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)     

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.     

Low and high affinity receptors mediate cellular uptake of heparanase

Heparanase is an endoglycosidase which cleaves heparan sulfate and hence participates in degradation and remodeling of the extracellular matrix. Importantly, heparanase activity correlated with the metastatic potential of tumor-derived cells, attributed to enhanced cell dissemination as a consequence of heparan sulfate cleavage and remodeling of the extracellular matrix barrier. Heparanase has been characterized as a glycoprotein, yet glycan biochemical analysis was not performed to date. Here, we applied the Qproteometrade mark GlycoArray kit to perform glycan analysis of heparanase, and compared the kit results with the more commonly used biochemical analyses. We employed fibroblasts isolated from patients with I-cell disease (mucolipidosis II), fibroblasts deficient of low density lipoprotein receptor-related protein and fibroblasts lacking mannose 6-phosphate receptor, to explore the role of mannose 6-phosphate in heparanase uptake. Iodinated heparanase has been utilized to calculate binding affinity. We provide evidence for hierarchy of binding to cellular receptors as a function of heparanase concentration. We report the existence of a high affinity, low abundant (i.e., low density lipoprotein receptor-related protein, mannose 6-phosphate receptor), as well as a low affinity, high abundant (i.e., heparan sulfate proteoglycan) receptors that mediate heparanase binding, and suggest that these receptors co-operate to establish high affinity binding sites for heparanase, thus maintaining extracellular retention of the enzyme tightly regulated.     

Role of protein phosphatases in insulin-like growth factor II (IGF II)-stimulated mannose 6-phosphate/IGF II receptor redistribution.

The regulated expression of mannose 6-phosphate/insulin-like growth factor II (M6P/IGF II) receptors in plasma membranes has previously been shown to be accompanied by marked changes in the phosphorylation state of the receptors (Corvera, S., Folander, K., Clairmont, K. B., and Czech, M. P. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 7567-7571). In the present study we show that protein phosphatase 2A dephosphorylates the human M6P/IGF II receptor in vitro. Incubation of human fibroblasts with okadaic acid, a specific inhibitor of this phosphatase, resulted in a depletion of M6P/IGF II receptors at the cell surface without affecting their internalization kinetics. The phosphorylation state of the remaining cell surface receptors was 3-fold increased. Thus, the endocytosis rate of M6P/IGF II receptors appears to be unaltered by increased phosphorylation. While the decreased cell surface expression of receptors was reversible upon removal of okadaic acid the IGF II-induced redistribution of M6P/IGF II receptors to the plasma membrane (Braulke, T., Tippmer, S., Neher, E., and von Figura, K. (1989) EMBO J. 8, 681-686) was irreversibly inhibited by the phosphatase inhibitor. Receptor redistribution in response to protein kinase C activation was not affected by okadaic acid. These results suggest that the cell surface expression of M6P/IGF II receptor can be regulated by phosphatase-dependent and -independent pathways. In addition, the phosphorylation state and the steady-state cell surface number of transferrin receptors were not affected by okadaic acid, whereas it impaired the IGF II-stimulated receptor redistribution similarly as for M6P/IGF II receptors. The data indicate that okadaic acid-sensitive protein phosphatases may play a general role in terms of IGF II-modulated receptor recycling.     

Effects of brefeldin A on the endocytic route. Redistribution of mannose 6-phosphate/insulin-like growth factor II receptors to the cell surface.

The effect of brefeldin A (BFA) on the trafficking of the mannose 6-phosphate/insulin-like growth factor II receptor within the endocytic route was analyzed. Treatment with BFA induced a redistribution of the receptor to the cell surface and increased both the binding and internalization of ligands 2-4-fold. The effect of BFA was dose- and time-dependent and reversible. Determinations of transport rates showed that BFA increases the internalization rate and the externalization rate of the receptor. This implies that the higher surface concentration is due to higher concentrations of receptor at the intracellular sites from where they recycle to the cell surface. The effect of BFA was additive to the redistribution induced by insulin-like growth factors I and II and was observed in all human and rodent cell lines analyzed. BFA increased also the cell surface expression of the Mr 46,000 mannose 6-phosphate receptor but not of the transferrin receptor. The results indicate that BFA interferes with the transport of mannose 6-phosphate receptors and affects the endocytosis of lysosomal enzymes by increasing the number of receptors available for recycling to the cell surface.     

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.     

Mannose 6-phosphate potentiates insulin-like growth factor II-stimulated inositol trisphosphate production in proximal tubular basolateral membranes

To ascertain whether mannose 6-phosphate affects insulin-like growth factor (IGF) II stimulation of phospholipase C activity in the basolateral membrane of the renal proximal tubular cell, we determined the effect of mannose 6-phosphate on IGF II-stimulated production of inositol trisphosphate (Ins-P3) in isolated basolateral membranes. Production of Ins-P3 measured in the presence of 10(-10), 10(-9), or 10(-8) M rat IGF II was potentiated approximately 2-fold by inclusion of 5 mM mannose 6-phosphate in incubations. Mannose 6-phosphate had no effect on Ins-P3 production in the absence of IGF II. Neither mannose 1-phosphate, mannose, glucose 6-phosphate, nor fructose 1-phosphate exerted similar potentiation. Enhancement of IGF II-stimulated Ins-P3 production required concentrations on the order of several millimolar mannose 6-phosphate. Total and specific binding of 10(-10) M 125I-IGF II to basolateral membranes was significantly increased by 5 mM mannose 6-phosphate. However, there was no significant effect on total or specific binding of 10(-9) or 10(-8) M 125I-IGF II. Our findings suggest that mannose 6-phosphate potentiates stimulation of phospholipase C by IGF II in the basolateral membrane of the renal proximal tubular cell and that potentiation is mediated via a mechanism in addition to enhanced binding of IGF II. Such potentiation could reflect a role for the mannose 6-phosphate moiety as a modulator of IGF II "signal" transmission in vivo.     

Mannose 6-phosphate/insulin like growth factor II receptor: the two types of ligands bind simultaneously to one receptor at different sites

Pentamannose 6-phosphate/trilysine substituted aprotinin (PMP-lys-aprotinin) and insulin like growth factor II (IGF II) were used as affinity ligands for the mannose 6-phosphate (M6P) and IGF II binding sites of the M6P/IGF II receptor. Both ligands were cross linked to intact receptor and tryptic fragments of the receptor. The pattern of receptor fragments with M6P and IGF II binding sites differed indicating that the two binding sites are located on different segments of the receptor. The receptor was incubated with [125I]IGF II and pentamannose 6-phosphate substituted bovine serum albumin (PMP-BSA). From these mixtures [125I]IGF II receptor complexes could be precipitated with antibodies against the PMP-BSA indicating that the M6P/IGF II receptor can bind simultaneously IGF II and M6P-containing ligands.     

I-cell disease-like phenotype in mice deficient in mannose 6-phosphate receptors

Mannose 6-phosphate receptor deficient mice were generated by crossing mice carrying null alleles for Igf2 and the 300 kDa and 46 kDa mannose 6-phosphate receptors, Mpr300 and Mpr46. Pre- and perinatal lethality of mice nullizygous for Igf2, Mpr300 and Mpr46 was increased. Triple deficient mice surviving the first postnatal day had normal viability and developed a phenotype resembling human I-cell disease. The triple deficient mice were characterized by dwarfism, facial dysplasia, waddling gait, dysostosis multiplex, elevated lysosomal enzymes in serum and histological signs of lysosomal storage predominantly in fibroblasts, but also in parenchymal cells of brain and liver. A paternally inherited Mpr300 wild type allele that is normally inactive in mice due to imprinting was reactivated in some tissues of mice lacking IGF II and MPR 46 and carrying a maternal Mpr300 null allele. Inspite of the partial reactivation the phenotype of these mice was similar to that of triple deficient mice.     

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.     

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.     

Synthesis and receptor binding affinity of carboxylate analogues of the mannose 6-phosphate recognition marker

The mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R) is involved in multiple physiological pathways including targeting of lysosomal enzymes, degradation of IGF2, and cicatrization through TGFbeta activation. To target potential therapeutics to this membrane receptor, four carboxylate analogues of mannose 6-phosphate (M6P) were synthesized. Three of them, two isosteric carboxylate analogues and a malonate derivative, showed a binding affinity for the M6P/IGF2R equivalent to or higher than that of M6P. Contrary to M6P, all these analogues were particularly stable in human serum. Moreover, these derivatives did not present any cytotoxic activity against two human cell lines. These analogues represent a new potential for the lysosomal targeting of enzyme replacement therapy in lysosomal diseases or to prevent the membrane-associated activities of the M6P/IGF2R.     

Binding of phosphorylated oligosaccharides to immobilized phosphomannosyl receptors

We have purified phosphomannosyl-enzyme receptors from bovine liver on an affinity column composed of glycoproteins isolated from Dictyostelium discoideum secretions. Binding of human fibroblast beta-hexosaminidase B to receptors reconstituted into phosphatidylcholine liposomes was 1) specifically inhibited by mannose 6-phosphate, but not mannose 1-phosphate or glucose 6-phosphate, and 2) had properties similar to the previously reported binding of enzyme to receptors on cell surfaces and isolated membranes. In order to determine the structural features of the phosphomannosyl recognition marker required for receptor recognition, we covalently coupled purified receptor to an agarose gel bead support for affinity chromatography of phosphorylated, high mannose-type oligosaccharides isolated from fibroblast secretions radiolabeled with [2-3H]mannose. Neutral oligosaccharides and oligosaccharides containing one or two phosphates in phosphodiester linkage were not retained by the receptor column. By contrast, oligosaccharides bearing one phosphomonoester moiety were retarded on the column; those bearing two phosphomonoesters were bound to the column and were eluted with 10 mM mannose 6-phosphate. The binding of the oligosaccharides to the immobilized receptor correlates with their ability to be pinocytosed by fibroblasts and shows that the preferred recognition marker for the phosphomannosyl-enzyme receptor is a high mannose-type oligosaccharide chain bearing two uncovered phosphomannosyl groups.     

Chromosomal mapping of the gene for the type II insulin-like growth factor receptor/cation-independent mannose 6-phosphate receptor in man and mouse

The gene for insulin-like growth factor II (IGF-II) receptor (IGF2R) that has recently been found, by DNA sequencing, to be identical to the cation-independent mannose 6-phosphate receptor (CIM6PR) has been mapped in the human and murine species. Cloned cDNAs for human and rat IGF-II receptors were used to probe Southern blots of somatic cell hybrid DNA and for in situ chromosomal hybridization. The genes are located in a region of other conserved syntenic genes on the long arm of human chromosome 6, region 6q25----q27, and mouse chromosome 17, region A-C. The CIM6PR/IGF2R locus in man is asyntenic with the genes encoding IGF-II (IGF2), the IGF-I receptor (IGF1R), and the cation-dependent mannose 6-phosphate receptor (CDM6PR).     

Mannose 6-phosphate-containing peptides activate phospholipase C in proximal tubular basolateral membranes from canine kidney

To ascertain whether mannose 6-phosphate-containing peptides that bind to the insulin-like growth factor II (IGF II)/mannose 6-phosphate receptor activate phospholipase C, we determined the effect of proliferin, transforming growth factor-beta 1 (TGF-beta 1) precursor, and beta-galactosidase on production of inositol trisphosphate (Ins-P3) in basolateral membranes isolated from the renal proximal tubule of dogs. Both proliferin and TGF-beta 1 precursor stimulated Ins-P3 production in a concentration-dependent manner. Maximal production was stimulated by approximately 10(-13) M of each peptide. beta-Galactosidase had no effect on Ins-P3 generation. Neither proliferin nor TGF-beta 1 precursor potentiated IGF II-stimulated Ins-P3 production. Mannose 6-phosphate itself had no effect on Ins-P3 generation. However, mannose 6-phosphate potentiated production stimulated by 10(-11) M proliferin or 10(-11) M TGF-beta 1 precursor while inhibiting production stimulated by 10(-14) M of either peptide. Addition of anti-mannose 6-phosphate receptor antibodies to basolateral membranes abolished proliferin and TGF-beta 1 precursor-stimulated Ins-P3 generation. We conclude that, in addition to IGF II, mannose 6-phosphate-containing ligands for the IGF II/mannose 6-phosphate receptor activate basolateral membrane phospholipase C. Such activation could reflect a common mechanism for signal transduction by these peptides mediated via the IGF II/mannose 6-phosphate 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.     

Biosynthesis and endocytosis of lysosomal enzymes in human colon carcinoma SW 1116 cells: impaired internalization of plasma membrane-associated cation-independent mannose 6-phosphate receptor.

The human colon adenocarcinoma cell lines SW 948, SW 1116, and SW 1222 were tested for their ability to sort and internalize lysosomal enzymes. The biosynthesis of the lysosomal enzymes cathepsin B, arylsulfatase A, and beta-hexosaminidase in these cell lines exhibits no significant differences to that in human fibroblasts. The intracellular targeting of newly synthesized hydrolases to the lysosomes relies in colon carcinoma cells on the mannose 6-phosphate receptor system. Both the cation-independent mannose 6-phosphate receptor (CI-MPR) and the cation-dependent mannose 6-phosphate receptor are expressed in all colon carcinoma cell lines investigated. Endocytosis of lysosomal enzymes via mannose 6-phosphate receptors is reduced in colon carcinoma cells as compared with human fibroblasts. SW 1116 cells were shown to be deficient in receptor-mediated endocytosis of mannose 6-phosphate containing ligands. Ligands of other endocytic receptors as well as the fluid-phase marker horseradish peroxidase were internalized at normal rates. While antibodies against CI-MPR bind to the surface of SW 1116 cells, these antibodies cannot be internalized. These data suggest that the cycling of CI-MPR is specifically impaired in SW 1116 cells.     

Targeted disruption of the M(r) 46,000 mannose 6-phosphate receptor gene in mice results in misrouting of lysosomal proteins.

Lysosomal enzymes containing mannose 6-phosphate recognition markers are sorted to lysosomes by mannose 6-phosphate receptors (MPRs). The physiological importance of this targeting mechanism is illustrated by I-cell disease, a fatal lysosomal storage disorder caused by the absence of mannose 6-phosphate residues in lysosomal enzymes. Most mammalian cells express two MPRs. Although the binding specificities, subcellular distribution and expression pattern of the two receptors can be differentiated, their coexpression is not understood. The larger of the two receptors with an M(r) of approximately 300,000 (MPR300), which also binds IGFII, appears to have a dominant role in lysosomal enzyme targeting, while the function of the smaller receptor with an M(r) of 46,000 (MPR46) is less clear. To investigate the in vivo function of the MPR46, we generated MPR46-deficient mice using gene targeting in embryonic stem cells. Reduced intracellular retention of newly synthesized lysosomal proteins in cells from MPR46 -/- mice demonstrated an essential sorting function of MPR46. The phenotype of MPR46 -/- mice was normal, indicating mechanisms that compensate the MPR46 deficiency in vivo.     

Binding of urokinase-type plasminogen activator receptor (uPAR) to the mannose 6-phosphate/insulin-like growth factor II receptor: contrasting interactions of full-length and soluble forms of uPAR

Urokinase-type plasminogen activator receptor (uPAR) binding by the mannose 6-phosphate/insulin-like growth factor II receptor (Man-6-P/IGF2R) is considered important to Man-6-P/IGF2R tumor suppressor function via regulation of cell surface proteolytic activity. Our goal was to map the uPAR binding site of the Man-6-P/IGF2R by analyzing the uPAR binding characteristics of a panel of minireceptors containing different regions of the Man-6-P/IGF2R extracytoplasmic domain. Coimmunoprecipitation assays revealed that soluble recombinant uPAR (suPAR) bound the Man-6-P/IGF2R at two distinct sites, one localized to the amino-terminal end of the Man-6-P/IGF2R extracytoplasmic domain (repeats 1-3) and the other to the more carboxyl-terminal end (repeats 7-9). These sites correspond with the positions of the two Man-6-P binding domains of Man-6-P/IGF2R. Indeed, the suPAR-Man-6-P/IGF2R interaction was inhibited by Man-6-P, and binding-competent su-PAR species represented a minor percentage (8-30%) of the suPAR present. In contrast, Man-6-P/IGF2R binding of endogenous, full-length uPAR solubilized from plasma membranes of the prostate cancer cell line, PC-3, was not inhibited by Man-6-P. Further studies showed that very little (<5%) endogenous uPAR was Man-6-P/IGF2R binding-competent. We conclude that, contrary to previous reports, the interaction between uPAR and Man-6-P/IGF2R is a low percentage binding event and that suPAR and full-length uPAR bind the Man-6-P/IGF2R by different mechanisms.     

Annexin VI is a mannose-6-phosphate-independent endocytic receptor for bovine β-glucuronidase

Endocytosis and transport of bovine liver β-glucuronidase to lysosomes in human fibroblasts are mediated by two receptors: the well-characterized cation-independent mannose 6-phosphate receptor (IGF-II/Man6PR) and an IGF-II/Man6PR-independent receptor, which recognizes a Ser-Trp*-Ser sequence present on the ligand. The latter receptor was detergent extracted from bovine liver membranes and purified. LC/ESI–MS/MS analysis revealed that this endocytic receptor was annexin VI (AnxA6). Several approaches were used to confirm this finding. First, the binding of bovine β-glucuronidase to the purified receptor from bovine liver membranes and His-tagged recombinant human AnxA6 protein was confirmed using ligand-blotting assays. Second, western blot analysis using antibodies raised against IGF-II/Man6PR-independent receptor as well as commercial antibodies against AnxA6 confirmed that the receptor and AnxA6 were indeed the same protein. Third, double immunofluorescence experiments in human fibroblasts confirmed a complete colocalization of the bovine β-glucuronidase and the AnxA6 receptor on the plasma membrane. Lastly, two cell lines were stably transfected with a plasmid containing the cDNA for human AnxA6. In both transfected cell lines, an increase in cell surface AnxA6 and in mannose 6-phosphate-independent endocytosis of bovine β-glucuronidase was detected. These results indicate that AnxA6 is a novel receptor that mediates the endocytosis of the bovine β-glucuronidase.