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.     

Interactions of the receptor for insulin-like growth factor II with mannose-6-phosphate and antibodies to the mannose-6-phosphate receptor

Recently, the sequence of the human receptor for insulin-like growth factor II (IGF-II) was found to be 80% identical [Morgan et al., (1987) Nature 329, 301-307] to the sequence of a partial clone of the bovine cation-independent mannose-6-phosphate receptor [Lobel et al., (1987) Proc. Natl. Acad. Sci. USA 84, 2233-2237]. In the present study, the purified receptor for insulin-like growth factor II (IGF-II) was found to react with two different polyclonal antibodies to the purified mannose-6-phosphate receptor. Moreover, mannose-6-phosphate was found to stimulate the binding of labeled IGF-II to the IGF-II receptor by two-fold. This effect had the same specificity and affinity as the reported binding of mannose-6-phosphate to its receptor; mannose-1-phosphate and mannose had no effect on the binding of labeled IGF-II to its receptor, and the half-maximally effective concentration of mannose-6-phosphate was 0.3 mM. Also, mannose-6-phosphate did not affect labeled IGF-II binding to the insulin receptor. These results support the hypothesis that a single protein of Mr-250,000 binds both IGF-II and mannose-6-phosphate. Furthermore, they indicate that mannose-6-phosphate can modulate the interaction of IGF-II to its receptor.     

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.     

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.     

A role for the insulin-like growth factor II/mannose-6-phosphate receptor in the insulin-induced inhibition of protein catabolism

Insulin and insulin-like growth factor (IGF)-I inhibit intracellular protein degradation in a variety of different cell types. In the present studies, the IGF-I-induced inhibition of protein metabolism in Chinese hamster ovary (CHO) cells was found to be blocked by polyclonal antibodies to the IGF-II/mannose-6-phosphate phosphate (Man-6-P) receptor, but not by control immunoglobulin. In contrast, these antibodies had no effect on the ability of IGF-I to stimulate glucose uptake in the same cells. The antibodies to the IGF-II/Man-6-P receptor also inhibited the effect of IGF-I and insulin on protein catabolism in human foreskin fibroblasts and human hepatoma cells, respectively. Moreover, CHO cells overexpressing a cDNA coding for the IGF-II/Man-6-P receptor were found to exhibit an increased effect of insulin on protein catabolism. In contrast, the insulin stimulation of glucose uptake is the same in these transfected cells as in the parental CHO cells. These results implicate the IGF-II/Man-6-P receptor in the insulin- and IGF-I-induced inhibition of protein catabolism.     

Epac- and Rap- independent ERK1/2 phosphorylation induced by Gs-coupled receptor stimulation in HEK293 cells

Serotonin activates Ras and Ras-dependent ERK1/2 phosphorylation in HEK293 cells expressing G(s)-coupled 5-HT(4) or 5-HT(7) serotonin receptors through unknown mechanisms. Both Epac/Rap-dependent and -independent pathways for Ras-dependent ERK1/2 activation have been suggested. Epac overexpression or Epac-specific 8-CPT-2'-O-Me-cAMP did not cause ERK1/2 phosphorylation, despite Rap activation. The data did not support a role for PLCepsilon or DAG-dependent Ras GEFs of the Ras-GRP family in Ras-dependent ERK1/2 phosphorylation. However, serotonin stimulated phosphorylation of endogenous and recombinant Ras-GRF1, increased [Ca(2+)](i) and caused Ca(2+)- and calmodulin-dependent ERK1/2 phosphorylation. Different signalling pathways seem to be utilised by G(s)-coupled receptors in various isolates of HEK293 cells.     

Specificity of binding of clathrin adaptors to signals on the mannose-6-phosphate/insulin-like growth factor II receptor.

Adaptors mediate the interaction of clathrin with select groups of receptors. Two distinct types of adaptors, the HA-II adaptors (found in plasma membrane coated pits) and the HA-I adaptors (localized to Golgi coated pits) bind to the cytoplasmic portion of the 270 kd mannose 6-phosphate (M6P) receptor-a receptor which is concentrated in coated pits on both the plasma membrane and in the trans-Golgi network. Neither type of adaptor appears to compete with the other for binding, suggesting that each type recognizes a distinct site on the M6P receptor tail. Mutation of the two tyrosines in the tail essentially eliminates the interaction with the HA-II plasma membrane adaptor, which recognizes a 'tyrosine' signal on other endocytosed receptors (for example, the LDL receptor and the poly Ig receptor). In contrast, the wild type and the mutant M6P receptor tail (lacking tyrosines) are equally effective at binding HA-I adaptors. This suggests that there is an HA-I recognition signal in another region of the M6P receptor tail, C-terminal to the tyrosine residues, which remains intact in the mutant. This signal is presumably responsible for the concentration of the M6P receptor, with bound lysosomal enzymes, into coated pits which bud from the trans-Golgi network, thus mediating efficient transfer of these enzymes to lysosomes.     

Regulation of amyloid precursor protein expression and secretion via activation of ERK1/2 by hepatocyte growth factor in HEK293 cells transfected with APP751

The increased intracellular levels and aberrant processing of the amyloid precursor protein (APP) are associated with beta-amyloid peptide (A beta) production, cerebrovascular amyloid deposition, and amyloid plaque formation. Here we report that APP level, soluble APP (sAPP) secretion, and A beta production in HEK293 cells transfected with either wild-type APP(751) or APP(751) carrying the Swedish mutation are all elevated by hepatocyte growth factor (HGF). We investigated the potential molecular mechanisms underlying the HGF effect. Our data show that HGF stimulated extended activation of extracellular signal-regulated protein kinases (ERK1/2). Pretreatment of cells with inhibitors (UO126 or PD98059) for MEK, the upstream kinase of ERK1/2, abolished ERK1/2 activation evoked by HGF, and abrogated HGF-induced increases in APP levels and sAPP secretion. In addition, transient expression of active MEK1 activated ERK1/2 and increased intracellular APP levels and sAPP secretion. Inhibition of ERK1/2 activity, however, failed to block HGF-stimulated A beta production. Consistently, transient expression of active MEK1 did not increase A beta accumulation. Taken together, these results suggest that: (1) HGF regulates the intracellular levels of APP and the secretion of sAPP and A beta; (2) the modulation of APP levels and sAPP secretion induced by HGF is mediated via the MEK1/ERK1/2 signaling pathway; (3) HGF-stimulated A beta production is independent of ERK activity and, therefore, independent of HGF-evoked elevation of intracellular APP levels.     

Chicken and Xenopus mannose 6-phosphate receptors fail to bind insulin-like growth factor II

The recent demonstration that a single mammalian receptor protein binds both mannose 6-phosphate (Man-6-P) and insulin-like growth factor II (IGF-II) with high affinity has suggested a multifunctional physiological role for this receptor, possibly including signal transduction. In order to better understand the functions of this receptor, we have investigated the properties of Man-6-P receptors from non-mammalian species. Receptors were affinity-purified from Triton X-100 extracts of total membranes from Xenopus and chicken liver as well as rat placenta using pentamannosyl 6-phosphate-Sepharose. The Man-6-P receptor was adsorbed to the pentamannosyl 6-phosphate-Sepharose and specifically eluted by Man-6-P in all three species, as evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver staining. When the purified receptors from these three species were cross-linked to 125I-IGF-II with disuccinimidyl suberate, only receptors isolated from rat membranes were affinity-labeled. To further evaluate the properties of these Man-6-P receptors, binding of 125I-rat-IGF-II and 125I-chicken Tyr-Gly-Thr-Ala-IGF-II to purified receptors from Xenopus, chicken, and rat was evaluated by polyethylene glycol precipitation. Only the rat Man-6-P receptor exhibited detectable binding of 125I-IGF-II. These data suggest that the emergence of a high affinity IGF-II binding site on the Man-6-P receptor occurred in evolution after the divergence of mammals from other vertebrates. Thus, the biological actions of IGF-II in chickens and frogs appear to be initiated by the type I IGF receptor.     

Both FGF23 and extracellular phosphate activate Raf/MEK/ERK pathway via FGF receptors in HEK293 cells

Fibroblast growth factor 23 (FGF23) is a phosphaturic hormone produced by bone and exerts its function in the target organs by binding the FGF receptor (FGFR) and Klotho. Since recent studies suggested that extracellular inorganic phosphate (Pi) itself triggers signal transduction and regulates gene expression in some cell types, we tested the notion that extracellular Pi induces signal transduction in the target cells of FGF23 also and influences its signaling, utilizing a human embryonic kidney cell line HEK293. HEK293 cells expressed low levels of klotho, and treatment with a recombinant FGF23[R179Q], a proteolysis‐resistant mutant of FGF23, resulted in phosphorylation of ERK1/2 and induction of early growth response‐1 (EGR1) expression. Interestingly, increased extracellular Pi resulted in activation of the Raf/MEK/ERK pathway and expression of EGR1, which involved type III sodium/phosphate (Na⁺/Pi) cotransporter PiT‐1. Since the effects of an inhibitor of Na⁺/Pi cotransporter on FGF23 signaling suggested that the signaling triggered by increased extracellular Pi shares the same downstream cascade as FGF23 signaling, we further investigated their convergence point. Increasing the extracellular Pi concentration resulted in the phosphorylation of FGF receptor substrate 2α (FRS2α), as did treatment with FGF23. Knockdown of FGFR1 expression diminished the phosphorylation of both FRS2α and ERK1/2 induced by the Pi. Moreover, overexpression of FGFR1 rescued the decrease in Pi‐induced phosphorylation of ERK1/2 in the cells where the expression of PiT‐1 was knocked down. These results suggest that increased extracellular Pi triggers signal transduction via PiT‐1 and FGFR and influences FGF23 signaling in HEK293 cells. J. Cell. Biochem. 111: 1210–1221, 2010. © 2010 Wiley‐Liss, Inc.     

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.     

Activation and nuclear translocation of PKCdelta,Pyk2 and ERK1/2 by gonadotropin releasing hormone in HEK293 cells

The mechanism of agonist-induced activation of Pyk2 and its relationship with ERK1/2 phosphorylation was analyzed in HEK293 cells stably expressing the gonadotropin releasing hormone (GnRH) receptor. GnRH stimulation caused rapid and sustained phosphorylation of ERK1/2 and Pyk2 that was accompanied by their nuclear translocation. Pyk2 was also localized on cell membranes and at focal adhesions. Dominant negative Pyk2 (PKM) had no effect on GnRH-induced ERK1/2 phosphorylation and c-fos expression. These actions of GnRH on ERK1/2 and Pyk2 were mimicked by activation of protein kinase C (PKC) and were abolished by its inhibition. GnRH caused translocation of PKC and δ, but not of , ι and λ, to the cell membrane, as well as phosphorylation of Raf at Ser338, a major site in the activation of MEK/ERK1/2. Stimulation of HEK293 cells by EGF caused marked ERK1/2 phosphorylation that was attenuated by the selective EGFR receptor (EGF-R) kinase inhibitor, AG1478. However, GnRH-induced ERK1/2 activation was independent of EGF-R activation. These results indicate that activation of PKC is responsible for GnRH-induced phosphorylation of both ERK1/2 and Pyk2, and that Pyk2 activation does not contribute to GnRH signaling. Moreover, GnRH-induced phosphorylation of ERK1/2 and expression of c-fos in HEK293 cells is independent of Src and EGF-R transactivation, and is mediated through the PKC/Raf/MEK cascade.     

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.     

Modulation of insulin-like growth factor-II/mannose 6-phosphate receptors and transforming growth factor-beta 1 during liver regeneration.

Transforming growth factor-beta 1 (TGF-beta 1) is a potent mito-inhibiting substance that is thought to play an important function in regulating hepatocyte proliferation during liver regeneration. In this investigation, we have shown by immunohistochemistry that hepatocytes containing significant intracellular concentrations of TGF-beta 1 12 h after a two-thirds partial hepatectomy. This increase in hepatocyte TGF-beta 1 concentration was initially confined to those cells that resided in the periportal region of the liver. The elevation of intracellular TGF-beta 1 was, however, transient, and within 36 h, the hepatocytes positive for TGF-beta 1 had changed in a wavelike fashion from the periportal to the pericentral region of the liver lobules. By 48 h, most hepatocytes no longer contained TGF-beta 1. Interestingly, this temporary increase in TGF-beta 1 always preceded the onset of hepatocyte replication by approximately 3-6 h. Since TGF-beta 1 mRNA has been shown to be absent from hepatocytes normally and throughout liver regeneration, these results imply that the increase in intracellular TGF-beta 1 resulted from an augmented uptake. We have further shown that the insulin-like growth factor-II/mannose 6-phosphate (IGF-II/Man-6-P) receptors were up-regulated during liver regeneration and that the increased expression of this receptor co-localized in those hepatocytes containing elevated concentrations of TGF-beta 1. The latent TGF-beta 1 phosphomannosyl glycoprotein complex has been shown to bind to the IGF-II/Man-6-P receptor. Therefore, our data are consistent with the hypothesis that this latent complex is internalized through the IGF-II/Man-6-P receptor to the intracellular acidic prelysosomal/endosomal compartments where the mature TGF-beta 1 molecule could be activated by dissociation from the latent complex.     

ERK/Egr-1 signaling pathway is involved in CysLT2 receptor-mediated IL-8 production in HEK293 cells

The CysLT₂ receptor is involved in myocardial ischemia/reperfusion injury, differentiation of colorectal cancers, bleomycin-induced pulmonary inflammation and fibrosis. However, the signal transduction of cysteinyl leukotriene receptor 2 (CysLT₂) in inflammatory responses remains to be clarified. In HEK293 cells stably expressing hCysLT₁, hCysLT₂ and rGPR17, we determined the signaling pathways for interleukin-8 (IL-8) production after CysLT₂ receptor activation. HEK293 cells were stably transfected with the recombinant plasmids of pcDNA3.1(+)-hCysLT₁, pcDNA3.1(+)-hCysLT₂ and pcDNA3.1-rGPR17. Leukotriene C₄ (LTC₄) and LTD₄ were used as the agonists to induce IL-8 production and the related changes in signal molecules. We found that LTC₄ and LTD₄ significantly induced IL-8 promoter activation in the HEK293 cells stably expressing hCysLT₂, but not in those expressing hCysLT₁ and rGPR17. In hCysLT₂-HEK293 cells, LTC₄ induced elevation of intracellular calcium, ERK1/2 phosphorylation and Egr-1 expression, and stimulated IL-8 expression and release. These responses were blocked by the selective CysLT₂ receptor antagonist HAMI3379. The ERK1/2 inhibitor U0126 inhibited Egr-1 and IL-8 expression as well as IL-8 release, but the JNK and p38 inhibitors did not have the inhibitory effects. Down-regulation of Egr-1 by RNA interference with its siRNA inhibited the LTC₄-induced IL-8 expression and release. In conclusion, these findings indicate the ERK-Egr-1 pathway of CysLT₂ receptors mediates IL-8 production induced by the pro-inflammatory mediators LTC₄ and LTD₄.     

Fibroblast chemotaxis and prolidase activity modulation by insulin-like growth factor II and mannose 6-phosphate

Chemotactic locomotion of fibroblasts requires extensive degradation of extracellular matrix components. The degradation is provided by a variety of proteases, including lysosomal enzymes. The process is regulated by cytokines. The present study shows that mannose 6-phosphate and insulin-like growth factor II (IGF-II) enhance fibroblast chemotaxis toward platelet-derived growth factor (PDGF). It is suggested that lysosomal enzymes (bearing mannose 6-phosphate molecules) are involved in chemotactic activity of the cells. The suggestion is supported by the observation that a-mannosidase and cathepsin D inhibitor - pepstatin are very potent inhibitors of fibroblast chemotaxis. Simultaneously, mannose 6-phosphate stimulates extracellular collagen degradation. The final step in collagen degradation is catalyzed by the cytosolic enzyme - prolidase. It has been found that mannose 6-phosphate stimulates also fibroblast prolidase activity with a concomitant increase in lysosomal enzymes activity. The present study demonstrates that the prolidase activity in fibroblasts may reflect the chemotactic activity of the cells and suggests that the mechanism of cell locomotion may involve lysosomal enzyme targeting, probably through IGF-II/mannose 6-phosphate receptor.