The insulin-like growth factor II (IGF-II)/mannose 6-phosphate receptor mediates IGF-II-induced motility in human rhabdomyosarcoma cells.

Insulin-like growth factor-II (IGF-II) is an autocrine growth and motility factor for human rhabdomyosarcoma. It interacts with three different receptors: the IGF-I, the IGF-II, and the insulin receptor. A specific function of the IGF-II receptor in mediating IGF-II responses has not been defined. In this report we investigate the mechanism of IGF-II-mediated motility in rhabdomyosarcoma cells. We demonstrate that IGF-II and [Leu27]IGF-II, an analog selective for the IGF-II receptor, stimulate motility at concentrations in which they interact only with their own receptor. An antibody that blocks the IGF-I receptor does not inhibit either peptide activity, while an antibody specific for the IGF-II receptor suppresses the IGF-II-induced motility. This antibody does not interfere with rhabdomyosarcoma cell proliferation. We conclude that in rhabdomyosarcoma cells IGF-II stimulates two different responses mediated by distinct receptors: 1) a mitogenic response through the type I receptor and 2) a motility response through the type II receptor.     

Biosynthesis of the insulin-like growth factor-II (IGF-II)/mannose-6-phosphate receptor in rat C6 glial cells: the role of N-linked glycosylation in binding of IGF-II to the receptor.

We examined the role of N-linked glycosylation of the insulin-like growth factor-II (IGF-II)/mannose 6-phosphate (Man-6-P) receptor in binding of [125I]IGF-II to the receptor. First we studied the synthesis and posttranslational processing of this receptor in rat C6 glial cells, which have abundant IGF-II/Man-6-P receptors. Cells were pulse labeled with [35S]methionine and lysed, and the IGF-II/Man-6-P receptor was immunoprecipitated using a specific IGF-II/Man-6-P receptor antibody (no. 3637). Analysis of the immunoprecipitate by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with reduction of disulfide bonds showed a 235-kDa receptor precursor that was processed into the mature 245-kDa IGF-II/Man-6-P receptor within 2 h of chase. Digestion of the 235-kDa precursor with endoglycosidase-H (Endo H) produced a 220-kDa form, whereas the mature 245-kDa receptor was relatively resistant to cleavage by Endo H. When cells were cultured in the presence of 2 microM monensin, the 235-kDa receptor was not further processed into the mature Endo H-resistant receptor form. In addition, the presence of swainsonine in C6 glial cell cultures led to the formation of a 240-kDa receptor hybrid molecule, which was cleaved by Endo H into a 225-kDa species. When tunicamycin was present during the pulse-chase labeling experiment, a 220-kDa receptor species accumulated. This species was 205 kDa by immunoblotting when SDS-PAGE was performed under nonreducing conditions. Pure IGF-II/Man-6-P receptor was digested with N-glycosidase-F, and the digest was immunoblotted with antiserum 3637 after SDS-PAGE under nonreducing conditions. Whereas undigested receptor was a single band of 215 kDa under nonreducing conditions, digested receptor was 205 kDa. The binding affinity of IGF-II for the digested receptor was the same as the binding affinity of IGF-II for the undigested receptor. In addition, affinity cross-linking experiments showed that [125I]IGF-II also bound to the unglycosylated receptor precursor that accumulated in the tunicamycin-treated cells, and the binding affinity of IGF-II for this species was indistinguishable from the binding affinity of IGF-II for the mature receptor. We conclude that IGF-II can bind to an IGF-II/Man-6-P receptor that lacks N-linked oligosaccharides.     

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.     

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.     

Developmental regulation of insulin-like growth factor-II/mannose 6-phosphate receptor mRNA in the rat.

This study examined levels of insulin-like growth factor-II/mannose 6-phosphate receptor (IGF-II/M6PR) mRNA in tissues of rats at different stages of growth. Northern blot analysis of total RNA from tissues of rats aged 2, 9, 21 and 42 days and from 21 day fetal rats was carried out using a cDNA probe to the IGF-II/M6PR. Northern blots showed this probe hybridized to a single 9kb band in all tissues tested. Highest hybridization signals were detected in fetal and neonatal tissues with levels rapidly decreasing after birth. For all age groups tested the highest signal was obtained with heart followed by muscle, lung, and kidney, with liver and brain showing lower levels of message. These results indicate that IGF-II/M6PR mRNA is developmentally regulated, and suggest a role for the IGF-II/M6PR in fetal and neonatal growth.     

Insulin-like growth factor-II/mannose 6-phosphate receptor is incapable of activating GTP-binding proteins in response to mannose 6-phosphate, but capable in response to insulin-like growth factor-II

We previously reported that insulin-like growth factor-II (IGF-II) stimulates both calcium influx and DNA synthesis by acting on the cell surface IGF-II receptor (IGF-IIR) in a manner sensitive to pertussis toxin, and recently demonstrated that IGF-II binding to the IGF-IIR gives rise to functional changes of purified Gi-2, a GTP-binding protein (G protein) in phospholipid vesicles as well as in broken cell membranes. On the other hand, a variety of evidence indicates that the IGF-IIR binds mannose 6-phosphate (man6P) with high affinity probably at a receptor extracellular region different from the IGF-II-binding site. In the present study, we examined whether man6P stimulation of the IGF-IIR evokes the activation of Gi-2 in phospholipid vesicles and in native cell membranes. In vesicles reconstituted with purified rat IGF-IIR and bovine Gi-2, man6P did not stimulate GDP dissociation from Gi-2 even in concentrations up to 10 mM, while IGF-II dose-dependently facilitated GDP release from Gi-2 with an EC50 of 6 nM. The stimulatory effect of IGF-II was not observed in vesicles reconstituted with Gi-2 alone. In addition, also in a native environment of cell membranes, man6P did not affect an endogenous 40-kDa protein or exogenously added purified Gi-2 as assessed with reduction of the pertussis toxin-catalyzed ADP-ribosylation. These results indicate that the IGF-IIR does not activate Gi-like proteins upon man6P binding in phospholipid vesicles and in native cellular membranes, whereas the receptor activates Gi-like proteins upon IGF-II binding in both environments. Thus, we postulate that the IGF-IIR dissimilarly responds to the two structurally unrelated ligands, IGF-II and man6P, in the linkage function with G proteins.     

Mannose 6-phosphate/insulin-like growth factor II receptor: distinct binding sites for mannose 6-phosphate and insulin-like growth factor II

Pentamannosyl phosphate substituted bovine serum albumin (PMP-BSA) and insulin like growth factor II (IGF II) bind specifically to immobilized mannose 6-phosphate/insulin like growth factor II receptor. An excess of IGF II inhibited binding of PMP-BSA by less than or equal to 20%, and an excess of PMP-BSA inhibited binding of IGF II by less than or equal to 10%. Polyclonal antibodies against the receptor purified from human liver inhibited preferentially the binding of PMP-BSA, and a monocloncal antibody 2C2 inhibited only the binding of IGF II to the receptor. Similar results were obtained for binding of PMP-BSA and IGF II to human skin fibroblasts. These results suggest that the binding sites for mannose 6-phosphate and IGF II reside in different portions of the receptor.     

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.     

Binding of insulin-like growth factor II (IGF-II) by human cation-independent mannose 6-phosphate receptor/IGF-II receptor expressed in receptor-deficient mouse L cells.

Mouse L cells deficient in expression of the murine cation-independent mannose 6-phosphate receptor/insulin-like growth factor II receptor (CI-MPR/IGF-IIR) were stably transfected with a plasmid containing the cDNA for the human receptor. Transfected cells expressed high levels of the human receptor which functioned in the transport of lysosomal enzymes and was capable of binding 125I-IGF-II, both at the cell surface and intracellularly. Cell surface binding of 125I-IGF-II by the receptor could be inhibited by pretreatment of cells with antibodies to the receptor or by coincubation with the lysosomal enzyme, beta-glucuronidase. Expression of the receptor conferred on transfected cells the ability to internalize and degrade 125I-IGF-II. Cells transfected with the parental vector and those expressing the human CI-MRP/IGF-IIR were found to express an atypical binding site for IGF-II that was distinct from the CI-MPR/IGF-IIR and the type I IGF-receptor. The availability of two cell lines, one of which overexpresses the human CI-MPR/IGF-IIR and one deficient in expression of the murine receptor, may help in the analysis of the role of the receptor in mediating the biological effects of IGF-II. They should also be useful in examining the significance of binding of ligands, such as transforming growth factor-beta 1 precursor and proliferin to this receptor.     

Interactions of cathepsin-D and insulin-like growth factor-II (IGF-II) on the IGF-II/mannose-6-phosphate receptor in human breast cancer cells and possible consequences on mitogenic activity of IGF-II

The lysosomal enzyme cathepsin-D (cath-D) and insulin-like growth factor-II (IGF-II), which share a common IGF-II/mannose-6-phosphate (M6P) transmembrane receptor, are both synthesized and secreted by breast cancer cells, upon which they might exert an intracrine/autocrine control on proliferation. We have evaluated the binding of 125I-immunopurified human cath-D in different breast cell membrane preparations. The concentration of high affinity M6P reversible binding sites (mean Kd, 0.85 nM) varied among the different breast cancer cells (0-0.82 pmol/mg membrane protein), but there was no correlation between the presence of steroid receptor and M6P-dependent binding. Cross-linking experiments with [125I]cath-D and [125I]IGF-II showed the formation of complexes with the 270,000 mol wt IGF-II/M6P receptor molecule which migrated, respectively, at 330,000 and 270,000 mol wt in 3-10% gradient sodium dodecyl sulfate-polyacrylamide gels. [125I]IGF-II cross-linking was increased by M6P (20% above control), whereas cath-D strongly inhibited IGF-II interaction by 80%. Conversely, IGF-II reduced [125I]cath-D cross-linking by 55%. Direct ligand binding on receptors transferred onto nitrocellulose sheets by Western blotting confirmed the interaction of both ligands on the same receptor molecule. By studying IGF-II's growth-promoting activity in these cells in a wide range of concentrations, we show that IGF-II triggers its mitogenic response via IGF-II/M6P receptor at low concentrations, whereas it is mainly acting via IGF-I receptor at high concentrations. Three lines of evidences lead us to that conclusion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the signal for rapid internalization of the bovine mannose 6-phosphate/insulin-like growth factor-II receptor.

The signal for rapid internalization of the mannose 6-phosphate/insulin-like growth factor II receptor has been localized to the sequence Tyr-Lys-Tyr-Ser-Lys-Val in positions 24-29 of its 163-residue cytoplasmic tail. Most of the activity of this signal is mediated by the carboxyl 4 amino acids, especially Tyr26 and Val29 (Canfield, W. M., Johnson, K. F., Ye, R. D., Gregory, W. and Kornfeld, S. (1991) J. Biol. Chem. 266, 5682-5688). In this study, we have tested the effect of a series of mutations on the internalization rate of a mutant receptor that contains a 29-amino acid cytoplasmic tail terminating with the 4-amino acid internalization sequence Tyr-Ser-Lys-Val. Replacement of Tyr26 with Phe or Trp gave rise to mutant receptors that were internalized at 10% the wild-type rate, while receptors with Ala, Leu, Ile, Val, or Asn at this position were totally inactive. Val29 could be replaced by other large hydrophobic residues (Phe, Leu, Ile, or Met) with no loss of activity, but the presence of Ala, Gly, Arg, Gln, or Tyr in this position inactivated the signal. Ser27 could be effectively replaced by many different amino acids, but not by Pro or Gly. However, Gly27 could be tolerated if the residues at positions 28 and 29 were also changed. A change in the 2-residue spacing between Tyr26 and Val29 destroyed the signal. These data show that the essential elements of this signal are an aromatic residue, especially a Tyr in the first position, separated from a large hydrophobic residue in the last position by 2 amino acids. The residues in positions 2 and 3 of the signal may have a modulating effect on its activity. The Tyr-Ser-Lys-Val signal could be moved to a more proximal region of the cytoplasmic tail with only a modest loss of activity. In addition, the signal could be effectively replaced by the putative 4-residue signals of seven other receptors and membrane proteins known to undergo rapid endocytosis, including the Tyr-Thr-Arg-Phe sequence of the transferrin receptor, a Type II membrane protein. These results are compatible with the 4-residue signals of this type being interchangeable, even among Type I and Type II membrane proteins.     

The mannose 6-phosphate/insulin-like growth factor-II receptor is a substrate of type V transforming growth factor-beta receptor.

The type V transforming growth factor beta (TGF-beta) receptor (TbetaR-V) is a ligand-stimulated acidotropic Ser-specific protein kinase that recognizes a motif of SXE/S(P)/D. This motif is present in the cytoplasmic domain of the mannose 6-phosphate/insulin-like growth factor-II (Man-6-P/IGF-II) receptor. We have explored the possibility that the Man-6-P/IGF-II receptor is a substrate of TbetaR-V. Purified bovine Man-6-P/IGF-II receptor was phosphorylated by purified bovine TbetaR-V in the presence of [gamma-32P]ATP and MnCl2 with an apparent Km of 130 nM. TGF-beta stimulated the phosphorylation of the Man-6-P/IGF-II receptor at 0 degrees C in mouse L cells overexpressing the Man-6-P/IGF-II receptor and in wild-type mink lung epithelial (Mv1Lu cells) metabolically labeled with [32P]orthophosphate. The in vitro and in vivo phosphorylation of the Man-6-P/IGF-II receptor occurred at the putative phosphorylation sites as revealed by phosphopeptide mapping and amino acid sequence analysis. TGF-beta stimulated Man-6-P/IGF-II receptor-mediated uptake (approximately 2-fold after 12 h treatment) of exogenous beta-glucuronidase in Mv1Lu cells and type II TGF-beta receptor (TbetaR-II)-defective mutant cells (DR26 cells) but not in type I TGF-beta receptor (TbetaR-I)-defective mutant cells (R-1B cells) and human colorectal carcinoma cells (RII-37 cells) expressing TbetaR-I and TbetaR-II but lacking TbetaR-V. These results suggest the Man-6-P/IGF-II receptor serves as an in vitro and in vivo substrate of TbetaR-V and that both TbetaR-V and TbetaR-I may play a role in mediating the TGF-beta-stimulated uptake of exogenous beta-glucuronidase.     

Developmental expression of the IGF-II/mannose 6-phosphate receptor

The first indication that the insulin-like growth factor-II/mannose 6-phosphate receptor (IGF-II/M6PR) is developmentally regulated came from studies of the serum form of the receptor in the rat. By immunoblotting, the circulating form of the receptor, which was 10 kDa smaller than the tissue receptor, was high in 19 day fetal and 3, 10, and 20 day postnatal sera and then declined sharply. We next used quantitative immunoblotting to measure the total tissue IGF-II/M6PR in the rat. The receptor levels were high in fetal tissues and in most tissues declined dramatically in late gestation and/or in the early postnatal period. The rank order of receptor expression was heart > placenta > lung = intestine > muscle = kidney > liver > brain. In heart, the receptor was 1.7% of total protein in the extract. More recently, we have examined the expression of IGF-II/M6PR mRNA using Northern blotting and a solution hybridization/RNase protection assay. The rank order of receptor mRNA concentration among fetal tissues agreed with the rank order of receptor protein. The concentration of receptor mRNA was significantly lower in postnatal tissue than in fetal tissue. Thus IGF-II/M6PR mRNA concentration is an important determinant of receptor protein in most tissues. What is the function of the IGF-II/M6PR in embryonic and fetal tissues? The M6PR in birds and frogs does not bind IGF-II. It is intriguing that the rat IGF-II/M6PR is prominent during the embryonic and fetal periods, times at which the differences between mammals, on the one hand, and frogs and birds, on the other, are most striking. Tissue remodeling is an important feature of embryonic and fetal development. Therefore, the well-established lysosomal enzyme targeting function of the receptor may be of particular importance. Since IGF-II can inhibit the cellular uptake of lysosomal enzymes via the IGF-II/M6PR, IGF-II may modulate this lysosomal enzyme targeting function. In addition, the receptor can provide a degradative pathway for IGF-II by receptor-mediated internalization. Thus the receptor could provide a check on the high levels of IGF-II known to be present in the fetus. Finally, the IGF-II/M6PR could directly signal certain biologic responses to IGF-II. © 1993 Wiley-Liss, Inc.     

The design, expression, and characterization of human insulin-like growth factor II (IGF-II) mutants specific for either the IGF-II/cation-independent mannose 6-phosphate receptor or IGF-I receptor.

Five mutants of recombinant insulin-like growth factor-II (rIGF-II) that bound with high affinity to either the IGF-II/cation-independent mannose 6-phosphate (IGF-II/CIM6-P) or the IGF-I receptor were prepared by site-directed mutagenic procedures, expressed as fusion proteins in the larva of Bombyx mori or Escherichia coli, purified to homogeneity, renatured, and characterized in terms of their receptor binding affinities and specificities as well as their biological activities. Class I mutants in which Phe26, Tyr27, and Val43 were substituted with Ser, Leu, and Leu, respectively, bound to enriched preparations of rat placental IGF-II/CIM6-P receptors with apparent equilibrium dissociation constants (Kd(app)) that were only slightly greater, i.e. 0.10, 0.05, and 0.06 nM, than that of rIGF-II (0.04 nM) or hIGF-II (0.03 nM). In contrast, replacing Phe26 with Ser resulted in 5- and 20-fold decreases in the affinities of this mutant for highly purified human placental IGF-I and insulin receptors, respectively. The affinities of the two other Class I mutants, [Leu27]- and [Leu43]rIGF-IIs, for these two receptors were reduced 80- to 220-fold. The affinities of Class II mutants, i.e. [Thr48,Ser49,Ile50]- and [Arg54,Arg55] rIGF-IIs, for IGF-I receptors were as potent as rIGF-II; however, they bound very poorly or not at all to the IGF-II/CIM6-P receptor. In the binding study of those mutant rIGF-IIs, IGF-II was observed to have an unexpectedly high affinity for pure human placental insulin receptor preparations. For example, the affinities of hIGF-II, rIGF-II, and two Class II rIGF-II mutants for the insulin receptor were only 3-, 9-, and 5-fold less, respectively, than that of porcine insulin. In two biological assay systems, i.e. the stimulation of DNA synthesis in Balb/c 3T3 cells and glycogen synthesis in HepG2 cells, the Kd(app) of the rIGF-II mutants for the IGF-I receptor but not the IGF-II/CIM6-P receptor correlated with their abilities to produce biological responses.     

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.     

The cytoplasmic tail of the mannose 6-phosphate/insulin-like growth factor-II receptor has two signals for lysosomal enzyme sorting in the Golgi

The mannose 6-phosphate/insulin-like growth factor-II (Man-6-P/IGF-II) receptor is known to cycle between the Golgi, endosomes, and the plasma membrane. In the Golgi the receptor binds newly synthesized lysosomal enzymes and transports them directly to an endosomal (prelysosomal) compartment without traversing the plasma membrane. Deletion of the carboxyl-terminal Leu-Leu-His-Val residues of the 163 amino acid cytoplasmic tail of the bovine Man-6-P/IGF-II receptor partially impaired this function, resulting in the diversion of a portion of the receptor-ligand complexes to the cell surface, where they were endocytosed. The same phenotype was observed when 134 residues of the cytoplasmic tail were deleted from the carboxyl terminus. Disruption of the Tyr24-Lys-Tyr-Ser-Lys-Val29 plasma membrane internalization signal alone had little effect on Golgi sorting, but when combined with either deletion resulted in a complete loss of this function. The mutant receptors retained the ability to recycle to the Golgi and bind cathepsin D. These results indicate that the cytoplasmic tail of the Man-6-P/IGF-II receptor contains two signals that contribute to Golgi sorting, presumably by interacting with the Golgi clathrin-coated pit adaptor proteins. The Leu-Leu-containing sequence represents a novel motif for mediating interaction with Golgi adaptor proteins.     

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.     

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.     

Kinetics of insulin-like growth factor II (IGF-II) interaction with domain 11 of the human IGF-II/mannose 6-phosphate receptor: function of CD and AB loop solvent-exposed residues

Ligands of the IGF-II/mannose 6-phosphate receptor (IGF2R) include IGF-II and mannose 6-phosphate modified proteins. Disruption of the negative regulatory effects of IGF2R on IGF-II-induced growth can lead to embryonic lethality and cancer promotion. Of the 15 IGF2R extracellular domains, domains 1-3 and 11 are known to have a conserved beta-barrel structure similar to that of avidin and the cation-dependent mannose 6-phosphate receptor, yet only domain 11 binds IGF-II with high specificity and affinity. In order to define the functional basis of this critical biological interaction, we performed alanine mutagenesis of structurally determined solvent-exposed loop residues of the IGF-II-binding site of human domain 11, expressed these mutant forms in Pichia pastoris, and determined binding kinetics with human IGF-II using isothermal calorimetry and surface plasmon resonance with transition state thermodynamics. Two hydrophobic residues in the CD loop (F1567 and I1572) were essential for binding, with a further non-hydrophobic residue (T1570) that slows the dissociation rate. Aside from alanine mutations of AB loop residues that decrease affinity by modifying dissociation rates (e.g. Y1542), a novel mutation (E1544A) of the AB loop enhanced affinity by threefold compared to wild-type. Conversion from an acidic to a basic residue at this site (E1544K) results in a sixfold enhancement of affinity via modification principally of the association rate, with enhanced salt-dependence, decreased entropic barrier and retained specificity. These data suggest that a functional hydrophobic binding site core is formed by I1572 and F1567 located in the CD loop, which initially anchors IGF-II. Within the AB loop, residues normally act to either stabilise or function as negative regulators of the interaction. These findings have implications for the molecular architecture and evolution of the domain 11 IGF-II-binding site, and the potential interactions with other domains of IGF2R.     

Insulin-like growth factor-II/cation-independent mannose 6-phosphate receptor mediates paracrine interactions during spermatogonial development

The insulin-like growth factor-II/cation-independent mannose 6-phosphate (IGF-II/M6P) receptor transduces signals after binding IGF-II or M6P-bearing growth factors. We hypothesized that this receptor relays paracrine signals between Sertoli cells and spermatogonia in the basal compartment of the seminiferous epithelium. For these studies spermatogonia were isolated from 8-day-old mice with purity >95% and viability >85% after overnight culture. The IGF-II/M6P receptors were present on the surface of spermatogonia, as detected by indirect immunofluorescence. We determined that both IGF-II and M6P-glycoproteins in Sertoli cell conditioned medium (SCM) modulate gene expression in isolated spermatogonia. The IGF-II produced dose-dependent increases in both rRNA and c-fos mRNA. These effects were mediated specifically by IGF-II/M6P receptors, as shown by studies using IGF-II analogues that are specific agonists for either IGF-I or IGF-II receptors. The SCM treatment also induced dose-dependent increases in rRNA levels, and M6P competition showed that this response required interaction with IGF-II/M6P receptors. The M6P-glycoproteins isolated from SCM by IGF-II/M6P receptor affinity chromatography increased spermatogonial rRNA levels at much lower concentrations than required by SCM treatment, providing further evidence for the paracrine activity of Sertoli M6P-glycoproteins. These results demonstrate that Sertoli cells secrete paracrine factors that modulate spermatogonial gene expression after interacting with cell-surface IGF-II/M6P receptors.