Structure and functional analysis of the IGF-II/IGF2R interaction

Embryonic development and normal growth require exquisite control of insulin-like growth factors (IGFs). In mammals the extracellular region of the cation-independent mannose-6-phosphate receptor has gained an IGF-II-binding function and is termed type II IGF receptor (IGF2R). IGF2R sequesters IGF-II; imbalances occur in cancers and IGF2R is implicated in tumour suppression. We report crystal structures of IGF2R domains 11-12, 11-12-13-14 and domains 11-12-13/IGF-II complex. A distinctive juxtaposition of these domains provides the IGF-II-binding unit, with domain 11 directly interacting with IGF-II and domain 13 modulating binding site flexibility. Our complex shows that Phe19 and Leu53 of IGF-II lock into a hydrophobic pocket unique to domain 11 of mammalian IGF2Rs. Mutagenesis analyses confirm this IGF-II 'binding-hotspot', revealing that IGF-binding proteins and IGF2R have converged on the same high-affinity site.     

A novel binding site for the human insulin-like growth factor-II (IGF-II)/mannose 6-phosphate receptor on IGF-II

The mammalian insulin-like growth factor (IGF)-II/cation-independent mannose 6-phosphate receptor (IGF2R) binds IGF-II with high affinity. By targeting IGF-II to lysosomal degradation, it plays a role in the maintenance of correct IGF-II levels in the circulation and in target tissues. Loss of IGF2R function is associated with tumor progression; therefore, the IGF2R is often referred to as a tumor suppressor. The interaction between IGF2R and IGF-II involves domains 11 and 13 of the 15 extracellular domains of the receptor. Recently, a hydrophobic binding region was identified on domain 11 of the IGF2R. In contrast, relatively little is known about the residues of IGF-II that are involved in IGF2R binding and the determinants of IGF2R specificity for IGF-II over the structurally related IGF-I. Using a series of novel IGF-II analogues and surface plasmon resonance assays, this study revealed a novel binding surface on IGF-II critical for IGF2R binding. The hydrophobic residues Phe(19) and Leu(53) are critical for IGF2R binding, as are residues Thr(16) and Asp(52). Furthermore, Thr(16) was identified as playing a major role in determining why IGF-II, but not IGF-I, binds with high affinity to the IGF2R.     

Recognition of Dictyostelium discoideum lysosomal enzymes is conferred by the amino-terminal carbohydrate binding site of the insulin-like growth factor II/mannose 6-phosphate receptor

The insulin-like growth factor-II/mannose 6-phosphate receptor (IGF-II/MPR) is a type I glycoprotein that mediates both the intracellular sorting of lysosomal enzymes bearing mannose 6-phosphate (Man-6-P) residues to the lysosome and the bioavailability of IGF-II. The extracytoplasmic region of the IGF-II/MPR contains 15 repeating domains; the two carbohydrate recognition domains (CRDs) have been localized to domains 1-3 and 7-9, and the high-affinity IGF-II binding site maps to domain 11. To characterize the carbohydrate binding properties of the IGF-II/MPR, regions of the receptor encompassing the individual CRDs were produced in a baculovirus expression system. Characterization of the recombinant proteins revealed that the pH optimum for carbohydrate binding is significantly more acidic for the carboxyl-terminal CRD than for the amino-terminal CRD (i.e., pH 6.4-6.5 vs 6.9). Equilibrium binding studies demonstrated that the two CRDs exhibit a similar affinity for Man-6-P. Furthermore, substitution of the conserved arginine residue in domain 3 (R435) or in domain 9 (R1334) with alanine resulted in a similar >1000-fold decrease in the affinity for the lysosomal enzyme, beta-glucuronidase. In contrast, the two CRDs differ dramatically in their ability to recognize the distinctive modifications (i.e., mannose 6-sulfate and Man-6-P methyl ester) found on Dictyostelium discoideum lysosomal enzymes: the amino-terminal CRD binds mannose 6-sulfate and Man-6-P methyl ester with a 14-55-fold higher affinity than the carboxyl-terminal CRD. Taken together, these results demonstrate that the IGF-II/MPR contains two functionally distinct CRDs.     

Computational model for the IGF-II/IGF2r complex that is predictive of mutational and surface plasmon resonance data

Insulin-like growth factors (IGFs) are key regulators of cell proliferation, differentiation, and transformation, and are thus pivotal in cancer, especially breast, prostate, and colon neoplasm. Their potent mitogenic and anti-apoptotic actions depend primarily on their availability to bind to the signaling IGF cell surface receptors. One mechanism by which IGF-II availability is thought to be modulated is by binding to the nonsignaling IGF-II receptor (IGF2R). This binding is essentially mediated by domain 11 in the multidomain IGF2R extracellular region. The crystal structure of domain 11 of the human IGF-II receptor (IGF2R-d11) has identified a putative IGF-II binding site, and a nuclear magnetic resonance (NMR) solution structure for the IGF-II ligand has also been characterized. These structures have now been used to model in silico the protein-protein interaction between IGF-II and IGF2R-d11 using the program 3D-Dock. Because the IGF-II data comprise an ensemble of 20 structures, all of which satisfy the NMR constraints, the docking procedure was applied to each member of the ensemble. Only those models in which residue Ile1572 of IGF2R-d11, known to be essential for the binding of IGF-II, was at the interface were considered further. These plausible complexes were then critically assessed using an array of analysis techniques including consideration of additional mutagenesis data. One model was strongly supported by these analyses and is discussed here in detail. Furthermore, we demonstrate in vitro experimental support for this model by studying the binding of chimeras of IGF-I and IGF-II to IGF2R fragments.     

Disruption of ligand binding to the insulin-like growth factor II/mannose 6-phosphate receptor by cancer-associated missense mutations.

The insulin-like growth factor II/mannose 6-phosphate receptor (IGF2R) carries out multiple regulatory and transport functions, and disruption of IGF2R function has been implicated as a mechanism to increase cell proliferation. Several missense IGF2R mutations have been identified in human cancers, including the following amino acid substitutions occurring in the extracytoplasmic domain of the receptor: Cys-1262 --> Ser, Gln-1445 --> His, Gly-1449 --> Val, Gly-1464 --> Glu, and Ile-1572 --> Thr. To determine what effects these mutations have on IGF2R function, mutant and wild-type FLAG epitope-tagged IGF2R constructs lacking the transmembrane and cytoplasmic domains were characterized for binding of insulin-like growth factor (IGF)-II and a mannose 6-phosphate-bearing pseudoglycoprotein termed PMP-BSA (where PMP is pentamannose phosphate and BSA is bovine serum albumin). The Ile-1572 --> Thr mutation eliminated IGF-II binding while not affecting PMP-BSA binding. Gly-1449 --> Val and Cys-1262 --> Ser each showed 30-60% decreases in the number of sites available to bind both (125)I-IGF-II and (125)I-PMP-BSA. In addition, the Gln-1445 --> His mutant underwent a time-dependent loss of IGF-II binding, but not PMP-BSA binding, that was not observed for wild type. In all, four of the five cancer-associated mutants analyzed demonstrated altered ligand binding, providing further evidence that loss of IGF2R function is characteristic of certain cancers.     

C-terminal domain of insulin-like growth factor (IGF) binding protein-6: structure and interaction with IGF-II

IGFs are important mediators of growth. IGF binding proteins (IGFBPs) 1-6 regulate IGF actions and have IGF-independent actions. The C-terminal domains of IGFBPs contribute to high-affinity IGF binding and modulation of IGF actions and confer some IGF-independent properties, but understanding how they achieve this has been constrained by the lack of a three-dimensional structure. We therefore determined the solution structure of the C-domain of IGFBP-6 using nuclear magnetic resonance (NMR). The domain consists of a thyroglobulin type 1 fold comprising an alpha-helix followed by a loop, a three-stranded antiparallel beta-sheet incorporating a second loop, and finally a disulfide-bonded flexible third loop. The IGF-II binding site on the C-domain was identified by examining NMR spectral changes upon complex formation. It consists of a largely hydrophobic surface patch involving the alpha-helix, the first beta-strand, and the first and second loops. The site was confirmed by mutagenesis of several residues, which resulted in decreased IGF binding affinity. The IGF-II binding site lies adjacent to surfaces likely to be involved in glycosaminoglycan binding of IGFBPs, which might explain their decreased IGF affinity when bound to glycosaminoglycans, and nuclear localization. Our structure provides a framework for understanding the roles of IGFBP C-domains in modulating IGF actions and conferring IGF-independent actions, as well as ultimately for the development of therapeutic IGF inhibitors for diseases including cancer.     

Interaction of insulin-like growth factor II (IGF-II) with multiple plasma proteins: high affinity binding of plasminogen to IGF-II and IGF-binding protein-3

In the circulation, most of the insulin-like growth factors (IGFs), IGF-binding proteins (IGFBPs), and IGFBP proteases are bound in high molecular mass complexes of > or =150 kDa. To investigate molecular interactions between proteins involved in IGF.IGFBP complexes, Cohn fraction IV of human plasma was subjected to IGF-II affinity chromatography followed by reversed-phase high pressure liquid chromatography and analysis of bound proteins. Mass spectrometry and Western blotting revealed the presence of IGFBP-3, IGFBP-5, transferrin, plasminogen, prekallikrein, antithrombin III, and the soluble IGF-II/mannose 6-phosphate receptor in the eluate. Furthermore, an IGFBP-3 protease cleaving also IGFBP-2 but not IGFBP-4 was co-purified from the IGF-II column. Inhibitor studies and IGFBP-3 zymography have demonstrated that the 92-kDa IGFBP-3 protease belongs to the class of serine-dependent proteases. IGF-II ligand blotting and surface plasmon resonance spectrometry have been used to identify plasminogen as a novel high affinity IGF-II-binding protein capable of binding to IGFBP-3 with 50-fold higher affinity than transferrin. In combination with transferrin, the overall binding constant of plasminogen/transferrin for IGF-II was reduced 7-fold. Size exclusion chromatography of the IGF-II matrix eluate revealed that transferrin, plasminogen, and the IGFBP-3 protease are present in different high molecular mass complexes of > or =440 kDa. The present data indicate that IGFs, low and high affinity IGFBPs, several IGFBP-associated proteins, and IGFBP proteases can interact, which may result in the formation of binary, ternary, and higher molecular weight complexes capable of modulating IGF binding properties and the stability of IGFBPs.     

Mechanisms for high affinity mannose 6-phosphate ligand binding to the insulin-like growth factor II/mannose 6-phosphate receptor

The two mannose 6-phosphate (Man-6-P) binding domains of the insulin-like growth factor II/mannose 6-phosphate receptor (Man-6-P/IGF2R), located in extracytoplasmic repeats 1-3 and 7-9, are capable of binding Man-6-P with low affinity and glycoproteins that contain more than one Man-6-P residue with high affinity. High affinity multivalent ligand binding sites could be formed through two possible mechanisms: the interaction of two Man-6-P binding domains within one Man-6-P/IGF2R molecule or by receptor oligomerization. To discriminate between these mechanisms, truncated FLAG epitope-tagged Man-6-P/IGF2R constructs, containing one or both of the Man-6-P binding domains, were expressed in 293T cells, and characterized for binding of pentamannose phosphate-bovine serum albumin (PMP-BSA), a pseudoglycoprotein bearing multiple Man-6-P residues. A construct containing all 15 repeats of the Man-6-P/IGF2R extracytoplasmic domain bound PMP-BSA with the same affinity as the full-length receptor (K(d) = 0.54 nm) with a curvilinear Scatchard plot. The presence of excess unlabeled PMP-BSA increased the dissociation rate of pre-formed (125)I-PMP-BSA/receptor complexes, suggesting negative cooperativity in multivalent ligand binding and affirming the role of multiple Man-6-P/IGF2R binding domains in forming high affinity binding sites. Truncated receptors containing only one Man-6-P binding domain and mutant receptor constructs, containing an Arg(1325) --> Ala mutation that eliminates binding to the repeats 7-9 binding domain, formed high affinity PMP-BSA binding, but with reduced stoichiometries. Collectively, these observations suggest that alignment of Man-6-P binding domains of separate Man-6-P/IGF2R molecules is responsible for the formation of high affinity Man-6-P binding sites and provide functional evidence for Man-6-P/IGF2R oligomerization.     

Real time kinetics of insulin-like growth factor II (IGF-II) interaction with the IGF-II/mannose 6-phosphate receptor: the effects of domain 13 and pH

The interaction of soluble forms of the human cation-independent insulin-like growth factor-II/mannose 6-phosphate receptor (IGF-IIR) with IGFs and mannosylated ligands was analyzed in real time. IGF-IIR proteins containing domains 1-15, 10-13, 11-13, or 11-12 were combined with rat CD4 domains 3 and 4. Following transient expression in 293T cells, secreted protein was immobilized onto biosensor chips. beta-Glucuronidase and latent transforming growth factor-beta1 bound only to domains 1-15. IGF-II bound to all constructs except a control, which contained a point mutation in domain 11. The affinity of domains 1-15, 10-13, 11-13, and 11-12 to IGF-II were 14, 120, 100, and 450 nm, respectively. Our data suggest that domain 13 acts as an enhancer of IGF-II affinity by slowing the rate of dissociation, but additional enhancement by domains other than 10-13 also occurs. As the receptor functions to transport ligands from either the trans-Golgi network or extracellular space to the endosomes, the interaction of IGF-IIR extracellular domains with IGF-II was analyzed over a pH range of 5.0-7.4. The constructs behaved differently in response to pH and in recovery after low pH exposure, suggesting that pH stability of the extracellular domains depends on domains other than 10-13.     

Endocytosis of insulin-like growth factor II by a mini-receptor based on repeat 11 of the mannose 6-phosphate/insulin-like growth factor II receptor

The mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF-II receptor) plays an important role in controlling the extracellular level of the insulin-like growth factor II (IGF-II) by mediating its binding at the cell surface and delivery to lysosomes. Loss of the receptor is associated with an accumulation of IGF-II, which can cause perinatal lethality if it is systemic, or local proliferation and tumorgenesis if it is spatially restricted. The extracytoplasmic domain of the receptor consists of 15 homologous repeats, of which repeat 11 carries the IGF-II-binding site of the multifunctional receptor. To investigate whether repeat 11 is sufficient to mediate binding and internalization of IGF-II, a construct consisting of repeat 11 fused to the transmembrane and cytoplasmic domain of the M6P/IGF-II receptor was transfected into mouse embryonic fibroblasts. The construct was expressed as a stable membrane protein which binds IGF-II with a 10-fold lower affinity as observed for the M6P/IGF-II receptor and is found at the cell surface and in endosomes. It mediates the internalization of IGF-II and its delivery to lysosomes, suggesting that it can function as a IGF-II mini-receptor controlling the extracellular IGF-II level.     

The chicken liver cation-independent mannose 6-phosphate receptor lacks the high affinity binding site for insulin-like growth factor II

The chicken liver cation-independent mannose 6-phosphate receptor has been purified to apparent homogeneity by affinity chromatography on pentamannose phosphate-Sepharose and tested for its ability to bind iodinated human IGF-I, human IGF-II, and chicken IGF-II. In contrast to the bovine, rat, and human cation-independent mannose 6-phosphate receptors, which bind human IGF-II and IGF-I with nanomolar and micromolar affinities, respectively, the chicken receptor failed to bind either radioligand at receptor concentrations as high as 1 microM. The bovine receptor binds chicken IGF-II with high affinity while the chicken receptor binds this ligand with only low affinity, which we estimate to be in the micromolar range. These data demonstrate that the chicken cation-independent mannose 6-phosphate receptor lacks the high affinity binding site for IGF-II. These results provide an explanation for the failure of previous investigators to identify the type II IGF receptor by IGF-II cross-linking to chicken cells and indicate that the mitogenic activity of IGF-II in chick embryo fibroblasts is most likely mediated via the type I IGF receptor.     

Structural determinants for high-affinity binding of insulin-like growth factor II to insulin receptor (IR)-A, the exon 11 minus isoform of the IR

The insulin receptor (IR) lacking the alternatively spliced exon 11 (IR-A) is preferentially expressed in fetal and cancer cells. The IR-A has been identified as a high-affinity receptor for insulin and IGF-II but not IGF-I, which it binds with substantially lower affinity. Several cancer cell types that express the IR-A also overexpress IGF-II, suggesting a possible autocrine proliferative loop. To determine the regions of IGF-I and IGF-II responsible for this differential affinity, chimeras were made where the C and D domains were exchanged between IGF-I and IGF-II either singly or together. The abilities of these chimeras to bind to, and activate, the IR-A were investigated. We also investigated the ability of these chimeras to bind and activate the IR exon 11+ isoform (IR-B) and as a positive control, the IGF-I receptor (IGF-1R). We show that the C domain and, to a lesser extent, the D domains represent the principal determinants of the binding differences between IGF-I and IGF-II to IR-A. The C and D domains of IGF-II promote higher affinity binding to the IR-A than the equivalent domains of IGF-I, resulting in an affinity close to that of insulin for the IR-A. The C and D domains also regulate the IR-B binding specificity of the IGFs in a similar manner, although the level of binding for all IGF ligands to IR-B is lower than to IR-A. In contrast, the C and D domains of IGF-I allow higher affinity binding to the IGF-1R than the analogous domains of IGF-II. Activation of IGF-1R by the chimeras reflected their binding affinities whereas the phosphorylation of the two IR isoforms was more complex.     

Dimerization of the insulin-like growth factor II/mannose 6-phosphate receptor

The insulin-like growth factor II/mannose 6-phosphate receptor (IGF2R) interacts with lysosomal enzymes through two binding domains in its extracytoplasmic domain. We report in the accompanying article (Byrd, J. C., and MacDonald, R. G. (2000) J. Biol. Chem. 275, 18638-18646) that only one of the two extracytoplasmic mannose 6-phosphate (Man-6-P) binding domains is necessary for high affinity Man-6-P ligand binding, suggesting that, like the cation-dependent Man-6-P receptor, oligomerization of the IGF2R contributes to high affinity interaction with lysosomal enzymes. In the present study, we have directly characterized both naturally occurring and engineered forms of the IGF2R for their ability to form oligomeric structures. Whereas gel filtration chromatography suggested that purified bovine IGF2R species exist in a monomeric form, native gel electrophoresis allowed for the separation of dimeric and monomeric forms of the receptors with distinct phosphomannosyl ligand binding characteristics. The ability of the IGF2R to form oligomeric complexes was confirmed and localized to the extracytoplasmic domain through the use of epitope-tagged soluble IGF2R constructs bearing deletions of the transmembrane and cytoplasmic domains. Finally, chimeric receptors were engineered containing the extracytoplasmic and transmembrane domains of the IGF2R fused to the cytoplasmic domain of the epidermal growth factor receptor with which dimerization of the chimeras could be monitored by measuring autophosphorylation. Collectively, these results show that the IGF2R is capable of forming oligomeric complexes, most likely dimers, in the absence of Man-6-P ligands.     

Structure of a functional IGF2R fragment determined from the anomalous scattering of sulfur

Insulin-like growth factor II receptor (IGF2R) is a multifunctional cell surface receptor implicated in tumour suppression. Its growth inhibitory activity has been associated with an ability to bind IGF-II. IGF2R contains 15 homologous extracellular domains, with domain 11 primarily responsible for IGF-II binding. We report a 1.4 A resolution crystal structure of domain 11, solved using the anomalous scattering signal of sulfur. The structure consists of two crossed beta-sheets forming a flattened beta-barrel. Structural analysis identifies the putative IGF-II binding site at one end of the beta-barrel whilst crystal lattice contacts suggest a model for the full-length IGF2R extracellular region. The structure factors and coordinates of IGF2R domain 11 have been deposited in the Protein Data Bank (accession codes 1GP0 and 1GP3).     

Insulin-like growth factor-II (IGF-II) inhibits both the cellular uptake of beta-galactosidase and the binding of beta-galactosidase to purified IGF-II/mannose 6-phosphate receptor

The insulin-like growth factor-II/mannose 6-phosphate receptor which targets acid hydrolases to lysosomes, has two different binding sites, one for the mannose 6-phosphate (Man-6-P) recognition marker on lysosomal enzymes and the other for insulin-like growth factor-II (IGF-II). We have asked whether IGF-II can regulate the cellular uptake of the lysosomal enzyme 125I-beta-galactosidase by modulating the binding of 125I-beta-galactosidase to the IGF-II/Man-6-P receptor. We first isolated high affinity 125I-beta-galactosidase by affinity chromatography on an IGF-II/Man-6-P receptor-Sepharose column. Specific uptake (mannose 6-phosphate-inhibitable) of 125I-beta-galactosidase in BRL 3A2 rat liver cells and in rat C6 glial cells was 3.7-4.8 and 4.0-8.0% of added tracer, respectively. The cell-associated 125I-beta-galactosidase in the uptake experiments largely represented internalized radioligand as measured by acid or mannose 6-phosphate washing. The uptake of 125I-beta-galactosidase was inhibited by an antiserum (No. 3637) specific for the IGF-II/Man-6-P receptor. Low concentrations of IGF-II also inhibited the uptake of 125I-beta-galactosidase. Maximal concentrations of IGF-II inhibited uptake by 73 +/- 8% (mean +/- S.D.) in C6 cells and by 77 +/- 6% in BRL 3A2 cells compared to the level of inhibition by mannose 6-phosphate. The relative potency of IGF-II, IGF-I, and insulin (IGF-II much greater than IGF-I; insulin, inactive) were characteristic of the relative affinities of the ligands for the IGF-II/Man-6-P receptor. IGF-II also partially inhibited the binding of 125I-beta-galactosidase to C6 and BRL 3A2 cells at 4 degrees C and inhibited the binding to highly purified IGF-II/Man-6-P receptor by 58 +/- 14%. We conclude that IGF-II inhibits the cellular uptake of 125I-beta-galactosidase and that this inhibition is partly explained by the ability of IGF-II to inhibit binding of 125I-beta-galactosidase to the IGF-II/Man-6-P receptor.     

The molecular basis of IGF-II/IGF2R recognition: a combined molecular dynamics simulation,free-energy calculation and computational alanine scanning study

Insulin-like growth factor-II (IGF-II) is a key regulator of cell growth, survival, migration and differentiation, and is thus pivotal in many cancers. An individual with a high IGF-II level is at high risk of developing cancer, whereas IGF2R is implicated as being important in tumor suppression. Thus, uncovering the essence of the IGF-II/IGF2R interaction is very important to understanding the origin of the tumor-suppressing effect of IGF2R. In this study, in order to investigate the interaction of the IGF-II/IGF2R complex and to characterize the binding hot spots of this interaction, a 10 ns molecular dynamics simulation combined with MM-PBSA/MM-GBSA computations and computational alanine scanning was performed on the IGF-II/IGF2R complex. From the results of the free-energy decomposition and the computational alanine scanning calculation, we identified the key residues in the IGF-II/IGF-2R interaction. The results from the calculation were consistent with reported experimental mutagenesis studies. The information on the interaction of IGF-II and IGF2R obtained is vital for understanding how the structure of IGF2R influences the function of IGF-II in growth and development. This study will also lead to new opportunities to develop molecular probes that can assist in diagnostic screening, and even novel approaches to controlling tumor development.     

The bovine mannose 6-phosphate/insulin-like growth factor II receptor. Localization of mannose 6-phosphate binding sites to domains 1-3 and 7-11 of the extracytoplasmic region.

The extracytoplasmic region of the 270-kDa mannose 6-phosphate/IGF-II receptor is composed of 15 repeating domains and is capable of binding 2 mol of mannose 6-phosphate (Man-6-P). To localize the Man-6-P binding domains, bovine receptor was subjected to partial proteolysis with subtilisin followed by affinity chromatography on pentamannosyl phosphate-agarose. Eleven proteolytic fragments ranging in apparent molecular mass from 53 to 206 kDa were isolated. Sequence analysis of six of the fragments localized their amino termini to either the beginning of domain 1 at the amino terminus of the molecule or the beginning of domain 7, according to the alignment of Lobel et al. (Lobel, P., Dahms, N. M., and Kornfeld, S. (1988) J. Biol. Chem. 263, 2563-2570). The smallest fragment, with an apparent molecular mass of 53 kDa, is predicted to encompass domains 1-3. Another fragment, with an apparent molecular mass of 82 kDa, is predicted to encompass domains 7-10 or 7-11. The Man-6-P binding site contained within domains 1-3 was further defined by expressing truncated forms of the receptor in Xenopus laevis oocytes and assaying their ability to bind phosphomannosyl residues. A soluble polypeptide containing domains 1-3 exhibited binding activity, whereas a polypeptide containing domains 1 and 2 did not. This indicates that domain 3 is a necessary component of one of the Man-6-P binding sites of the receptor.     

Structural insights into the interaction of insulin-like growth factor 2 with IGF2R domain 11

The insulin-like growth factor II/mannose-6-phosphate receptor (IGF2R) mediates trafficking of mannose-6-phosphate (M6P)-containing proteins and the mitogenic hormone IGF2. IGF2R also plays an important role as a tumor suppressor, as mutation is frequently associated with human carcinogenesis. IGF2 binds to domain 11, one of 15 extracellular domains on IGF2R. The crystal structure of domain 11 and the solution structure of IGF2 have been reported, but, to date, there has been limited success when using crystallography to study the interaction of IGFs with their binding partners. As an approach to investigate the interaction between IGF2 and IGF2R, we have used heteronuclear NMR in combination with existing mutagenesis data to derive models of the domain 11-IGF2 complex by using the program HADDOCK. The models reveal that the molecular interaction is driven by critical hydrophobic residues on IGF2 and IGF2R, while a ring of flexible, charged residues on IGF2R may modulate binding.     

Domain interactions of the mannose 6-phosphate/insulin-like growth factor II receptor

The mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R) forms oligomeric structures important for optimal function in binding and internalization of Man-6-P-bearing extracellular ligands as well as lysosomal biogenesis and growth regulation. However, neither the mechanism of inter-receptor interaction nor the dimerization domain has yet been identified. We hypothesized that areas near the ligand binding domains of the receptor would contribute preferentially to oligomerization. Two panels of minireceptors were constructed that involved truncations of either the N- or C-terminal regions of the M6P/IGF2R encompassing deletions of various ligand binding domains. alpha-FLAG or alpha-Myc-based immunoprecipitation assays showed that all of the minireceptors tested were able to associate with a full-length, Myc-tagged M6P/IGF2R (WT-M). In the alpha-FLAG but not alpha-Myc immunoprecipitation assays, the degree of association of a series of C-terminally truncated minireceptors with WT-M showed a positive trend with length of the minireceptor. In contrast, length did not seem to affect the association of the N-terminally truncated minireceptors with WT-M, except that the 12th extracytoplasmic repeat appeared exceptionally important in dimerization in the alpha-FLAG assays. The presence of mutations in the ligand-binding sites of the minireceptors had no effect on their ability to associate with WT-M. Thus, association within the heterodimers was not dependent on the presence of functional ligand binding domains. Heterodimers formed between WT-M and the minireceptors demonstrated high affinity IGF-II and Man-6-P-ligand binding, suggesting a functional association. We conclude that there is no finite M6P/IGF2R dimerization domain, but rather that interactions between dimer partners occur all along the extracytoplasmic region of the receptor.     

Reptilian MPR 300 is also the IGF-IIR: Cloning, sequencing and functional characterization of the IGF-II binding domain

The mammalian cation-independent mannose 6-phosphate/insulin-like growth factor (IGF)-II receptor binds IGF-II with high affinity. Ligands transported by the MPR 300/IGF-IIR include IGF-II and mannose 6-phosphate-modified proteins. By targeting IGF-II to lysosomal degradation, it plays a key role in the maintenance of correct IGF-II levels in the circulation and in target tissues. Although, from our studies we found homologous receptor in calotes but its functional significance was not known. We present here the first report on the calotes MPR 300/IGF-IIR binds IGF-II with K_d of 12.02 nM; these findings provide new and strong evidence that MPR 300/IGF-IIR in Calotes versicolor binds IGFII with high affinity.