Insulin-like growth factor-II/mannose-6-phosphate receptors are increased in hepatocytes from regenerating rat liver

Insulin-like growth factor-II (IGF-II) receptor levels were determined in hepatocytes from sham-operated and two thirds hepatectomized rats. [125I]IGF-II binding to confluent cultures increased 2-fold in cells isolated 24 and 48 h after hepatectomy compared to that in cells from sham-operated rats. Receptor levels increased from 1.74 +/- 0.14 x 10(4)/cell (sham-operated) to 3.60 +/- 0.31 x 10(4)/cell (hepatectomized; P = 0.002), with no change in affinity of IGF-II binding (Ka = 6.4 x 10(9) M-1). As previously reported, receptor levels increased in cells plated at low density, but this effect was decreased in cells from hepatectomized rats (80% increase) compared to that in cells from control rats (300% increase). Serum IGF-I levels decreased 50% 24 h after partial hepatectomy (P less than 0.001), but returned to normal levels by 48 h. However, IGF-I synthesis was not decreased in hepatocytes isolated 24 h after partial hepatectomy, suggesting that decreased serum levels are due to decreased liver mass. Circulating IGF-II levels were not altered by partial hepatectomy, and IGF-II production was not detected in hepatocytes from sham-operated or hepatectomized rats. Transforming growth factor-beta is thought to terminate hepatocyte proliferation upon complete liver regeneration. In hepatocytes from sham-operated or hepatectomized rats transforming growth factor-beta totally blocked DNA synthesis, but had no effect on elevated IGF-II receptor levels after partial hepatectomy. Concomitant with increased IGF-II receptor levels, hepatocytes from hepatectomized rats were more sensitive to IGF-II stimulation of DNA synthesis. [3H]Thymidine incorporation in the presence of epidermal growth factor (50 ng/ml) was stimulated 66% by IGF-II (300 ng/ml) in control cells compared with 220% in cells from hepatectomized animals. These results suggest that IGF-II and the IGF-II receptor may play a role in liver regeneration.     

Effects of mannose-6-phosphate on receptor-mediated endocytosis of insulin-like growth factor-II

The insulin-like growth factor-II (IGF-II) and lysosomal enzymes containing a mannose-6-phosphate (M6P) recognition site bind to different sites of the same receptor molecule. We have observed that M6P increases the receptor-mediated uptake of IGF-II into IM-9 cells. We now confirm this phenomenon in a different line, the H-35 rat hepatoma cells, and present additional characterization of the underlying mechanisms. When incubated in the presence of radiolabeled IGF-II, H-35 cells accumulated, in a time-dependent fashion, radioactivity that was resistant to removal by trypsin digestion at 15 C, indicating that it was endocytosed. In the presence of 3 mM M6P, endocytosed counts were approximately 2-fold higher after 5 min of incubation, an enhancement that peaked at 10 min, then declined, but was still evident after 40 min (1.5-fold). The rate of release of cell-associated IGF-II, degraded or intact, as measured in a chase experiment, was not affected by M6P. These observations indicate that M6P increased accumulation of IGF-II by accelerating its rate of endocytosis rather than by interfering with IGF-II degradation or with the recycling of intact hormone-receptor complexes to the cell surface. Electrophoresis after affinity cross-linking of labeled cells demonstrated that the enhancement in radioactivity could be located at a molecular size of approximately 250 kDa, corresponding to IGF-II-receptor complexes. Preincubation with M6P did not significantly alter the specific binding of IGF-II to the cell surface of H-35 cells, as measured by a binding assay at 4 C. Finally, pretreatment with cycloheximide for up to 8 h, to remove all newly synthesized lysosomal enzymes bound to the M6P/IGF-II receptor, did not affect IGF-II endocytosis beyond what could be accounted for by some loss of receptor, suggesting that the observed effect of M6P is due to the binding of M6P itself to the receptor and not to displacement of lysosomal enzymes. We conclude that simultaneous occupancy of the M6P/IGF-II receptor by ligands on both binding sites enhances its rate of endocytosis.     

Activation of osteoblast insulin-like growth factor-II/cation-independent mannose-6-phosphate receptors by specific phosphorylated sugars and antibodies induce insulin-like growth factor-II effects.

The phosphorylated monosaccharide, mannose-6-phosphate (M6P), causes a dose-dependent stimulation of alkaline phosphatase production by osteoblasts. The concentrations tested ranged from 0.1 to 30 mM. A maximal effect was reproducibly seen at 10-30 mM, and represented a 30% stimulation over control cells. Glucose-6-phosphate and fructose-1-phosphate also stimulated osteoblast alkaline phosphatase production, but not to the same extent as M6P. Sugar residues such as mannose, mannose-1-phosphate, and fructose-6-phosphate had no effect. The stimulatory effect of M6P is similar to that seen with insulin-like growth factor II(IGF-II). However, increasing doses of IGF-II did not further stimulate or add to the effect of 10 mM M6P. These data indicate that the mechanism for the transduction of the stimulatory signal may be similar for both IGF-II and M6P. They do not address, however, the possibility of separate or similar binding sites for the two agents. A specific polyclonal antibody to the IGF-II/cation-independent mannose-6-phosphate receptor (IGF-II/CI-MPR) elicits the same effects as M6P and IGF-II in these bone cells. Non-immune serum used as a control does not have any effect. These results suggest that activation of the osteoblast IGF-II/CI-MPR by either M6P or a specific antibody can evoke a biological response similar to that observed with IGF-II.     

Secretion of soluble insulin-like growth factor-II/mannose 6-phosphate receptor by rat tissues in culture.

The insulin-like growth factor-II/mannose 6 phosphate receptor, which binds both IGF-II and mannose 6-phosphate containing proteins, such as lysosomal enzymes, has been detected in the serum of several species. Neither the source nor the role of this soluble, truncated form of the membrane receptor has been determined. This study has examined the ability of a variety of rat tissues in culture to synthesize and release soluble receptor. Explants (1 mm3) of 21-day fetal, neonatal and adult rat tissues were cultured in serum-free medium and the conditioned medium analyzed for the presence of receptor. Using IGF-II binding and immunochemical techniques, receptor was detected in media from heart, kidney, liver, lung and muscle explants. [35S]cysteine labeling indicated that de novo synthesis of the soluble receptor occurs in the cultured explants and can be inhibited by cycloheximide. This rat tissue explant culture system demonstrates that soluble receptor is released by a number of tissues, and should provide a useful model for further investigations into its function and regulation.     

Expression of the insulin-like growth factor-II/mannose-6-phosphate receptor in multiple human tissues during fetal life and early infancy.

The insulin like growth factor-II/mannose-6-phosphate (IGF-II/M6P) receptor has been detected in many cells and tissues. In the rat, there is a dramatic developmental regulation of IGF-II/M6P receptor expression, the receptor being high in fetal and neonatal tissues and declining thereafter. We have systematically studied the expression of the human IGF-II/M6P receptor protein in tissues from 10 human fetuses and infants (age 23 weeks gestation to 24 months postnatal). We have asked 1) whether there is differential expression among different organs, and 2) whether or not the human IGF-II/M6P receptor is developmentally regulated from 23 weeks gestation to 24 months postnatal. Protein was extracted from human tissues using a buffer containing 2% sodium dodecyl sulfate and 2% Triton X-100. Aliquots of the protein extracts were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting using an anti-IGF-II/M6P receptor antiserum (no. 66416) and 125I-protein A or an immunoperoxidase stain. IGF-II/M6P receptor immunoreactivity was detected in all tissues studied with the highest amount of receptor being expressed in heart, thymus, and kidney and the lowest receptor content being measured in brain and muscle. The receptor content in ovary, testis, lung, and spleen was intermediate. The apparent molecular weight of the IGF-II/M6P receptor (220,000 kilos without reduction of disulfide bonds) varied among the different tissues: in brain the receptor was of lower molecular weight than in other organs. Immunoquantitation experiments employing 125I-protein A and protein extracts from human kidney at different ages revealed a small, albeit not significant, difference of the receptor content between fetal and postnatal tissues: as in other species, larger amounts of receptor seemed to be present in fetal than in postnatal organs. In addition, no significant difference of the receptor content between human fetal liver and early postnatal liver was measured employing 125I-protein A-immunoquantitation in three fetal and five postnatal liver tissue samples. The distribution of IGF-binding protein (IGEBP) species, another abundant and major class of IGF binding principles, was also measured in human fetal and early postnatal lung, liver, kidney, muscle, and brain using Western ligand blotting with 125I-IGF-II: as with IGF-II/M6P receptor immunoreactivity there was differential expression of the different classes of IGFBPs in the various organs.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mannose-6-phosphate enhances cross-linking efficiency between insulin-like growth factor-II (IGF-II) and IGF-II/mannose-6-phosphate receptors in membranes.

Endogenous and exogenous phosphomannosyl ligands inhibit binding of insulin-like growth factor-II (IGF-II) to the IGF-II/mannose-6-phosphate receptor (IGF-II/Man-6-P receptor). In the present study, the mechanism of this antagonism was examined using a [125I]IGF-II cross-linking assay with disuccinimidyl suberate in cell membranes. Treatment with 5 mM Man-6-P enhanced [125I]IGF-II cross-linking to the receptor. The magnitude of the Man-6-P enhancement differed depending on the source of the membranes, ranging from a 30% increase in JEG-3 human choriocarcinoma up to a 560% increase in B16-F1 mouse melanoma. Man-6-P stimulated [125I]IGF-II-receptor cross-linking in H-35 hepatoma membranes by about 80%, even at concentrations of labeled IGF-II (greater than or equal to 10 nM) that nearly saturated the receptors. Thus, in addition to its effect on IGF-II-binding affinity, Man-6-P caused a 1.5- to 2-fold increase in cross-linking efficiency within the IGF-II-receptor complex. Furthermore, Man-6-P enhanced [125I]IGF-II cross-linking to the H-35 receptor by a constant (approximately 80%) increment 1) when the cross-linking reaction was conducted in buffers of different pH over the range 6.8-8.0, or 2) using cross-linking agents differing in spacer arm length from 6.4-16.1 A. Washing membranes before assay with either Man-6-P (pH 7.4) or 0.5 M NaCl (pH 4.5) reduced the subsequent Man-6-P enhancement of [125I]IGF-II-receptor cross-linking, suggesting that this phenomenon was actually due to displacement of inhibitory phosphomannosyl ligands bound endogenously to the Man-6-P sites of the receptor. In support of this hypothesis, Man-6-P produced a minimal (8-14%) enhancement of [125I]IGF-II-receptor cross-linking in membranes from I-cell fibroblasts lacking such phosphomannosyl ligands. Thus, phosphomannosyl ligands bound to the IGF-II/Man-6-P receptor decrease both IGF-II-binding affinity and IGF-II-receptor cross-linking efficiency. Membrane-associated receptors appear to exist in experimentally and perhaps functionally distinct populations, depending on occupancy of the Man-6-P-binding sites.     

The insulin-like growth factor II/mannose-6-phosphate receptor is present in monkey serum

We recently reported that the insulin-like growth factor II (IGF-II)/mannose-6-phosphate (Man-6-P) receptor is present in fetal and postnatal rat serum and that its serum content declined dramatically postnatally between days 20 and 40 . We now provide evidence that the IGF-II/Man-6-P receptor is also present in monkey serum. Serum was gel filtered on Sephadex G-200, and the column fractions were assayed for binding of radiolabeled IGF-II. There was significant binding of [125I]IGF-II to the void volume fractions in addition to binding to the 150K and 40K carrier proteins. Binding to the void volume fractions was greatest in cord serum and decreased with age. Competitive binding studies with [125I]IGF-II and the void volume pools from monkey serum demonstrated that IGF-I competed less potently than IGF-II, and insulin did not compete. Radiolabeled IGF-I did not bind specifically to the void volume pools. Chemical cross-linking of [125I]IGF-II to aliquots of the void volume pools from monkey cord serum samples and analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of dithiothreitol demonstrated a specific band at about 240K. Western blotting using a specific antiserum (no. 3637) against rat IGF-II/Man-6-P receptor was performed on aliquots of the Sephadex G-200 void volume pools of monkey serum. A band of approximately the same size as that found with human fibroblast members (approximately 215 K without dithiothreitol) was detected. The IGF-II/Man-6-P receptor band was more intense in cord serum than in the postnatal samples. When cord serum Sephadex G-200 pools were gel filtered on Sephadex G-50 in 1 mol/L acetic acid to separate binding components from free IGF, and IGF-II was measured by RRA, approximately 20% of the circulating IGF-II was found to be associated with this IGF-II/Man-6-P receptor in monkey serum. We conclude that the IGF-II/Man-6-P receptor present in serum may be a significant carrier for IGF-II in the monkey.     

Identification and in situ localization of the insulin-like growth factor-II/mannose-6-phosphate (IGF-II/M6P) receptor in the rat gastrointestinal tract: comparison with the IGF-I receptor.

In the present study we investigated pharmacological, biochemical, and immunological characteristics as well as the tissue distribution of the insulin-like growth factor-II/mannose-6-phosphate (IGF-II/M6P) receptor in the rat gastrointestinal tract, and compared the data with those from corresponding experiments for the IGF-I receptor. Competitive binding and affinity cross-linking studies with [125I]IGF-II, and [125I]IGF-I respectively, in rat jejunum yielded results analogous to those previously obtained for IGF-II/M6P and IGF-I receptors in intestinal epithelial membranes and other tissues. Furthermore, the IGF-II/M6P receptor antibody no. 3637 completely inhibited the association of [125I]IGF-II with receptor protein but nonimmune antibody did not, providing additional evidence for the presence of the IGF-II/M6P receptor in the rat gut. Also, analysis of the IGF-II/M6P receptor by immunoblotting using antiserum no. 3637 identified a specific band of mol wt 220.000 throughout the gastrointestinal tract with the highest content of immunoreactivity being present in colon and ileum. Autoradiographic mapping of the distribution of IGF-receptors in the rat gut showed that the expression of IGF-II/M6P receptors was in general 2-3 times greater than that of IGF-I receptors. IGF-II/M6P receptors were found 1) in greatest densities in colon and ileum, 2) more abundantly in the mucosa than in the muscularis propria, and 3) predominantly in the luminal part of the mucosal epithelial cells. Radioimmunocytochemistry employing anti-IGF-II/M6P receptor antibody no. 3637 and [125I]protein A demonstrated an IGF-II/M6P receptor distribution analogous to that shown by autoradiography with [125I]IGF-II). IGF-I receptors were present 1) in greatest densities in ileum and colon, 2) more abundantly in the muscularis propria than in the mucosa, and 3) within the mucosa in greater densities in the lamina propria than in the surface epithelium. For both receptor types densities were greater in crypt than in villous epithelial cells. We conclude: 1) the presence of IGF-II/M6P receptors throughout the rat gastrointestinal tract points to an important role for IGF-II in this organ, 2) the finding of different patterns of distribution for IGF-II/M6P and IGF-I receptors supports the concept of their different principal functions, 3) a high degree of expression of both receptor types in crypt epithelium suggests an essential role for both IGF receptors in the regulation of cell mitogenesis and growth.     

Insulin injection increases the levels of serum receptors for transferrin and insulin-like growth factor-II/mannose-6-phosphate in intact rats

Previous work indicates that a serum form of the insulin-like growth factor-II/mannose-6-phosphate (IGF-II/Man-6-P) receptor that circulates in mammals is a truncated form of the cellular receptor which lacks its cytoplasmic domain. In this study the effects of insulin administration on the levels of the serum forms of IGF-II/Man-6-P and transferrin receptors were measured. After sc injection of insulin into rats fasted overnight, the amount of IGF-II/Man-6-P receptor in serum increases to approximately twice that observed in untreated animals within 30 min, then decreases to a level lower than the initial level by 60 min before returning to control values by 90 min. The time course of the initial increase in serum receptor levels in response to insulin is similar to that observed for the decrease in blood glucose concentrations. Measurements of serum enzyme activities (creatine kinase and lactate dehydrogenase) during this time show no significant increase in response to insulin treatment. Furthermore, the increase in serum IGF-II/Man-6-P receptors is proportional to the amount of insulin injected over the range tested. In diabetic rats the serum IGF-II/Man-6-P receptor concentration is decreased to approximately 80% as much serum receptor as that in normal age-matched rats. The acute response of serum IGF-II/Man-6-P receptor levels to insulin administration is similar in both time course and extent of change to the increase in isolated fat cells of cell surface receptor levels due to insulin action. These results suggest the hypothesis that insulin stimulates the movement of cellular IGF-II/Man-6-P receptors to the cell surface, where they are proteolytically cleaved and released into serum.     

Radioimmunoassay of soluble insulin-like growth factor-II/mannose 6-phosphate receptor: developmental regulation of receptor release by rat tissues in culture.

The soluble form of the insulin-like growth factor-II/mannose 6-phosphate receptor, which has been detected in serum from a variety of species, is synthesized and released by rat tissue explants in culture. Investigations into its regulation and function, however, have been hampered by inadequate means of quantification. In this paper a RIA that enables sensitive and specific quantification of soluble receptor from serum and conditioned medium is described. In order to further clarify the source of soluble receptor in serum and to determine whether release of receptor from rat tissues is under developmental regulation, receptor release from rat explants was examined and compared to serum levels of receptor. Heart, skeletal muscle, kidney, and liver explants from fetal, neonatal, weanling, and adult rats were cultured in serum-free medium, and the conditioned medium was analyzed by RIA for the presence of receptor. Soluble receptor release was highest for fetal and neonatal tissues, with weanling and adult tissues showing dramatically decreased levels. Release of soluble receptor varied between tissues, with the tissue-specific pattern altering during development, such that while heart and muscle appeared as major tissues of release in fetal animals, the liver was the tissue of highest release in adult animals. It is concluded that release of soluble receptor by rat tissues is developmentally regulated, with levels of receptor in serum reflecting the pattern of release from tissues. The RIA will prove a useful tool for further examination of the regulation and function of the soluble insulin-like growth factor-II/mannose 6-phosphate receptor.     

Purification and immunological characterization of the rat liver insulin-like growth factor-II receptor

Rat liver microsomal insulin-like growth factor-II (IGF-II) receptor has been purified to homogeneity using a single step affinity chromatographic procedure on agarose-IGF-II with elution at pH 4. Determined by either IGF-II binding or a direct RIA for receptor, purification of 2000-fold was obtained. The mean recovery was 28% for five such preparations. Sodium dodecyl sulfate-electrophoresis and autoradiography of purified receptor, radioiodinated receptor, and affinity-labeled receptor all indicated a molecular mass of approximately 250K. Scatchard analysis of IGF-II binding to purified receptor, solubilized microsomal membranes, or plasma membranes showed a single class of binding site with an affinity constant of 6 X 10(10) liter/mol in all cases. Potent antibodies to the purified receptor were raised in rabbits, capable of inhibiting 50% of IGF-II binding at dilutions of 1:170,000 and also of fully precipitating IGF-II-prelabeled receptor at 1:50,000. Both types of antibodies reacted with IGF-II receptors in rat adipose tissue, brain, heart, kidney, lung, and spleen. However, little cross-reactivity was seen with other species. Comparison of the ability of receptor antibodies to inhibit IGF-II binding to microsomal and plasma membranes indicated a specific immunological difference between the IGF-II receptors in the two membrane preparations.     

The insulin-like growth factor II/mannose-6-phosphate receptor is present in fetal and maternal sheep serum

A large mol wt binding protein for insulin-like growth factor II (IGF-II) has been described in fetal sheep serum. We now provide evidence to demonstrate that this binding protein is the IGF-II/mannose-6-phosphate (Man-6-P) receptor. Serum and plasma were gel filtered on Sephadex G-200, and the column fractions were assayed for binding of radiolabeled IGF-II. There was significant binding of [125I]IGF-II to the void volume fractions in addition to binding to the 150K and 40K carrier proteins. Binding to the void volume fractions was increased in fetal serum as well as maternal serum and dramatically decreased in the nonpregnant adult. Competitive binding studies with [125I]IGF-II and the void volume pools from fetal and maternal sheep serum demonstrated that IGF-I competed less potently than IGF-II, and insulin did not compete. There was no specific binding of [125I]IGF-I to the void volume pools of either fetal or maternal samples. Chemical cross-linking of [125I]IGF-II to aliquots of the void volume pools from fetal and maternal sheep serum samples and analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of dithiothreitol demonstrated a specific band at about 240K. Western blotting using a specific antiserum (no. 3637) against rat IGF-II/Man-6-P receptor was performed on aliquots of the Sephadex G-200 void volume pools of fetal, maternal, uterine vein, and adult sheep serum; a band of approximately 210K (without dithiothreitol) was seen. The IGF-II/Man-6-P receptor band was more intense in fetal serum than in either maternal or adult nonpregnant sheep serum. There was also increased binding of [125I]IGF-II in the 40K region of the Sephadex G-200 column fractions in the maternal serum compared to that in serum from nonpregnant adult ewes. When fetal, maternal, and adult nonpregnant sheep serum Sephadex G-200 pools were gel filtered on Sephadex G-50 in 1 mol/liter acetic acid to separate bound from free IGF, and IGF-II was measured by RRA, approximately 50% of the circulating IGF-II was associated with this IGF-II/Man-6-P receptor in fetal serum compared to 7% in maternal serum and 3% in adult nonpregnant serum. These data demonstrate that the IGF-II/Man-6-P receptor circulates in fetal sheep serum as well as maternal serum and appears to be a significant carrier for IGF-II in fetal sheep.     

Retinoic acid alters the intracellular trafficking of the mannose-6-phosphate/insulin-like growth factor II receptor and lysosomal enzymes

Previously, we showed that retinoic acid (RA) binds to the mannose-6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R) with high affinity, suggesting that M6P/IGF2R may be a receptor for RA. Here, we show that RA, after 2–3 h of incubation with cultured neonatal-rat cardiac fibroblasts, dramatically alters the intracellular distribution of M6P/IGF2R as well as that of cathepsin B (a lysosomal protease bearing M6P). Immunofluorescence techniques indicate that this change in intracellular distribution is characterized by a shift of the proteins from the perinuclear area to cytoplasmic vesicles. The effect of RA was neither blocked by an RA nuclear receptor antagonist (AGN193109) nor mimicked by a selective RA nuclear-receptor agonist (TTNPB). Furthermore, the RA-induced translocation of cathepsin B was not observed in M6P/IGF2R-deficient P388D1 cells but occurred in stably transfected P388D1 cells expressing the receptor, suggesting that the effect of RA might be the result of direct interaction with M6P/IGF2R, rather than the result of binding to the nuclear receptors. These observations not only support the idea that M6P/IGF2R mediates an RA-response pathway but also indicate a role for RA in control of intracellular trafficking of lysosomal enzymes. Therefore, our observations may have important implications for the understanding of the diverse biological effects of retinoids.     

Insulin-like growth factor-II/mannose 6-phosphate receptor overexpression reduces growth of choriocarcinoma cells in vitro and in vivo

The IGF-II/mannose-6 phosphate receptor (IGF-II/M6PR) interacts with multiple tumor growth factors, including IGF-II and latent TGFbeta1. The IGF-II/M6PR has been proposed to be a tumor growth suppressor, a hypothesis supported by our previous finding that decreased IGF-II/M6PR expression enhances tumor growth. In this study, we further demonstrate that IGF-II/M6PR overexpression, resulting from cDNA transfection of JEG-3 choriocarcinoma cells, leads to a decreased cellular growth rate in vitro and decreased tumor growth in nude mice. Examination of several IGF-II/M6PR ligands in receptor-overexpressing cells showed no change in endogenous IGF-II or secretion of procathepsins D and L but an increase in latent TGFbeta1 secretion and activation. Cells transfected with cDNA for a truncated, soluble form of the receptor, previously shown to inhibit IGF-II-stimulated DNA synthesis, displayed a very slow growth rate in vitro and in nude mice but showed no alteration in TGFbeta1 levels. This suggests that, in IGFII/M6PR-transfected cells, increased levels of soluble IGF-II/M6PR may play a role in growth inhibition. Overall, the findings in this study are consistent with the hypothesis that the IGF-II/M6PR suppresses tumor growth.     

Endocytosis of receptor-bound insulin-like growth factor II is enhanced by mannose-6-phosphate in IM9 cells

The insulin-like growth factor II (IGF-II), and glycoproteins containing mannose 6-phosphate (M6P), bind to two different sites of the same receptor molecule (Morgan et al, Nature 329:301, 1987). To study the interactions between the two ligands on their common receptor in intact cells, we examined the effect of free M6P on IGF-II binding and endocytosis in the IM9 human lymphoblastoid cell line. M6P, up to a 3 mM concentration, had no effect on the binding of IGF-II to the cell surface receptor of intact IM9 cells at 4 degrees C. By contrast, when IM9 cells were incubated with 125I-IGF-II at 37 degrees C, 1mM M6P increased cell-associated radioactivity by twofold. The increase was resistant to acid wash at 4 degrees C, and therefore assumed to represent endocytosed IGF-II. Acid-washable radioactivity was no different, confirming that, in intact cells, M6P does not affect IGF-II surface binding. In addition, preincubation of cells with M6P at 37 degrees C for up to 3 hours did not change the abundance of receptor on the cell surface, as measured by a subsequent 4 degrees C binding assay. We conclude that M6P causes a shift of IGF-II-occupied receptors form the cell surface to intracellular locations without affecting surface binding of this ligand in IM9 cells. The effect could be produced by the binding of M6P itself, or by the displacement of endogenous phosphomannosylated ligands.     

Cloning and expression of the cDNA of chicken cation-independent mannose-6-phosphate receptor.

We cloned and sequenced the 8767-bp full-length cDNA for the chicken cation-independent mannose-6-phosphate receptor (CI-MPR), of interest because, unlike its mammalian homologs, it does not bind insulin-like growth factor II (IGF-II). The cDNA encodes a protein of 2470 aa that includes a putative signal sequence, an extracytoplasmic domain consisting of 15 homologous repeat sequences, a 23-residue transmembrane sequence, and a 161-residue cytoplasmic sequence. Overall, it shows 60% sequence identity with human and bovine CI-MPR homologs, and all but two of 122 cysteine residues are conserved. However, it shows much less homology in the N-terminal signal sequence, in repeat 11, which is proposed to contain the IGF-II-binding site in mammalian CI-MPR homologs, and in the 14-aa residue segment in the cytoplasmic sequence that has been proposed to mediate G-protein-coupled signal transduction in response to IGF-II binding by the human CI-MPR. Transient expression in COS-7 cells produced a functional CI-MPR which exhibited mannose-6-phosphate-inhibitable binding and mediated endocytosis of recombinant human beta-glucuronidase. Expression of the functional chicken CI-MPR in mice lacking the mammalian CI-MPR should clarify the controversy over the physiological role of the IGF-II-binding site in mammalian CI-MPR homologs.     

Endocytosis of receptor-bound insulin-like growth factor II is enhanced by mannose-6-phosphate in IM9 cells

The insulin-like growth factor II (IGF-II), and glycoproteins containing mannose 6-phosphate (M6P), bind to two different sites of the same receptor molecule (Morgan et al, Nature 329:301, 1987). To study the interactions between the two ligands on their common receptor in intact cells, we examined the effect of free M6P on IGF-II binding and endocytosis in the IM9 human lymphoblastoid cell line. M6P, up to a 3 mM concentration, had no effect on the binding of IGF-II to the cell surface receptor of intact IM9 cells at 4 degrees C. By contrast, when IM9 cells were incubated with 125I-IGF-II at 37 degrees C, 1 mM M6P increased cell-associated radioactivity by twofold. The increase was resistant to acid wash at 4 degrees C, and therefore assumed to represent endocytosed IGF-II. Acid-washable radioactivity was no different, confirming that, in intact cells, M6P does not affect IGF-II surface binding. In addition, preincubation of cells with M6P at 37 degrees C for up to 3 hours did not change the abundance of receptor on the cell surface, as measured by a subsequent 4 degrees C binding assay. We conclude that M6P causes a shift of IGF-II-occupied receptors form the cell surface to intracellular locations without affecting surface binding of this ligand in IM9 cells. The effect could be produced by the binding of M6P itself, or by the displacement of endogenous phosphomannosylated ligands.