Transcobalamin II receptor interacts with megalin in the renal apical brush border membrane.

Purified human transcobalamin II receptor (TC II-R) binds to megalin, a 600 kDa endocytic receptor with an association constant, K(a), of 66 n M and bound(max) of 1.1 mole of TC II-R/mole of megalin both in the presence and absence of its ligand, transcobalamin II (TC II). Immunoprecipitation followed by immunoblotting of Triton X-100 extracts of the apical brush border membrane (BBM) from rabbit renal cortex revealed association of these two proteins. (35)[S]-TC II complexed with cobalamin (Cbl; Vitamin B(12)) bound to Sepharose-megalin affinity matrix and the binding was enhanced 5-fold when TC II-R was prebound to megalin. Megalin antiserum inhibited both the TC II-R-dependent and -independent binding of (35)[S]-TC II-Cbl to megalin, while TC II-R antiserum inhibited only the TC II-R-dependent binding. In rabbits with circulating antiserum to megalin, renal apical BBM megalin was present as an immune complex, but its levels were not altered. However, the protein levels of both TC II-R and the cation-independent mannose 6-phosphate receptor (CIMPR) were drastically reduced and the urinary excretion of TC II, albumin, and other low-molecular weight proteins was significantly increased. These results suggest that megalin contains a distinct single high-affinity binding site for TC II-R and their association in the native renal BBM is important for tubular reabsorption of many proteins, including TC II.

Recognition of Dictyostelium discoideum lysosomal enzymes is conferred by the amino-terminal carbohydrate binding site of the insulin-like growth factor II/mannose 6-phosphate receptor.

The insulin-like growth factor-II/mannose 6-phosphate receptor (IGF-II/MPR) is a type I glycoprotein that mediates both the intracellular sorting of lysosomal enzymes bearing mannose 6-phosphate (Man-6-P) residues to the lysosome and the bioavailability of IGF-II. The extracytoplasmic region of the IGF-II/MPR contains 15 repeating domains; the two carbohydrate recognition domains (CRDs) have been localized to domains 1-3 and 7-9, and the high-affinity IGF-II binding site maps to domain 11. To characterize the carbohydrate binding properties of the IGF-II/MPR, regions of the receptor encompassing the individual CRDs were produced in a baculovirus expression system. Characterization of the recombinant proteins revealed that the pH optimum for carbohydrate binding is significantly more acidic for the carboxyl-terminal CRD than for the amino-terminal CRD (i.e., pH 6.4-6.5 vs 6.9). Equilibrium binding studies demonstrated that the two CRDs exhibit a similar affinity for Man-6-P. Furthermore, substitution of the conserved arginine residue in domain 3 (R435) or in domain 9 (R1334) with alanine resulted in a similar >1000-fold decrease in the affinity for the lysosomal enzyme, beta-glucuronidase. In contrast, the two CRDs differ dramatically in their ability to recognize the distinctive modifications (i.e., mannose 6-sulfate and Man-6-P methyl ester) found on Dictyostelium discoideum lysosomal enzymes: the amino-terminal CRD binds mannose 6-sulfate and Man-6-P methyl ester with a 14-55-fold higher affinity than the carboxyl-terminal CRD. Taken together, these results demonstrate that the IGF-II/MPR contains two functionally distinct CRDs.

Mutational analysis of the binding site residues of the bovine cation-dependent mannose 6-phosphate receptor.

Mannose 6-phosphate receptors (MPRs) deliver soluble acid hydrolases to the lysosome in higher eukaryotic cells. The two MPRs, the cation-dependent MPR (CD-MPR) and the insulin-like growth factor II/cation-independent MPR, carry out this process by binding with high affinity to mannose 6-phosphate residues found on the N-linked oligosaccharides of their ligands. To elucidate the key amino acids involved in conveying this carbohydrate specificity, site-directed mutagenesis studies were conducted on the extracytoplasmic domain of the bovine CD-MPR. Single amino acid substitutions of the residues that form the binding pocket were generated, and the mutant constructs were expressed in transiently transfected COS-1 cells. Following metabolic labeling, mutant CD-MPRs were tested for their ability to bind pentamannosyl phosphate-containing affinity columns. Of the eight amino acids mutated, four (Gln-66, Arg-111, Glu-133, and Tyr-143) were found to be essential for ligand binding. In addition, mutation of the single histidine residue, His-105, within the binding site diminished the binding of the receptor to ligand, but did not eliminate the ability of the CD-MPR to release ligand under acidic conditions.

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.

Biosynthesis and secretion of the mannose 6-phosphate receptor and its ligands in polarized Caco-2 cells.

We have analyzed the transport of newly synthesized mannose 6-phosphate (Man-6-P)-bearing proteins (i.e., lysosomal enzymes) in the polarized human colon adenocarcinoma cell line, Caco-2, by subjecting filter-grown cells to a pulse-chase labeling protocol using [(35)S]methionine, and the resulting cell lysate, apical medium, and basolateral medium were immunoprecipitated with insulin-like growth factor II/Man-6-P receptor (IGF-II/MPR)-specific antisera. The results showed that the majority of secreted lysosomal enzymes accumulated in the apical medium at >2 h of chase and that this polarized distribution was facilitated by the IGF-II/MPR selectively endocytosing lysosomal enzymes from the basolateral surface. Treatment with various agents known to affect vesicular transport events demonstrated that incubations at 16 degrees C or incubations with brefeldin A inhibited the secretion of lysosomal enzymes from both the apical and basolateral surface, whereas treatment with nocodazole selectively blocked apical secretion. In contrast, incubation with NH4Cl or nocodazole had a stimulatory effect on basolateral secretion. Taken together, these results demonstrate that the sorting of Man-6-P-containing proteins into the apical and basolateral secretory pathways is regulated by distinct components of the intracellular trafficking machinery.

The rate of internalization of the mannose 6-phosphate/insulin-like growth factor II receptor is enhanced by multivalent ligand binding.

The cation-independent mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF-II receptor) undergoes constitutive endocytosis, mediating the internalization of two unrelated classes of ligands, mannose 6-phosphate (Man-6-P)-containing acid hydrolases and insulin-like growth factor II (IGF-II). To determine the role of ligand valency in M6P/IGF-II receptor-mediated endocytosis, we measured the internalization rates of two ligands, beta-glucuronidase (a homotetramer bearing multiple Man-6-P moieties) and IGF-II. We found that beta-glucuronidase entered the cell approximately 3-4-fold faster than IGF-II. Unlabeled beta-glucuronidase stimulated the rate of internalization of 125I-IGF-II to equal that of 125I-beta-glucuronidase, but a bivalent synthetic tripeptide capable of occupying both Man-6-P-binding sites on the M6P/IGF-II receptor simultaneously did not. A mutant receptor with one of the two Man-6-P-binding sites inactivated retained the ability to internalize beta-glucuronidase faster than IGF-II. Thus, the increased rate of internalization required a multivalent ligand and a single Man-6-P-binding site on the receptor. M6P/IGF-II receptor solubilized and purified in Triton X-100 was present as a monomer, but association with beta-glucuronidase generated a complex composed of two receptors and one beta-glucuronidase. Neither IGF-II nor the synthetic peptide induced receptor dimerization. These results indicate that intermolecular cross-linking of the M6P/IGF-II receptor occurs upon binding of a multivalent ligand, resulting in an increased rate of internalization.

Characterization of truncated and glycosylation-deficient forms of the cation-dependent mannose 6-phosphate receptor expressed in baculovirus-infected insect cells.

A soluble truncated form of the cation-dependent mannose 6-phosphate receptor (CD-MPR) encoding only the extracytoplasmic region, Stop155, and a truncated glycosylation-deficient form of the CD-MPR, Asn81/Stop155, which has been modified to contain only one N-linked glycosylation site at position 81 instead of five, were purified from baculovirus-infected High Five insect cells. The glycosylated recombinant proteins were functional in ligand binding and acid-dependent dissociation as assessed by pentamannosyl phosphate-agarose affinity chromatography. Gel filtration, sucrose gradients, and cross-linking experiments revealed that both Stop155 and Asn81/Stop155 are dimeric, demonstrating that the transmembrane and cytoplasmic region of the receptor as well as N-linked oligosaccharides at positions 31, 57, and 87 are not required for dimerization. The Kd of Stop155 and Asn81/Stop155 for the lysosomal enzyme, beta-glucuronidase, was 0.2 and 0.3 nM, respectively. These values are very similar to those reported for the full-length CD-MPR, demonstrating that the extracellular region of the CD-MPR is sufficient for high-affinity binding and that oligosaccharides at positions 31, 57, and 87 do not influence ligand binding.

The two mannose 6-phosphate binding sites of the insulin-like growth factor-II/mannose 6-phosphate receptor display different ligand binding properties.

The two mannose 6-phosphate (Man-6-P) binding sites of the insulin-like growth factor-II/mannose 6-phosphate receptor (IGF-II/MPR) have been localized to domains 1-3 and 7-9, and studies have shown that Arg435 in domain 3 and Arg 1334 in domain 9 are essential for Man-6-P binding. To determine whether the IGF-II/MPR containing a single Man-6-P binding site is functional, clonal mouse L cell lines stably transfected with either mutant bovine IGF-II/MPR cDNA, containing substitutions at position 435 and/or 1334, or the wild type receptor cDNA were assayed for their ability to sort lysosomal enzymes to the lysosome. Mutant receptors containing a single Man-6-P binding site were approximately 50% less efficient than the wild type receptor in the overall targeting of lysosomal enzymes to the lysosome. Mutant receptors containing a substitution at Arg1334 (Dom9(Ala)), in contrast to those containing a substitution at Arg435 (Dom3(Ala)), were unable to target cathepsin D and beta-hexosaminidase to the lysosome. Equilibrium binding assays using 125I-labeled beta-glucuronidase demonstrated that Dom3(Ala) and Dom9(Ala) had a Kd of 2.0 and 4.3 nM, respectively. In addition, Dom3(Ala), unlike Dom9(Ala), was unable to completely dissociate from ligand under acidic pH conditions. These data indicate that the two Man-6-P binding sites of the IGF-II/MPR are not functionally equivalent.

Molecular basis of lysosomal enzyme recognition: three-dimensional structure of the cation-dependent mannose 6-phosphate receptor.

Targeting of newly synthesized lysosomal hydrolases to the lysosome is mediated by the cation-dependent mannose 6-phosphate receptor (CD-MPR) and the insulin-like growth factor II/cation-independent mannose 6-phosphate receptor (IGF-II/CI-MPR). The two receptors, which share sequence similarities, constitute the P-type family of animal lectins. We now report the three-dimensional structure of a glycosylation-deficient, yet fully functional form of the extracytoplasmic domain of the bovine CD-MPR (residues 3-154) complexed with mannose 6-phosphate at 1.8 A resolution. The extracytoplasmic domain of the CD-MPR crystallizes as a dimer, and each monomer folds into a nine-stranded flattened beta barrel, which bears a striking resemblance to avidin. The distance of 40 A between the two ligand-binding sites of the dimer provides a structural basis for the observed differences in binding affinity exhibited by the CD-MPR toward various lysosomal enzymes.

Bipolar functional expression of transcobalamin II receptor in human intestinal epithelial Caco-2 cells.

Transcobalamin II (TC II) receptor is expressed in the apical and basolateral membranes of human intestinal mucosa and in post-confluent human intestinal epithelial Caco-2 cells with a 6-7-fold enrichment in basolateral membranes. Caco-2 cells grown on culture inserts bound (at 5 degrees C) 30 and 180 fmol of the ligand, TC II-[57Co]cobalamin (Cbl), to the apical and the basolateral surfaces, respectively. Within 5 h at 37 degrees C, all apically bound Cbl was internalized and subsequently transcytosed as TC II-Cbl. In contrast, all basolateral surface-bound Cbl was internalized and retained by the cells, but transferred from TC II to other cellular proteins. Chloroquine or leupeptin had no effect on the apical to basolateral transcytosis of either [57Co]Cbl or 125I-TC II. In contrast, following basolateral internalization of the ligand, both chloroquine and leupeptin inhibited the intracellular degradation of 125I-TC II, which resulted in secretion of 60-65% of TC II-Cbl complex into the basolateral medium. When 125I-TC II-Cbl was orally administered to rats, intact labeled TC II was detected in the portal blood 4 and 8 h later. These studies suggest that TC II-Cbl is processed when presented to the (a) apical/luminal side by a hitherto unrecognized non-lysosomal pathway in which both TC II and Cbl are transcytosed and (b) basolateral side by the lysosomal pathway in which TC II is degraded and the released Cbl is utilized.

Insulin-like growth factor-II/mannose-6-phosphate receptor expression during early heart development.

Expression of the insulin-like growth factor-II/mannose-6-phosphate (ICF-II/ M6P) receptor was examined during the major stages of heart morphogenesis in the chicken embryo. By using an affinity-purified antibody, Western blot analysis of total embryonic proteins from stages 5-24 revealed little if any IGF-II/M6P receptor protein until stage 7, approximately 8 hours prior to the appearance of the rudimentary myocardial tubes. Thereafter, receptor accumulation increased until stage 14, after which receptor protein levels remained constant, up to 7 days in ovo. Immunohistochemical localization revealed that, among all embryonic tissues at stages 10-24, the predominant site of receptor expression was the developing myocardium. Receptor expression was also immunohistochemically evaluated in a defined in vitro model of cardiogenesis in which explanted precardiac mesoderm is induced to undergo differentiation by co-explanted endoderm. In this system, as in vivo, IGF-II/M6P receptors were only detected after precardiac mesoderm had differentiated into a synchronously contractile multilayer which expressed cardiac alpha-actin. These findings indicate that the IGF-II/M6P receptor has an important role during early heart development.

Expression of insulin-like growth factor (IGF)-I receptors, IGF-II/cation-independent mannose 6-phosphate receptors (CI-MPRs), and cation-dependent MPRs in polarized human intestinal Caco-2 cells.

We have analyzed the surface distribution and functional expression of the insulin-like growth factor I (IGF-I) receptor and the IGF-II/cation-independent mannose 6-phosphate (IGF-II/CI-MPR) in the polarized human colon adenocarcinoma cell line, Caco 2. Domain-selective biotinylation of the apical and basolateral surfaces of Caco-2 cells grown on filter supports revealed a 3-4-fold enrichment of these receptors on basolateral membranes. In addition, the biotinylation studies revealed the presence of the cation-dependent MPR on both membrane surfaces, with a 3.4-fold enrichment on basolateral membranes. Binding of 125I-IGF-I at 4 degrees C confirmed similar higher levels of expression of the IGF-I receptor at the basolateral surface than at the apical surface. Cell surface-specific binding of the iodinated lysosomal enzyme beta-glucuronidase was detected at 4 degrees C on both plasma membrane domains. However, significant uptake of beta-glucuronidase at 37 degrees C was observed only from the basolateral surface. These results indicate that the MPRs and the IGF-I receptor are expressed in a polarized fashion in Caco-2 cells and that the IGF-II/CI-MPR present on apical membranes, unlike the IGF-II/CI-MPR expressed on the basolateral surface, is not functional in endocytosing lysosomal enzymes.

Employer-specific versus community-wide report cards: is there a difference?

This article describes preliminary results from a natural experiment that tested the impact of report cards on employees. As part of the 1995 enrollment process, some members of the State of Minnesota Employee Group Insurance Program received report cards on the plans offered to them, and others did not. Both groups of employees had a chance to review a second community-wide report card covering all Minnesota plans that had been distributed by an independent organization through local newspapers. Both groups were surveyed before and after they made their health plan selections. We compare the likelihood of seeing, the intensity of reading, and the perceived helpfulness of the first, employer-specific report card with the second, community-wide report card for consumers who make plan selections.

Expression of IGF-II and IGF binding proteins in differentiating human intestinal Caco-2 cells.

The mitogenic and metabolic effects of insulin-like growth factor-II (IGF-II) can be modulated by six distinct IGF binding proteins (IGFBPs). As a first step toward understanding the role of IGFs and their binding proteins in intestinal epithelial cell differentiation, the expression of IGF-II and IGFBPs was characterized in the human colon adenocarcinoma Caco-2 cell line. Northern blot analysis revealed two IGF-II transcripts of 5.4 and 4.5 kb, and ribonuclease protection assays indicated that IGF-II mRNA levels are regulated during Caco-2 differentiation. A specific radioimmunoassay detected IGF-II in serum-free conditioned medium, the level of which was three- to fivefold higher in proliferating cells than in differentiated cells. Immunoprecipitation and ligand blot analyses of conditioned medium demonstrated that IGFBP-2, IGFBP-3, IGFBP-4, and IGFBP-6 are synthesized by Caco-2 cells, with IGFBP-2 and IGFBP-4 being the major IGFBPs secreted, and that the levels of IGFBP-2 and IGFBP-6 decreased as differentiation proceeded. These results indicate that the expression of IGF-II, IGFBP-2, and IGFBP-6 is regulated in a differentiation-dependent manner in Caco-2 cells.

Regulation of lysosomal and ubiquitin degradative pathways in differentiating human intestinal Caco-2 cells.

The expression of various components of the lysosomal and ubiquitin-dependent degradative pathways was characterized in an in vitro model of differentiating enterocytes, the human colon adenocarcinoma Caco-2 cell line. The activities of the cell-associated lysosomal enzymes alpha-D-mannosidase, beta-hexosaminidase, beta-glucuronidase, and beta-galactosidase increased approximately 2- to 4-fold as differentiation proceeded. In contrast, the protein levels of the two mannose 6-phosphate receptors (MPRs), the insulin-like growth factor II/cation-independent MPR (IGF-II/CI-MPR) and the cation-dependent MPR (CD-MPR), did not change significantly during Caco-2 differentiation. In addition, quantitative Western blot analyses revealed that on a molar basis the CD-MPR is 3.5 times more abundant than the IGF-II/CI-MPR in Caco-2 cells. Since only limited secretion of lysosomal enzymes was observed throughout differentiation, the level of expression of the MPRs was sufficient to target the increased levels of lysosomal enzymes to the lysosome. Unlike the expression of lysosomal enzymes, Western blot analysis demonstrated an approximately 40% and approximately 30% decrease, respectively, in the steady-state levels of free and conjugated ubiquitin during Caco-2 differentiation. Taken together, these results show that the ubiquitin-dependent proteolytic pathway is regulated differently than the lysosomal degradative pathway during Caco-2 differentiation.

Expression and characterization of functional bovine cation-dependent mannose 6-phosphate receptors in baculovirus-infected insect cells.

A recombinant baculovirus containing the cDNA for the full-length bovine cation-dependent mannose 6-phosphate receptor (CD-MPR) was generated by homologous recombination. Spodoptera frugiperda (Sf9) and Trichoplusia ni 5B1-4 (High Five) insect cells, which do not contain endogenous MPRs as determined by Western blot analyses and pentamannosyl phosphate-agarose affinity chromatography, were infected with recombinant baculovirus. The infected cells expressed 26-, 29-, 32-, 35-, and 39-kDa species that were immunologically reactive with antisera raised against the native bovine CD-MPR and quantitative Western analysis demonstrated 1-2 x 10(6) CD-MPR molecules/cell, a level which is comparable to that expressed normally in a variety of cell lines and tissues. Digestion with endo-beta-N-acetylglucosaminidase H indicated that these multiple species were due to the presence of either 0, 1, 2, 3, or 4 N-linked oligosaccharide chains, respectively, and that the majority (87%) of these carbohydrates were of the high-mannose type. The receptor produced was biologically active since it bound specifically to a pentamannosyl phosphate-agarose column and exhibited acid-dependent ligand dissociation. In addition, studies using a homobifunctional cross-linking agent on the purified CD-MPR suggested that, like the receptor expressed in mammalian cells, the insect-produced CD-MPR existed as a dimer in the membrane.

Effect of processing inhibitors on cobalamin (vitamin B12) transcytosis in polarized opossum kidney cells.

The main objectives of the current study were to investigate the effect of tunicamycin and other posttranslational processing inhibitors on the apical brush border expression of intrinsic factor-cobalamin receptor (IFCR) and the apical to basolateral transcytosis of cobalamin (Cbl). Because of the high and selective expression of IFCR in the apical brush border membrane of opossum kidney (OK) cells (K. S. Ramanujam, S. Seetharam, N. Dahms, and B. Seetharam, (1991) J. Biol. Chem. 266, 13135-13140), we have used cultured OK cells to address these issues. When polarized OK cells grown on culture inserts were incubated with tunicamycin, deoxynojirimycin, swainsonine, or cerulenin, the surface binding of the ligand, intrinsic factor-[56Co]Cbl was inhibited by tunicamycin but not by the other inhibitors. However, Cbl transcytosis was inhibited by both tunicamycin and cerulenin but not with deoxynojirimycin or swainsonine. Incubation of cells with tunicamycin decreased the half-life of IFCR from 48 to 24 h, thus causing faster degradation and depletion of the surface receptor. Incubation of cells with cerulenin resulted in the intralysosomal retention of internalized Cbl. Mature receptor labeled with either [35S]methionine or [3H]mannose was sensitive to digestion with both endoglycosidase H and peptide N-glycosidase F and revealed the presence of two or three N-linked oligosaccharides of the high mannose or hybrid type. Metabolic labeling of OK cells with [3H]palmitic acid revealed that IFCR was palmitoylated and the label was sensitive to treatment with hydroxylamine. Based on these results we suggest that IFCR expression in the apical membrane and Cbl transcytosis in polarized OK cells are regulated by core N-glycosylation but not by further processing of the terminal sugars. In addition, we also suggest that the inhibition of Cbl transcytosis by cerulenin is due to inhibition of postinternalization events.

The bovine mannose 6-phosphate/insulin-like growth factor II receptor. Localization of the insulin-like growth factor II binding site to domains 5-11.

The mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF-II receptor) binds two distinct ligands, mannose 6-phosphate (Man-6-P) and insulin-like growth factor II (IGF-II). The extracytoplasmic region of the receptor is composed of 15 homologous repeating domains and domains 1-3 and 7-9 have been shown to contain the two Man-6-P binding sites. To determine the location of the single IGF-II binding site, truncated forms of the M6P/IGF-II receptor were expressed transiently in COS-1 cells and assayed for their ability to bind iodinated human recombinant IGF-II. The binding of [125I]IGF-II to the receptors in the presence or absence of excess unlabeled IGF-II, IGF-I, or insulin was determined by incubation with homobifunctional cross-linking agents followed by SDS-polyacrylamide gel electrophoresis. These binding studies demonstrated that a construct encoding domains 5-11 bound 0.9 mol of IGF-II/mol of receptor, whereas a construct encoding domains 5-10 exhibited no detectable binding to IGF-II. These results indicate that the IGF-II binding site of the M6P/IGF-II receptor is contained within domains 5-11 and that residues in domain 11 play an important role in IGF-II binding.