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.     

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)     

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.     

Structural basis for recognition of phosphorylated high mannose oligosaccharides by the cation-dependent mannose 6-phosphate receptor.

Mannose 6-phosphate receptors (MPRs) play an important role in the targeting of newly synthesized soluble acid hydrolases to the lysosome in higher eukaryotic cells. These acid hydrolases carry mannose 6-phosphate recognition markers on their N-linked oligosaccharides that are recognized by two distinct MPRs: the cation-dependent mannose 6-phosphate receptor and the insulin-like growth factor II/cation-independent mannose 6-phosphate receptor. Although much has been learned about the MPRs, it is unclear how these receptors interact with the highly diverse population of lysosomal enzymes. It is known that the terminal mannose 6-phosphate is essential for receptor binding. However, the results from several studies using synthetic oligosaccharides indicate that the binding site encompasses at least two sugars of the oligosaccharide. We now report the structure of the soluble extracytoplasmic domain of a glycosylation-deficient form of the bovine cation-dependent mannose 6-phosphate receptor complexed to pentamannosyl phosphate. This construct consists of the amino-terminal 154 amino acids (excluding the signal sequence) with glutamine substituted for asparagine at positions 31, 57, 68, and 87. The binding site of the receptor encompasses the phosphate group plus three of the five mannose rings of pentamannosyl phosphate. Receptor specificity for mannose arises from protein contacts with the 2-hydroxyl on the terminal mannose ring adjacent to the phosphate group. Glycosidic linkage preference originates from the minimization of unfavorable interactions between the ligand and receptor.     

Localization of the carbohydrate recognition sites of the insulin-like growth factor II/mannose 6-phosphate receptor to domains 3 and 9 of the extracytoplasmic region

The insulin-like growth factor II/mannose 6-phosphate receptor is a multifunctional receptor that binds to a diverse array of mannose 6-phosphate (Man-6-P) modified proteins as well as nonglycosylated ligands. Previous studies have mapped its two Man-6-P binding sites to a minimum of three domains, 1-3 and 7-9, within its 15-domain extracytoplasmic region. Since the primary amino acid determinants of carbohydrate recognition by the insulin-like growth factor II/mannose 6-phosphate receptor are predicted by sequence alignment to the cation-dependent mannose 6-phosphate receptor to reside within domains 3 and 9, constructs encoding either domain 3 alone or domain 9 alone were expressed in a Pichia pastoris expression system and tested for their ability to bind several carbohydrate ligands, including Man-6-P, pentamannosyl phosphate, the lysosomal enzyme, beta-glucuronidase, and the carbohydrate modifications (mannose 6-sulfate and Man-6-P methyl ester) found on Dictyostelium discoideum lysosomal enzymes. Although both constructs were functional in ligand binding and dissociation, these studies demonstrate the ability of domain 9 alone to fold into a high affinity (K(d) = 0.3 +/- 0.1 nm) carbohydrate-recognition domain whereas the domain 3 alone construct is capable of only low affinity binding (K(d) approximately 500 nm) toward beta-glucuronidase, suggesting that residues in adjacent domains (domains 1 and/or 2) are important, either directly or indirectly, for optimal binding by domain 3.     

Differential sorting of lysosomal enzymes in mannose 6-phosphate receptor-deficient fibroblasts.

In higher eukaryotes, the transport of soluble lysosomal enzymes involves the recognition of their mannose 6-phosphate signal by two receptors: the cation-independent mannose 6-phosphate/insulin-like growth factor II receptor (CI-MPR) and the cation-dependent mannose 6-phosphate receptor (CD-MPR). It is not known why these two different proteins are present in most cell types. To investigate their relative function in lysosomal enzyme targeting, we created cell lines that lack either or both MPRs. This was accomplished by mating CD-MPR-deficient mice with Thp mice that carry a CI-MPR deleted allele. Fibroblasts prepared from embryos that lack the two receptors exhibit a massive missorting of multiple lysosomal enzymes and accumulate undigested material in their endocytic compartments. Fibroblasts that lack the CI-MPR, like those lacking the CD-MPR, exhibit a milder phenotype and are only partially impaired in sorting. This demonstrates that both receptors are required for efficient intracellular targeting of lysosomal enzymes. More importantly, comparison of the phosphorylated proteins secreted by the different cell types indicates that the two receptors may interact in vivo with different subgroups of hydrolases. This observation may provide a rational explanation for the existence of two distinct mannose 6-phosphate binding proteins in mammalian cells.     

Brefeldin A affects the cellular distribution of endocytic receptors differentially.

The cell surface expression of three endocytic receptors was studied in human hepatoma Hep G2 cells treated with brefeldin A (BFA). Ligand binding and cell surface iodination revealed that BFA increased the number of mannose 6-phosphate/insulin-like growth factor II receptors twofold and decreased the amount of asialoglycoprotein and transferrin receptors by 40-60%. The altered expression of receptors at the cell surface was paralleled by changes in the respective ligand uptake. The implications of this finding on our understanding of intracellular trafficking are discussed.     

Interactions of recombinant and platelet transforming growth factor-beta 1 precursor with the insulin-like growth factor II/mannose 6-phosphate receptor

Recombinant transforming growth factor (TGF)-beta 1 precursor was recently found to contain mannose 6-phosphate (Purchio et al., 1988, J. Biol. Chem. 263, 14211-14215). In the present study, recombinant TGF-beta 1 precursor was shown to bind to the insulin-like growth factor (IGF)-II/mannose 6-phosphate (man6P) receptor on the plasma membrane of cells since: 1) Insulin, which induces an increase in cell surface IGF-II/man6P receptors on adipocytes, caused a 2.7-fold increase in TGF-beta 1 precursor binding to adipocytes; 2) Chinese hamster ovary cells selected for overexpression of the IGF-II/man6P receptor exhibited an increased binding of TGF-beta 1 precursor in comparison to the parental cells; and 3) the binding of 125I-TGF-beta 1 precursor to these transfected cells and adipocytes was largely inhibited by man6P. After 15 minutes at 37 degrees C, 75% of the recombinant TGF-beta 1 precursor was found to be internalized in the transfected cells. Additional studies with latent TGF-beta 1 isolated from platelets indicated that this material could also bind to the isolated IGF-II/man6P receptor.     

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.     

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).     

Receptor-mediated endocytosis of α-galactosidase A in human podocytes in Fabry disease

Injury to the glomerular podocyte is a key mechanism in human glomerular disease and podocyte repair is an important therapeutic target. In Fabry disease, podocyte injury is caused by the intracellular accumulation of globotriaosylceramide. This study identifies in the human podocyte three endocytic receptors, mannose 6-phosphate/insulin-like growth II receptor, megalin, and sortilin and demonstrates their drug delivery capabilities for enzyme replacement therapy. Sortilin, a novel α-galactosidase A binding protein, reveals a predominant intracellular expression but also surface expression in the podocyte. The present study provides the rationale for the renal effect of treatment with α-galactosidase A and identifies potential pathways for future non-carbohydrate based drug delivery to the kidney podocyte and other potential affected organs.     

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.     

叶酸缺乏致胎鼠宫内发育迟缓及肝脏胰岛素生长因子系统表达变化

目的：叶酸是一种水溶性B族维生素,在体内氨基酸与核苷酸代谢中起重要作用,是胎儿生长发育所必须的营养素。本文通过建立叶酸缺乏的孕鼠模型,探讨叶酸缺乏对胎鼠宫内发育的影响,并研究胎鼠肝脏组织中胰岛素生长因子（IGF）系统的表达变化。方法：雌性C57BL／6J小鼠叶酸缺乏组6只、正常对照组6只,分别饲以不舍叶酸和含2mg叶酸／kg的纯合饲料。四周后与雄鼠交配,于怀孕第13．5天（13．5dpc）对孕鼠剖腹取胎,观察和评价胎鼠发育指标,并对宫内发育迟缓（IUGR）比率进行统计。用Real-timePCR法检测胎鼠肝脏组织中胰岛素生长因子I（IGFI）、胰岛素生长因子I受体（IGFIR）、胰岛素生长因子II（IGFII）、胰岛素生长因子II受体（IGFIIR）、胰岛素生长因子结合蛋白1（IGFBP-1）和胰岛素生长因子结合蛋白3（IGFBP-3）mRNA的相对表达水平。结果：叶酸缺乏组雌鼠合笼前每日体重增长量降低,13．5dpc胎鼠吸收胎和死胎比率升高,胎重下降,IUGR比率显著升高,差异有统计学意义（P〈0．05）;叶酸缺乏组胎鼠肝脏组织中IGFII和IGFIIRmRNA的相对表达水平均低于正常对照组（P〈0．05）,IGFI、IGFIR、IGFBP-1和IGFBP-3mRNA的相对表达水平两组间没有差异（P〉0．05）。结论：叶酸缺乏会导致小鼠孕中期胎鼠IUGR比率升高及胎肝IGFII和IGFIIRmRNA的表达水平降低,提示叶酸缺乏对IGF系统基因的调控,可能与胎鼠I-UGR发生机制有关。     

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.     

P-type lectins

The two members of the P-type lectin family, the cation-dependent mannose 6-phosphate receptor (CD-MPR) and the insulin-like growth factor II/mannose 6-phosphate receptor (IGF-II/MPR), are distinguished from all other lectins by their ability to recognize phosphorylated mannose residues. The P-type lectins play an essential role in the generation of functional lysosomes within the cells of higher eukaryotes by directing newly synthesized lysosomal enzymes bearing the mannose 6-phosphate (M6P) signal to lysosomes. At the cell surface, the IGF-II/MPR also binds to the nonglycosylated polypeptide hormone, IGF-II, targeting this potent mitogenic factor for degradation in lysosomes. Moreover, in recent years, the multifunctional nature of the IGF-II/MPR has become increasingly apparent, as the list of extracellular ligands recognized by this receptor has grown to include a diverse spectrum of M6P-containing proteins as well as nonglycosylated ligands, implicating a role for the IGF-II/MPR in a number of important physiological pathways. Recent investigations have provided valuable insights into the molecular basis of ligand recognition by the MPRs as well as the complex intracellular trafficking pathways traversed by these receptors. This review provides a current view on the structures, functions, and medical relevance of the P-type lectins.     

The insulin-like growth factor type 1 and insulin-like growth factor type 2/mannose-6-phosphate receptors independently regulate ERK1/2 activity in HEK293 cells

Insulin-like growth factor types 1 and 2 (IGF-1; IGF-2) and insulin-like peptides are all members of the insulin superfamily of peptide hormones but bind to several distinct classes of membrane receptor. Like the insulin receptor, the IGF-1 receptor is a heterotetrameric receptor tyrosine kinase, whereas the IGF-2/ mannose 6-phosphate receptor is a single transmembrane domain protein that is thought to function primarily as clearance receptors. We recently reported that IGF-1 and IGF-2 stimulate the ERK1/2 cascade by triggering sphingosine kinase-dependent "transactivation" of G protein-coupled sphingosine-1-phosphate receptors. To determine which IGF receptors mediate this effect, we tested seven insulin family peptides, IGF-1, IGF-2, insulin, and insulin-like peptides 3, 4, 6, and 7, for the ability to activate ERK1/2 in HEK293 cells. Only IGF-1 and IGF-2 potently activated ERK1/2. Although IGF-2 was predictably less potent than IGF-1 in activating the IGF-1 receptor, they were equipotent stimulators of ERK1/2. Knockdown of IGF-1 receptor expression by RNA interference reduced the IGF-1 response to a greater extent than the IGF-2 response, suggesting that IGF-2 did not signal exclusively via the IGF-1 receptor. In contrast, IGF-2 receptor knockdown markedly reduced IGF-2-stimulated ERK1/2 phosphorylation, with no effect on the IGF-1 response. As observed previously, both the IGF-1 and the IGF-2 responses were sensitive to pertussis toxin and the sphingosine kinase inhibitor, dimethylsphingosine. These data indicate that endogenous IGF-1 and IGF-2 receptors can independently initiate ERK1/2 signaling and point to a potential physiologic role for IGF-2 receptors in the cellular response to IGF-2.     

Binding of 125I-insulin-like growth factor-II to cells cultured in fetal bovine serum: a complication

Insulin-like growth factor II is an important fetal mitogen in mice and humans and its biological activity is regulated in a complex manner. The peptide interacts with three membrane-bound receptors, with a superfamily of insulin-like growth factor binding proteins and with the proteoglycan, glypican-3. Recently, the blood protein, vitronectin, has been identified as a novel insulin-like growth factor II-binding protein. Many studies have used cell lines maintained in fetal bovine serum to identify cell surface insulin-like growth factor II binding sites. We now describe a complication associated with the interpretation of such in vitro studies. Fetal bovine serum-derived vitronectin adheres very tightly to tissue culture dishes. When cells that have been maintained in fetal bovine serum are incubated with 125I-insulin-like growth factor II, a substantial fraction of the 125I-insulin-like growth factor II apparently associated with the cell surfaces may represent radioliogand bound by the fetal bovine serum-derived vitronectin. This may result in over-estimation of cell surface insulin-like growth factor II binding sites.     

Insulin stimulates the assembly of cytosolic clathrin onto adipocyte plasma membranes

The effects of insulin on the subcellular distribution of the heavy chain of clathrin and on the insulin-like growth factor II (IGF-II) mannose 6-phosphate receptor were investigated in isolated rat adipocytes. Plasma membranes, intracellular membranes, and cytosol were separated by differential centrifugation, and the concentration of clathrin and receptor in each fraction was quantified by sequential immunoblotting with monoclonal and polyclonal antibodies against these proteins. A 3-fold increase in the amount of clathrin heavy chain associated with isolated plasma membranes was found after treatment of cells with low concentrations of insulin. This effect was complete within 2 min of stimulation at 37 degrees C and was abolished at 5-10 degrees C. The insulin-mediated increase in the cell surface concentration of receptors for IGF-II/mannose 6-phosphate displayed a similar time course and temperature dependence. A concomitant decrease in the concentration of IGF-II/mannose 6-phosphate receptors in intracellular membranes was observed. In contrast, no significant changes in the concentration of clathrin in this fraction could be detected. Instead, a marked decrease in the level of unassembled cytosolic clathrin was observed in insulin-treated cells compared with controls. These results suggest that insulin induces an increase in the assembly of cytosolic clathrin onto the plasma membrane in conjunction with its ability to increase the concentration of receptors on the cell surface.     

Insulin receptor isoform A, a newly recognized, high-affinity insulin-like growth factor II receptor in fetal and cancer cells

Insulin-like growth factor II (IGF-II) is a peptide growth factor that is homologous to both insulin-like growth factor I (IGF-I) and insulin and plays an important role in embryonic development and carcinogenesis. IGF-II is believed to mediate its cellular signaling via the transmembrane tyrosine kinase type 1 insulin-like growth factor receptor (IGF-I-R), which is also the receptor for IGF-I. Earlier studies with both cultured cells and transgenic mice, however, have suggested that in the embryo the insulin receptor (IR) may also be a receptor for IGF-II. In most cells and tissues, IR binds IGF-II with relatively low affinity. The IR is expressed in two isoforms (IR-A and IR-B) differing by 12 amino acids due to the alternative splicing of exon 11. In the present study we found that IR-A but not IR-B bound IGF-II with an affinity close to that of insulin. Moreover, IGF-II bound to IR-A with an affinity equal to that of IGF-II binding to the IGF-I-R. Activation of IR-A by insulin led primarily to metabolic effects, whereas activation of IR-A by IGF-II led primarily to mitogenic effects. These differences in the biological effects of IR-A when activated by either IGF-II or insulin were associated with differential recruitment and activation of intracellular substrates. IR-A was preferentially expressed in fetal cells such as fetal fibroblasts, muscle, liver and kidney and had a relatively increased proportion of isoform A. IR-A expression was also increased in several tumors including those of the breast and colon. These data indicate, therefore, that there are two receptors for IGF-II, both IGF-I-R and IR-A. Further, they suggest that interaction of IGF-II with IR-A may play a role both in fetal growth and cancer biology.