, it remains uncertain whether this bivalent binding activ- ity 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. [24], who found that a multivalent phosphomannosy- lated ligand cross-bridged the dimeric receptor to pro- mote optimal internalization. This conclusion was reinforced by Byrd et al. [34], who analyzed mutant receptors bearing a substitution of Arg for Ala at posi- tion 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 con- clusion 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. [16] 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 A ˚ , indicating that a single diphosphorylated oligosaccha- ride, with a maximum distance of approximately 30 A ˚ [35], could not bind to the Man-6-P binding domains of both repeats 3 and 9 simultaneously [16]. The pres- ent study was designed to test whether 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 coopera- tion of both monomers. For this purpose, we devel- oped a quantitative assay for heterodimer formation that was based on immunoprecipitation of differen- tially epitope-tagged receptors. The availability of the I1572T mutant allowed us to address the initial question of whether a nonfunctional 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 immunoprecipita- tion between differentially epitope-tagged mini-recep- tors was feasible. These assays also indicated that immunoprecipitation was not preferential to the epitope used to tag the receptor because 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, approximately 50% of the Myc-tagged mini- receptors were co-precipitated from a cell lysate pre- pared from cells co-transfected with equal amounts of the tagged mini-receptor cDNA. This strongly sug- gests, but does not prove, that the mini-receptors asso- ciated 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 rela- tive to the structure of the receptor is that the mini- receptors were in the form of dimers. Byrd et al. [34] 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 recep- tor binding sites [35], these data suggested that oligo- merization of the receptor contributes to high-affinity binding. In addition, native gel electrophoresis demon- M. A. Hartman et al. Ligand binding by the dimeric M6P ⁄ IGF2R FEBS Journal 276 (2009) 1915–1929 ª 2009 The Authors Journal compilation ª 2009 FEBS 1923 strated that the receptor could be separated into monomeric and dimeric forms in the presence or absence of Man-6-P-containing ligands [25]. York et al. [24] demonstrated, by sucrose gradient sedimen- tation and gel filtration, that the receptor bound to a multivalent Man-6-P-containing ligand, b-glucuroni- dase, exhibited a sedimentation coefficient and Stokes radius that were consistent with a complex of two receptor molecules plus one molecule of ligand. How- ever, when these experiments were performed with IGF-II, the receptor appeared to exist as a monomer. Internalization experiments performed with [ 125 I]IGF- II in the presence of b-glucuronidase revealed that b-glucuronidase accelerated the rate of IGF-II uptake, suggesting that intermolecular cross-linking of recep- tors enhanced receptor endocytosis [24]. 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. The present study, employing soluble forms of the receptor [25,26,34], indicates that the ectodomain of the receptor is capable of dimer forma- tion in the absence of ligand. Affinity cross-linking of [ 125 I]IGF-II to a mutant repeat 11 mini-receptor revealed that the I1572T muta- tion completely abolished IGF-II binding [31]. This finding was confirmed by Linnell et al. [22] by utilizing surface plasmon resonance of a truncated receptor containing extracytoplasmic repeats 10–13, which con- tained 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 b-barrel structure [36]. 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 (i.e. having identical epitope tags, both of which are teth- ered 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 func- tional state. The most interesting and unexpected finding of the present study is that asymmetric heterodimers (i.e. hav- ing 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. By contrast, we found that, if the heterodimer is tethered to the resin by the mutant partner, the amount of binding observed is substan- tially less than expected. This complex binding behav- ior observed with IGF-II binding must only be a local effect because 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 asym- metric 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 distor- tion of the IGF-II binding pocket and a consequent reduced ability to bind IGF-II. This effect is likely not due to reduced contact between repeats 11 and 13 because 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 extracyto- plasmic domain results in the inability to form appro- priate contacts between dimeric partners. Follow-up experiments using structural approaches are required to address this possibility. Localization of the two Man-6-P binding domains was previously reported by Westlund et al. [30], who subjected the M6P ⁄ IGF2R to partial proteolytic diges- tion using subtilisin. They determined that repeats 1–3 and 7–10 can independently bind Man-6-P-containing ligands. Dahms et al. [19] 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 recep- tor’s high-affinity Man-6-P binding sites. The structure of repeats 1–3 of the bovine M6P ⁄ IGF2R in the pres- ence of Man-6-P was solved by Olson et al. [18]. Their Ligand binding by the dimeric M6P ⁄ IGF2R M. A. Hartman et al. 1924 FEBS Journal 276 (2009) 1915–1929 ª 2009 The Authors Journal compilation ª 2009 FEBS [...]... to the two Man-6-P binding sites of the receptor [17] 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 appears that the binding energy of the site must be decreased with the. .. 2-hydroxyl group of the mannose ring but these represent only two out of more than a dozen noncovalent interactions between ligand and receptor within the binding pocket This leads to the question as to whether the RfiA 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... 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. . .Ligand binding by the dimeric M6P ⁄ IGF2R M A Hartman et al 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 cation-dependent mannose 6-phosphate receptor and Arg426 of the human M6P ⁄ IGF2R) forms one or two critical hydrogen bonds with the. .. 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 c-counter Specific [125I]IGF-II binding was determined by subtracting c.p.m ligand bound in replicate reactions carried out in the presence of 1 lm IGF-II Binding of [125I]PMP-BSA... prevented the co-precipitation of endogenous phos- phomannosylated ligands The resin was collected by centrifugation at 13 000 g for 20 s 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 for 16 h at 4 °C The addition of IGF-I... incorporate the R426A mutation responsible for altering the ectodomain repeat 3 Man-6-P 1926 binding site using the Megaprimer approach [41] This first round of amplification involved producing the mutation, by amplifying from that site (nucleotide 1425) to the 3¢-end of the mini-receptor (nucleotide 2487) This ‘megaprimer’ was then used in a second round of amplification with the 5¢-primer used previously The. .. PMPBSA binding In summary, the major findings obtained in the present study are consistent with a dimer model for M6P ⁄ IGF2R oligomerization because all co-immunoprecipitations resulted in the 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, whereas high-affinity binding of multivalent Man-6-P ligands... [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 prismÔ (Graphpad Software, San Diego, CA, USA) 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... Byrd JC & MacDonald RG (2000) Mechanisms for high affinity mannose 6-phosphate ligand binding to the insulin-like growth factor II ⁄ mannose 6-phosphate receptor J Biol Chem 275, 18638–18646 Tong PY, Gregory W & Kornfeld S (1989) Ligand interactions of the cation-independent mannose 6-phosphate receptor The stoichiometry of mannose 6-phosphate binding J Biol Chem 264, 7962–7969 Brown J, Esnouf RM, Jones . if the heterodimer is tethered by the wild-type partner. By contrast, we found that, if the heterodimer is tethered to the resin by the mutant partner, the. 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