Insulin-induced redistribution of the insulin-like growth factor II/mannose 6-phosphate receptor in intact rat liver

The ability of acute insulin treatment to elicit a redistribution of the liver insulin-like growth factor-II/ mannose 6-phosphate (IGF-II/M6P) receptor has been studied in rats, using cell fractionation. Injection of insulin (0.4-50 microg) led to a time- and dose-dependent decrease in IGF-II binding activity in Golgi-endosomal (GE) fractions, along with an increase in activity in the plasma membrane (PM) fraction; only receptor number was affected. Quantitative subfractionation of the microsomal fraction on sucrose density gradients showed that IGF-II binding activity distributed similarly to galactosyltransferase (a Golgi marker), at slightly higher densities than in vivo internalized (125)I-insulin, and at lower densities than 5' nucleotidase and alkaline phosphodiesterase (two plasma membrane markers). Insulin treatment led to a slight time-dependent and reversible shift of IGF-II binding activity toward higher densities. Subfractionation of the GE fraction on Percoll gradients showed that IGF-II binding activity was broadly distributed, with about 60% at low densities coinciding with galactosyltransferase and early internalized (125)I-insulin and with 40% at high densities in the region of late internalized (125)I-insulin. Insulin treatment caused a time-dependent and reversible shift of the distribution of IGF-II binding activity toward low densities. On SDS-PAGE, the size of the affinity-labeled IGF-II/M6P receptor was comparable in GE and PM fractions (about 255 kDa), but on Western blots receptor size was slightly lower in the latter (245 kDa) than in the former (255 kDa). Insulin treatment did not affect the size, but modified the abundance of the IGF-II/M6P receptor in a manner similar to that of IGF-II binding. In vivo chloroquine treatment fully suppressed the changes in IGF-II binding activity in liver GE and PM fractions observed in insulin-treated rats. We conclude that insulin elicits a time-dependent and reversible redistribution of liver IGF-II receptors from Golgi elements and endosomes to the plasma membrane, presumably via early endosomes.     

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.     

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.     

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.     

Soluble insulin-like growth factor II/mannose 6-phosphate receptor inhibits DNA synthesis in insulin-like growth factor II sensitive cells

The soluble form of the insulin-like growth factor II (IGF-II)/mannose 6-P (IGF-II/M6P) receptor is released by cells in culture and circulates in the serum. It retains its ability to bind IGF-II and blocks IGF-II-stimulated DNA synthesis in isolated rat hepatocytes. Because these cells are not normally stimulated to divide by IGF-II in vivo, the effect of soluble IGF-II/M6P receptor on DNA synthesis has been further investigated in two cell lines sensitive to IGF-II; mouse 3T3(A31) fibroblasts, stimulated by low levels of IGF-II following priming by epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) and Buffalo rat liver (BRL) cells, which secrete IGF-II and proliferate in the absence of exogenous growth factors. Soluble IGF-II/M6P receptor (0.2-2.0 microgram/ml) purified from a rat hepatoma cell line inhibited DNA synthesis (determined by dThd incorporation) in both cell lines. Basal DNA synthesis was very low in serum-free 3T3 cells, but high in serum-free BRL cells, possibly as a result of autocrine IGF-II production. The inhibitory effect was reversible in cells preincubated with soluble receptor prior to incubation with growth factors and could also be overcome by excess IGF-II. Soluble receptor was more potent in IGF-II-stimulated 3T3 cells and serum-free BRL cells than in BRL cells incubated with serum. Mean inhibition by four preparations of soluble receptor (1 microgram/ml) was 34.7% +/- 4.4% in BRL cells stimulated with fetal calf serum (FCS) (5%) compared to 54.8% +/- 4.2% in serum-free BRL cells (P = 0.05) and 60.6% +/- 6.5% (P = 0.02) in 3T3 cells stimulated by PDGF, EGF, and IGF-II. Soluble receptor had no effect on DNA synthesis in 3T3 cells stimulated with IGF-I. These results demonstrate that soluble receptor, at physiological concentrations, can block proliferation of cells by IGF-II and could therefore play a role in blocking tumor growth mediated by IGF-II.     

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.     

Mannose 6-phosphate/insulin-like growth factor II-binding proteins in human serum and urine. Their relation to the mannose 6-phosphate/insulin-like growth factor II receptor.

Human serum and urine contain polypeptides which bind mannose 6-phosphate (M6P) and insulin-like growth factor II (IGF II) and crossreact with antibodies against the M6P/IGF II receptor. These polypeptides are considered to be fragments of the M6P/IGF II receptor. The major Mr approx. 205,000 fragment in serum and urine is about 10 kDa smaller in size than the membrane-associated receptor and is accompanied by minor forms with Mr values ranging from 104,000 to 180,000. The presence of receptor fragments in biological fluids indicates that shedding is one of the mechanisms contributing to the turnover of the M6P/IGF II receptor and that receptor fragments are part of the heterogenous group of serum proteins whic bind IGF II.     

Isolation and characterization of mannose 6-phosphate/insulin-like growth factor II receptor from bovine serum.

A convenient means was devised for the purification of milligram quantities of a soluble form of the mannose 6-phosphate/insulin-like growth factor II receptor (Man-6-P/IGF II receptor). The receptor was purified to near homogeneity from bovine serum by affinity chromatography on agarose-pentamannosephosphate in the absence of detergent. Approximately 2.5 mg of receptor were obtained from 500 ml of fetal calf serum. The concentration of receptor in serum decreased sharply with development. Fetal calf serum Man-6-P/IGF II receptor was immunologically similar to detergent-solubilized, membrane-bound Man-6-P/IGF II receptor from bovine liver. N-Terminal sequence analysis revealed that the purified serum receptor, but not the solubilized, membrane-associated receptor, contains stoichiometric amounts of bound IGF II. The results of sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel chromatography studies suggest that the fetal calf serum receptor (in contrast to the solubilized, membrane-bound bovine testis receptor) does not aggregate. The affinity of the fetal calf serum receptor for bovine testis beta-galactosidase approximated one-half that observed for solubilized, membrane-bound bovine testis receptor.     

Dominant-negative effect of truncated mannose 6-phosphate/insulin-like growth factor II receptor species in cancer

Oligomerization of the mannose 6-phosphate/insulin-like growth factor?II receptor (M6P/IGF2R) is important for optimal ligand binding and internalization. M6P/IGF2R is a tumor suppressor gene that exhibits loss of heterozygosity and is mutated in several cancers. We tested the potential dominant-negative effects of two cancer-associated mutations that truncate M6P/IGF2R in ectodomain repeats 9 and 14. Our hypothesis was that co-expression of the truncated receptors with the wild-type/endogenous full-length M6P/IGF2R would interfere with M6P/IGF2R function by heterodimer interference. Immunoprecipitation confirmed formation of heterodimeric complexes between full-length M6P/IGF2Rs and the truncated receptors, termed Rep9F and Rep14F. Remarkably, increasing expression of either Rep9F or Rep14F provoked decreased levels of full-length M6P/IGF2Rs in both cell lysates and plasma membranes, indicating a dominant-negative effect on receptor availability. Loss of full-length M6P/IGF2R was not due to increased proteasomal or lysosomal degradation, but instead arose from increased proteolytic cleavage of cell-surface M6P/IGF2Rs, resulting in ectodomain release, by a mechanism that was inhibited by metal ion chelators. These data suggest that M6P/IGF2R truncation mutants may contribute to the cancer phenotype by decreasing the availability of full-length M6P/IGF2Rs to perform tumor-suppressive functions such as binding/internalization of receptor ligands such as insulin-like growth factor II.     

The insulin-like growth factor II/mannose-6-phosphate receptor

Recent evidence from molecular cloning, biochemical and immunological experiments has established that the cation-independent mannose-6-phosphate (Man-6-P) receptor and insulin-like growth factor-II (IGF-II) receptor are the same protein. Although the role of the IGF-II/Man-6-P receptor as a transporter of hydrolytic enzymes in the biogenesis of lysosomes is certain, elucidation of the receptor's structure has not yet provided major insights into the function of IGF-II binding. Mutually exclusive binding of IGF-II and naturally occurring phosphomannosyl ligands to distinct but proximal sites on the receptor suggests that the IGF-II/Man-6-P receptor cannot simultaneously fulfill the functional requirements of both IGF-II and lysosomal enzymes. Does the receptor transduce on intracellular signal in order to mediate the biological effects of IGF-II? If so, then the receptor must interact with an effector molecule, perhaps a G protein, in the mechanism of IGF-II action. Further information from ligand binding and especially mutagenesis experiments will be needed to elucidate the potentially multiple functions of the IGF-II/Man-6-P receptor.     

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 kangaroo cation-independent mannose 6-phosphate receptor binds insulin-like growth factor II with low affinity.

The mammalian cation-independent mannose 6-phosphate receptor (CI-MPR) binds mannose 6-phosphate-bearing glycoproteins and insulin-like growth factor (IGF)-II. However, the CI-MPR from the opossum has been reported to bind bovine IGF-II with low affinity (Dahms, N. M., Brzycki-Wessell, M. A., Ramanujam, K. S., and Seetharam, B. (1993) Endocrinology 133, 440-446). This may reflect the use of a heterologous ligand, or it may represent the intrinsic binding affinity of this receptor. To examine the binding of IGF-II to a marsupial CI-MPR in a homologous system, we have previously purified kangaroo IGF-II (Yandell, C. A., Francis, G. L., Wheldrake, J. F., and Upton, Z. (1998) J. Endocrinol. 156, 195-204), and we now report the purification and characterization of the CI-MPR from kangaroo liver. The interaction of the kangaroo CI-MPR with IGF-II has been examined by ligand blotting, radioreceptor assay, and real-time biomolecular interaction analysis. Using both a heterologous and homologous approach, we have demonstrated that the kangaroo CI-MPR has a lower binding affinity for IGF-II than its eutherian (placental mammal) counterparts. Furthermore, real-time biomolecular interaction analysis revealed that the kangaroo CI-MPR has a higher affinity for kangaroo IGF-II than for human IGF-II. The cDNA sequence of the kangaroo CI-MPR indicates that there is considerable divergence in the area corresponding to the IGF-II binding site of the eutherian receptor. Thus, the acquisition of a high-affinity binding site for regulating IGF-II appears to be a recent event specific to the eutherian lineage.     

Regulation of mannose 6-phosphate/insulin-like growth factor II receptor distribution by activators and inhibitors of protein kinase C

The tumor-promotor phorbol dibutyrate (PDBt) increases the binding of a neoglycoprotein containing mannose 6-phosphate (Man6P) and of insulin-like growth factor II (IGF-II) to the Man6P/IGF-II receptor at the cell surface. This effect is dependent on time and concentration and is also seen with synthetic 1-oleoyl-2-acetyl-sn-glycerol, but not with 4 alpha-phorbol, an inactive tumor-promoter. The increase is due to a 3-4-fold increase in the number of cell-surface, receptors, accompanied by a 1.6-fold increase in ligand-binding affinity. The internalization rate of the Man6P/IGF-II receptor is not affected by PDBt, suggesting that the redistribution of these receptors to the cell surface is due to an accelerated externalization rate. The redistribution of Man6P/IGF-II receptors did not impair the sorting of newly synthesized Man6P-containing ligands while uptake of these ligands is 2-4-fold increased. Inactivation or down regulation of protein kinase C decreased the binding of the Man6P-containing neoglycoprotein to 65% of controls. Incubation of cells with Man6P, IGF-I, IGF-II or epidermal growth factor induces a rapid redistribution of Man6P/IGF-II receptors to the plasma membrane [Braulke, T., Tippmer, S., Neher, E. & von Figura, K. (1989) EMBO J. 8, 681-686]. Incubation with PDBt prevented the effect of growth factors but not that of Man6P on receptor redistribution. Inactivation of protein kinase C did not affect the Man6P/IGF-II receptor redistribution induced by Man6P and growth factors. These data suggest that Man6P, growth factors and activation of protein kinase C by phorbol esters and diacylglycerols modulate Man6P/IGF-II receptor cell-surface binding by at least two independent mechanisms, receptor redistribution as well as an increase of binding affinity, which might be involved in regulation of endocytosis of ligands.     

Regulation of the mannose 6-phosphate/IGF II receptor expression at the cell surface by mannose 6-phosphate, insulin like growth factors and epidermal growth factor.

Mannose 6-phosphate, insulin like growth factors I and II (IGF I, IGF II), insulin and epidermal growth factor (EGF) induce a 1.5- to 2-fold increase of mannose 6-phosphate binding sites at the cell surface of human skin fibroblasts. The increase is completed within 10-15 min, is dose and temperature dependent, reversible and transient even in the presence of the effectors. It is due to a redistribution of mannose 6-phosphate/IGF II receptors from internal membranes to the cell surface, while the affinity of the receptors is not affected. Combinations of mannose 6-phosphate with IGF I, IGF II or EGF stimulate the redistribution of the receptor to the cell surface in an additive manner, while combinations of the growth factors result in a non-additive stimulation of redistribution. The redistribution is not dependent on extracellular calcium and appears also to be independent of changes of free intracellular calcium. Pre-treatment of fibroblasts with cholera toxin or pertussis toxin increases the number of cell surface receptors 2- and 1.5-fold, respectively. Neither of the toxins affects the redistribution of mannose 6-phosphate/IGF II receptors induced by the growth factors, while both toxins abolish the receptor redistribution induced by mannose 6-phosphate. These results suggest a multiple regulation of the cell surface expression of mannose 6-phosphate/IGF II receptors by Gs- and Gi-like proteins sensitive to cholera toxin and pertussis toxin and by stimulation of mannose 6-phosphate/IGF II, IGF I and EGF receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Extracellular release as the major degradative pathway of the insulin-like growth factor II/mannose 6-phosphate receptor.

The presence of a soluble, truncated form of the IGF-II/Man-6-P receptor in serum has suggested that cleavage from the cell surface may be an initial step in the degradation of this protein (MacDonald, R. G., Tepper, M. A., Clairmont, K. B., Perregaux, S. B., and Czech, M. P. (1989) J. Biol. Chem. 264, 3256-3261). In order to test this hypothesis, we pulse-labeled cultured BRL-3A rat liver cells with [35S]methionine and [35S]cysteine and measured the fate of labeled receptor at various times after incubation with unlabeled amino acids. It was found that the appearance of labeled IGF-II/Man-6-P receptor in the medium accounts quantitatively for the loss of labeled receptor from the BRL-3A cells. In similar experiments with Chinese hamster ovary cells, L6 rat myoblasts, and chick embryo fibroblasts, labeled receptor from the cell membranes decreases with a time course corresponding to the appearance of soluble receptor in the medium. The release of labeled receptor into the medium can be blocked by the addition of the protease inhibitors aprotinin, chymostatin, or phenylmethylsulfonyl fluoride, but not antipain, leupeptin, and benzamidine. The results are consistent with the hypothesis that the degradation and loss of cellular IGF-II/Man-6-P receptors occurs by a nonlysosomal mechanism involving their proteolysis and removal into the extracellular fluid.     

Identification of residues essential for carbohydrate recognition by the insulin-like growth factor II/mannose 6-phosphate receptor.

Two distinct mannose 6-phosphate (Man-6-P) receptors (MPRs), the cation-dependent MPR (CD-MPR) and the insulin-like growth factor II/MPR (IGF-II/MPR), recognize a diverse population of Man-6-P-containing ligands. The IGF-II/MPR is a type I transmembrane glycoprotein with a large extracytoplasmic region composed of 15 repeating domains that display sequence identity to each other and to the single extracytoplasmic domain of the CD-MPR. A structure-based sequence alignment of the two distinct Man-6-P-binding sites of the IGF-II/MPR with the CD-MPR implicates several residues of IGF-II/MPR domains 3 and 9 as essential for Man-6-P binding. To test this hypothesis single amino acid substitutions were made in constructs encoding either the N- or the C-terminal Man-6-P-binding sites of the bovine IGF-II/MPR. The mutant IGF-II/MPRs secreted from COS-1 cells were analyzed by pentamannosyl phosphate-agarose affinity chromatography, identifying four residues (Gln-392, Ser-431, Glu-460, and Tyr-465) in domain 3 and four residues (Gln-1292, His-1329, Glu-1354, and Tyr-1360) in domain 9 as essential for Man-6-P recognition. Binding affinity studies using the lysosomal enzyme, beta-glucuronidase, confirmed these results. Together these analyses provide strong evidence that the two Man-6-P-binding sites of the IGF-II/MPR are structurally similar to each other and to the CD-MPR and utilize a similar carbohydrate recognition mechanism.     

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.     

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.     

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.     

Serum form of the rat insulin-like growth factor II/mannose 6-phosphate receptor is truncated in the carboxyl-terminal domain

The insulin-like growth factor (IGF)-II/mannose 6-phosphate (Man-6-P) receptor present in mammalian tissues as an apparent molecular mass = 250 kDa glycoprotein has recently been detected in fetal rat serum in a lower molecular mass form (240 kDa). In the present studies the serum receptor was affinity labeled with 125I-IGF-II after its adsorption onto pentamannosyl 6-phosphate-Sepharose, demonstrating that it can also bind both ligands simultaneously. The receptors in both serum and fresh plasma exhibited the lower molecular mass compared to tissue receptors, indicating this form circulates in vivo. In order to probe the structural basis of the serum receptor's lower mass, we raised antipeptide antibodies against cytoplasmic and extracellular domains of the tissue form of the rat receptor deduced from complementary DNA clones (MacDonald, R. G., Pfeffer, S. R., Coussens, L., Tepper, M. A., Brocklebank, C. M., Mole, J. E., Anderson, J. K., Chen, E., Czech, M. P., and Ullrich, A. (1988) Science 239, 1134-1137). Peptide 22C, Glu-Glu-Glu-Thr-Asp-Glu-Asn-Glu-Thr-Glu-Trp-Leu-Met-Glu-Glu-Ile-Gln-Val- Pro-Ala - Pro-Arg, located in the cytoplasmic domain 32 residues carboxyl-terminal to the transmembrane region, and peptide 13D, Tyr-Tyr-Leu-Asn-Val-Cys-Arg-Pro-Leu-Asn-Pro-Val-Pro-Gly-Cys-Asp, located 1476 residues amino-terminal to the transmembrane domain were synthesized and used as immunogens in rabbits. IGF-II/Man-6-P receptors were first immunoprecipitated from either rat serum or a Triton X-100 extract of rat placental plasma membranes using a polyclonal antireceptor antibody. The immunoadsorbed receptors were then reduced, alkylated, electrophoresed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotted onto nitrocellulose, and probed with antipeptide antibodies. Anti-13D revealed the major receptor band in all the membrane and serum samples tested as well as several minor species of lower apparent mass in serum. Fetal and neonatal rat sera contained 3-4 times as much of the receptor as adult serum. In contrast, anti-22C recognized the membrane IGF-II/Man-6-P receptor but failed to recognize any of the serum receptor species. These results indicate that the serum IGF-II/Man-6-P receptor is truncated or altered in its cytoplasmic domain, consistent with the hypothesis that it is derived from cells by proteolytic cleavage.