Incorporation of mannose 6-phosphate receptors into liposomes. Receptor topography and binding of alpha-mannosidase.

A receptor that binds the lysosomal enzyme alpha-mannosidase via mannose 6-phosphate moieties (mannose 6-phosphate receptor) was purified from Swarm-rat chondrosarcoma and bovine liver microsomal membranes. Receptor-reconstituted liposomes were prepared by dialysis of taurodeoxycholate-dispersed lipids with purified mannose 6-phosphate receptor. Liposomes appeared by electron microscopy as 60-120 nm unilamellar vesicles. Receptor-reconstituted liposomes retained the ability to bind alpha-mannosidase specifically. Binding was saturable with an apparent Kd of 1 nM and was competitively inhibited by mannose 6-phosphate (Ki 2mM). Liposomes containing entrapped 125I-bovine serum albumin were used to demonstrate that treatment with 0.045% taurodeoxycholate rendered liposomes permeable to macromolecules without solubilizing the membrane. Receptor orientation in the liposome membrane was established by measuring binding of ligand to intact and detergent-treated liposomes. Unlike coated vesicles, which contain cryptic mannose 6-phosphate receptors [Campbell, Fine, Squicciarini & Rome (1983) J. Biol. Chem. 258, 2526-2533], treatment of liposomes with detergent revealed no additional cryptic binding sites. In addition, treatment of liposomes with 0.75% trypsin abolished total receptor binding activity. The results suggest that the receptor is inserted with its binding site facing the outside of the liposome.     

46-kDa mannose 6-phosphate-specific receptor: purification, subunit composition, chemical modification

A cation-dependent mannose 6-phosphate-specific receptor has recently been isolated from murine P388D1 macrophages and bovine liver (B. Hoflack & S. Kornfeld, (1985) J. Biol. Chem. 260, 12008-12014). The receptor purified from human liver has a subunit molecular size of 43 kDa, is rich in hydrophobic and charged amino acids and contains threonine at the N-terminus. The receptors from human and rat liver are antigenically related. Both are immunologically distinct from the cation-independent 215-kDa mannose 6-phosphate-specific receptor from human liver. Cross-linking experiments indicate that the cation-dependent receptor exists in solution as a tetramer. Modification of arginine and histidine residues, reduced drastically the binding of the receptor to immobilized ligands. Presence of mannose 6-phosphate during modification of arginine residues protected the binding properties of the receptor, suggesting that arginine is a constituent of the mannose 6-phosphate binding site of the receptor. The significance of the inability of histidine-modified receptors to bind ligands remains to be established.     

Biochemical evidence that the type II insulin-like growth factor receptor is identical to the cation-independent mannose 6-phosphate receptor

Cloning and sequencing of the human type II insulin-like growth factor (IGF) receptor cDNA revealed an 80% deduced amino acid sequence homology with the bovine cation-independent mannose 6-phosphate (Man-6-P) receptor, suggesting identity of the two receptors (Morgan, D. O., Edman, J. C., Standring, D. N., Fried, V. A., Smith, M. C., Roth, R. A., and Rutter, W. J. (1987) Nature 329, 301-307). We have performed biochemical experiments that support this proposal. Rat liver type II IGF receptor, purified by the conventional method of IGF-II affinity chromatography, bound quantitatively to a beta-galactosidase affinity column and was eluted with Man-6-P. Bovine liver Man-6-P receptor, prepared by the conventional method of affinity chromatography on phosphomannan-Sepharose, bound IGF-II with high affinity (Kd = 1 nM). Affinity cross-linking of 125I-IGF-II to the Man-6-P receptor and analysis by sodium dodecyl sulfate-gel electrophoresis showed that beta-galactosidase, but not Man-6-P, inhibited the formation of the 250-kDa 125I-IGF-II-receptor complex. The inhibition by beta-galactosidase was prevented by coincubation with Man-6-P. 125I-IGF-II did not bind to the 46-kDa cation-dependent Man-6-P receptor. For immunologic studies we purified type II IGF receptors and Man-6-P receptors in parallel from rat placental membranes using either IGF-II- or beta-galactosidase affinity chromatography. A panel of five antisera that previously had been raised against either type II IGF receptor or Man-6-P receptor behaved identically toward type II IGF receptor versus Man-6-P receptor in ligand blocking and immunoprecipitation assays. Our data support the conclusion that the type II IGF receptor and the cation-independent Man-6-P receptor are the same protein and that the IGF-II and Man-6-P-binding sites are distinct.     

A macrophage receptor for liver lysosomal beta-glucosidase

beta-Glucosidase was purified from lysosomal membranes isolated from rat liver. Binding and uptake of the purified beta-glucosidase were mediated via an apparently single binding site on rat peritoneal macrophages. The number of sites and the Kd were 4.20 X 10(4)/cell and 1.00 X 10(-7) M, respectively. Neither of the processes was inhibited by ligands for mannose/fucose receptors, mannose 6-phosphate receptors, or scavenger receptors, or by other glycoproteins and sugar compounds. A portion of the beta-glucosidase taken up into the macrophages was degraded rapidly. These results suggested that liver lysosomal beta-glucosidase was endocytosed via a receptor not previously described.     

Proliferin secreted by cultured cells binds to mannose 6-phosphate receptors

Proliferin is a prolactin-related glycoprotein secreted by proliferating mouse cell lines and by mouse placenta. In an attempt to identify target sites for proliferin action, we looked for proliferin receptors in murine fetal and maternal tissues during pregnancy using proliferin purified from the conditioned medium of a constructed Chinese hamster ovary cell line carrying amplified copies of proliferin cDNA. Purified proliferin bound to membrane preparations from fetal or maternal liver and from placenta with a Kd of 1 to 2 nM. The amount of proliferin bound per microgram of membrane protein varied markedly during pregnancy; maximal binding to day 16 fetal liver membranes was approximately 25 times that to liver membranes from adult animals. Binding to fetal and maternal receptors was specifically and completely inhibited by mannose 6-phosphate, with half-maximal inhibition at 10 microM. Furthermore, non-glycosylated proliferin did not inhibit the binding of the glycosylated protein. A approximately 300 Kd proliferin receptor was purified from the liver of pregnant mice using a proliferin affinity column and elution with mannose 6-phosphate. This receptor reacted with antibodies directed against the rat cation-independent mannose 6-phosphate receptor. We conclude that 1) proliferin secreted by cultured cell binds to cation-independent mannose 6-phosphate receptors and therefore may be a lysosomal protein or targeted to lysosomes, and 2) the concentration or activity of mannose 6-phosphate receptors in murine fetal and maternal liver and in placenta is regulated during pregnancy.     

Localization of transferrin receptors and insulin-like growth factor II receptors in vesicles from 3T3-L1 adipocytes that contain intracellular glucose transporters

Transferrin receptors in detergent extracts of subcellular membrane fractions prepared from 3T3-L1 adipocytes were measured by a binding assay. There was a small but significant increase (1.2-fold) in the amount of receptor in a crude plasma membrane fraction and a 40% decrease in the number of transferrin receptors in microsomal membranes prepared from insulin-treated cells, when compared with corresponding fractions from control cells. Intracellular vesicles containing insulin-responsive glucose transporters (GT) have been isolated by immunoadsorption from the microsomal fraction (Biber, J. W., and G. E. Lienhard. 1986. J. Biol. Chem. 261:16180-16184). All of the transferrin receptors in this fraction were localized in these vesicles; however, because the GT vesicles contain approximately 30-fold fewer transferrin receptors than GT, on the average only one vesicle in three contains a transferrin receptor. The binding of 125I-pentamannose 6-phosphate BSA to 3T3-L1 adipocytes at 4 degrees C was used to monitor surface insulin-like growth factor II (IGF-II)/mannose 6-phosphate receptors. Exposure of cells to insulin at 37 degrees C for 5 min resulted in a 2.5-4.5-fold increase in surface receptors. There was a corresponding 20% decrease in the amount of IGF-II receptors in the microsomal membranes prepared from insulin-treated cells, as assayed by immunoblotting. Moreover, the IGF-II receptors and GT were located in the same intracellular vesicles, since antibodies to the carboxyterminal peptide of either protein immunoadsorbed vesicles containing 70-95% of both proteins initially present in the microsomal fraction. In conjunction with other studies, these results indicate that in 3T3-L1 adipocytes, three membrane proteins (the GT, the transferrin receptor, and the IGF-II receptor) respond similarly to insulin, by redistributing to the surface from intracellular compartment(s) in which they are colocalized.     

Expression of human cation-independent mannose 6-phosphate receptor cDNA in receptor-negative mouse P388D1 cells following gene transfer

We recently reported the cDNA cloning, sequence, and expression of the human cation-independent mannose 6-phosphate receptor (hCI-MPR) (Oshima, A., Nolan, C. M., Kyle, J. W., Grubb, J. H., and Sly, W. S. (1988) J. Biol. Chem. 263, 2553-2562). The sequence of the hCI-MPR was virtually identical to that of the human insulin-like growth factor II receptor cDNA (Morgan, D. O., Edman, J. C., Standring, D. N., Fried, V. A., Smith, M. C., Roth, R. A., and Rutter, W. J. (1987) Nature 329, 301-307). To test the role of the putative bifunctional receptor in intracellular sorting of acid hydrolases, we studied its effect on lysosomal enzyme transport following gene transfer to receptor-negative cells. Receptor-negative mouse P388D1 cells were transfected with a cDNA construct containing the entire coding sequence of hCI-MPR under the control of the mouse metallothionine I promoter. Stable transformants were isolated and characterized. The expressed hCI-MPR was localized in membranes including the plasma membrane, bound mannose 6-phosphate containing ligands, and mediated endocytosis which could be specifically blocked by mannose 6-phosphate. We next measured the effect of the expressed hCI-MPR on intracellular and secreted acid hydrolases. The intracellular activity of the lysosomal marker enzymes beta-glucuronidase and beta-hexosaminidase increased up to 2-fold following transformation. In addition, expression of the receptor greatly reduced the fraction of acid hydrolases secreted. These phenotypic changes in the transformed cell lines support the proposed role of the cation-independent mannose 6-phosphate receptor in intracellular sorting and targeting of lysosomal enzymes.     

Cation-independent mannose 6-phosphate and 78 kDa receptors for lysosomal enzyme targeting are located in different cell compartments

The distribution of the cation-independent mannose 6-phosphate and 78 kDa receptors was studied in postnuclear subcellular fractions from two rat liver cell lines. ELISA assays revealed that the mannose 6-phosphate receptor is enriched in the light buoyant Percoll fractions that contain Golgi structures and early endosomes. Most of the 78 kDa receptor is localized in a heavy fraction at the bottom of the Percoll gradient and smaller amounts in the endosomal fractions. The high-density compartment is denser than lysosomes, contains LAMP2 but not LIMPII or acid hydrolases, and is not disrupted with glycyl-l-phenylalanine 2-naphthylamide, a substrate for cathepsin C that selectively disrupts lysosomes. Immunofluorescence microscopy studies indicate no colocalization of the 78 kDa receptor with the mannose 6-phosphate receptor or LIMPII. Mannose 6-phosphate-independent endocytosed beta-glucuronidase was found in the lysosomal, the early and late endosomal fractions. These fractions were immunoadsorbed in columns containing antibodies against the 78 kDa receptor. Only the endocytosed beta-glucuronidase present in the early and late endosomal fractions is associated to immunoadsorbed vesicles. In these vesicles, LAMP2 was detected but no LIMPII or the mannose 6-phosphate receptor. Results obtained suggest that the 78 kDa receptor is found along the endocytic pathway, but in vesicles different from the cation-independent mannose 6-phosphate receptor.     

Mannose 6-phosphate Receptor (MPR 300) Proteins from Goat and Chicken Bind Human IGF-II

Mannose 6-phosphate receptor proteins (MPR 300 and 46) in mammals have been shown to mediate transport of lysosomal enzymes to lysosomes intracellularly. Both receptors are also expressed on the plasma membrane. Only MPR 300 protein on the plasma membrane has been shown to be a multifunctional protein which in addition to binding mannose 6-phosphate containing proteins also binds human insulin-like growth factor-II (IGF-II) causing its internalization [Hille-Rehfeld, A. (1995) Mannose 6-phosphate receptors in sorting and transport of lysosomal enzymes. Biochim. Biophys. Acta. 1241: 177–194]. This property has been shown to be exhibited by other mammalian receptors but not by the chicken and frog receptors. In a recent study however it was shown that the fish embryo MPR 300 binds human IGF-II. [Mendez, E., Planas, J.V., Castillo, J., Navarro, I. and Gutierrez, J. (2001) Identification of a type II insulin-like growth factor receptor in fish embryos. Endocrinology, 142: 1090–1097]. In the present study, we demonstrate that the purified goat and chicken liver receptors bind human IGF-II by employing cross-linking experiments (purified receptors and radiolabeled IGF-II) and by ligand blotting (using purified receptors and biotinylated IGF-II). Further CEF cells (chicken embryonic fibroblasts) that are known to contain the putative MPR 300 protein were employed to demonstrate that the CEF cell receptor binds human IGF-II.     

Processing of the phosphorylated recognition marker in lysosomal enzymes. Characterization and partial purification of a microsomal alpha-N-acetylglucosaminyl phosphodiesterase

N-Acetylglucosamine(1)phospho(6)mannose groups recently identified in lysosomal enzymes were proposed to be precursors of the recognition markers terminating with mannose 6-phosphate (Tabas, I., and Kornfeld, S. (1980) J. Biol. Chem. 225, 6633-6639; Hasilik, A., Klein, U., Waheed, A., Strecker, G., and von Figura, K. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 7074-7078). To study the presumptive enzyme removing N-acetylglucosamine from the diester, an assay was developed using a radioactive oligosaccharide containing diester groups of the above structure. An alpha-N-acetylglucosaminyl phosphodiesterase cleaving this substrate in vitro was found in human placenta and in rat liver. The enzyme was solubilized from the microsomal fraction of human placenta and more than 800-fold purified with 75% yield. It is distinct from the lysosomal alpha-N-acetylglucosaminidase by the criteria of immunological cross-reactivity, substrate specificity, and heat stability. The partially purified enzyme cleaves alpha-N-acetylglucosamine phosphodiester bonds in oligosaccharides from lysosomal enzymes, in lysosomal enzymes, and in UDP-N-acetylglucosamine. We propose that the microsomal alpha-N-acetylglucosaminyl phosphodiesterase is involved in the processing of the phosphorylated recognition marker in lysosomal enzymes.     

Phosphomannosyl-enzyme receptors in rat liver. Subcellular distribution and role in intracellular transport of lysosomal enzymes

beta-Hexosaminidase B purified from human fibroblast secretions was used as a ligand to study phosphomannosyl-enzyme receptors in membranes from rat tissues. Enzyme binding to rat liver membranes was saturable, competitively inhibited by mannose 6-phosphate, not dependent on calcium, and destroyed by prior treatment of the hexosaminidase with either alkaline phosphatase or endoglycosidase H. Most (90%) of the phosphomannosyl-enzyme receptors were found in endoplasmic reticulum, Golgi apparatus, and lysosomes; 9.5% in the plasma membrane, and less than 1% in nuclei and mitochondria. Receptors were vesicle-enclosed in all fractions except plasma membrane. Receptors in the endoplasmic reticulum apparently were occupied by endogenous ligands, but most receptors in lysosomes and plasma membrane were unoccupied. Most of the endogenous beta-hexosaminidase was in lysosomes and was released from vesicles by detergent treatment. Displacement of the residual receptor-bound endogenous beta-hexosaminidase (mostly in endoplasmic reticulum and Golgi apparatus) from detergent-treated membranes by mannose 6-phosphate released high uptake enzyme with properties expected for phosphomannosyl-enzymes. Mannose 6-phosphate-inhibitable enzyme receptor activity was found in nine rat organs and correlated roughly with their lysosomal enzyme content. These data support a general model for lysosomal enzyme transport in which the phosphomannosyl-enzyme receptor acts as a vehicle for delivery of newly synthesized acid hydrolases from the endoplasmic reticulum to lysosomes.     

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.     

The mannose 6-phosphate receptor of Chinese hamster ovary cells. Compartmentalization of acid hydrolases in mutants with altered receptors

The localization of acid hydrolases was examined in Chinese hamster ovary cells with defective mannose 6-phosphate receptors; these mutants had been shown to exhibit reduced uptake and altered binding of exogenously added acid hydrolase (Robbins, A. R., Myerowitz, R., Youle, R. J., Murray, G. J., and Neville, D. M., Jr. (1981) J. Biol. Chem. 256, 10618-10622). Cells were grown in the presence of [3H]mannose, alpha-L-iduronidase and beta-hexosaminidase were immunoprecipitated sequentially, electrophoresed on polyacrylamide gels containing sodium dodecyl sulfate, and detected by fluorography. About 55% of the alpha-L-iduronidase and beta-hexosaminidase synthesized by the mutants in 12 h was found in the growth medium; parental cells secreted only approximately 15%. The mutants also secreted 2 to 6 times more alpha-mannosidase, beta-glucuronidase, and alpha-L-fucosidase than the parent as determined by measurements of enzyme activity. Intracellular levels of these enzymes were reduced in the mutants. The mutants secreted acid hydrolases in the precursor forms, within the cells these enzymes resided in lysosomes and were processed normally; thus, the mutants appeared aberrant only with respect to distribution of hydrolases between intracellular and extracellular compartments. [35S]methionine-labeled beta-hexosaminidase and alpha-L-iduronidase secreted by the mutants were taken up normally by both human fibroblasts and wild type CHO cells, and this uptake was inhibited by mannose 6-phosphate. Thus, the elevated secretion of acid hydrolases was not due to alteration of the mannose 6-phosphate recognition marker on the enzymes, but appears to result from alterations in the mannose 6-phosphate receptor.     

Evidence for the presence of muscarinic acetylcholine receptors in bovine brain coated vesicles

Evidence obtained from quinuclidinylbenzilate binding determinations suggested that muscarinic acetylcholine receptor molecules are present in purified bovine brain coated vesicles. Immunoprecipitates formed from coated vesicles with polyclonal antibodies to clathrin bound on the surface of fixed Staphylococcus aureus cells also showed quinuclidinylbenzilate binding activity. The high purity of coated vesicles was established by assays for biochemical markers and by electron microscopy. Muscarinic receptor sites for quinuclidinylbenzilate binding to coated vesicles displayed a Kd of 25 pM and a Bmax of about 191 fmol/mg of protein. Binding competition experiments using atropine, N-methylscopolamine, oxotremorine, and carbamylcholine confirmed the typical muscarinic nature of the binding site. Ranking order of potency for the receptors was: atropine greater than N-methylscopolamine greater than oxotremorine greater than carbachol Analysis of data using a two-site model revealed 13% high-affinity sites for oxotremorine, 66% high-affinity sites for carbachol, and 62% for the antagonist N-methylscopolamine. Heterogeneity of binding affinities for muscarinic drugs detected in the coated vesicles may be related to the presence of coated vesicle subpopulations in brain tissue, (Kohtz, D. S., Kohtz, J. D., Schook, W. J., and Puszkin, S. (1985) J. Cell Biol. 101, 48a; Pfeffer, S. R., and Kelly, R. B. (1985) Cell 40, 949-957).     

Purification and protein sequence analysis of rat liver prolactin receptor

Prolactin receptors were purified from rat liver membranes by single-step immunoaffinity chromatography using a specific monoclonal antibody to the rat liver prolactin receptor. Scatchard analysis of 125I-human growth hormone binding to the purified receptor revealed two classes of specific binding sites with Ka = 18.5 x 10(9) and 1.2 x 10(9) M-1. Considering that both classes of binding sites are responsible for high affinity prolactin binding, the partially purified receptor preparation had a binding activity of 1.69 nmol/mg protein, representing 1000-fold purification over microsomal receptors with a recovery of 52%. From three separate purifications, 6 mg of partially purified prolactin receptor were obtained with a purity of approximately 4 to 6.5%. Thus, the use of monoclonal antibody for affinity chromatography resulted in a large improvement of prolactin receptor purification compared to previous hormone affinity chromatography (300-fold purification, 15% recovery). The purified receptor was run on preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis, and a homogeneous preparation of prolactin receptor was obtained by electroelution from gel slices corresponding to Mr 38,000-43,000. Immunoblot analysis using a radiolabeled monoclonal antibody revealed two separate but closely located bands of Mr 42,000 and 40,000 in microsomal, partially purified, and electroeluted preparations. The homogeneous receptor protein was extensively digested with L-1-tosylamido-2-phenylethyl chloromethyl ketone trypsin, and 10 internal amino acid sequences of the rat liver prolactin receptor were determined by gas-phase sequence analysis. Oligonucleotide probes were prepared against two of these internal sequences, and a prolactin receptor cDNA was isolated from a rat liver library using one of these probes (Boutin, J. M., Jolicoeur, C., Okamura, H., Gagnon, J., Edery, M., Shirota, M., Banville, D., Dusanter-Fourt, I., Djiane, J., and Kelly, P. A. (1988) Cell 53, 69-77). The amino acid sequence deduced from the cDNA reveals three potential sites of N-linked glycosylation, two of which were confirmed during protein sequencing. The prolactin receptor was characterized by affinity labeling with 125I-human growth hormone. Cross-linking of microsomes revealed a single band for the hormone-receptor complex with Mr 62,000. On the other hand, cross-linking of Triton X-100-solubilized or partially purified receptor with labeled hormone resulted in the appearance of two bands with Mr 62,000 and 102,000, suggesting the existence of a subunit structure of the prolactin receptor, or alternatively, the existence of two types of prolactin receptor.(ABSTRACT TRUNCATED AT 400 WORDS)     

The glucose-6-phosphate transport is not mediated by a glucose-6-phosphate/phosphate exchange in liver microsomes

A phosphate-linked antiporter activity of the glucose-6-phosphate transporter (G6PT) has been recently described in liposomes including the reconstituded transporter protein. We directly investigated the mechanism of glucose-6-phosphate (G6P) transport in rat liver microsomal vesicles. Pre-loading with inorganic phosphate (Pi) did not stimulate G6P or Pi microsomal inward transport. Pi efflux from pre-loaded microsomes could not be enhanced by G6P or Pi addition. Rapid G6P or Pi influx was registered by light-scattering in microsomes not containing G6P or Pi. The G6PT inhibitor, S3483, blocked G6P transport irrespectively of experimental conditions. We conclude that hepatic G6PT functions as an uniporter.     

Proof that the endogenous, heat-stable glucocorticoid receptor-activating factor is thioredoxin

Extraction of rat liver cytosol with charcoal inactivates glucocorticoid-binding capacity and receptors can be reactivated to the steroid-binding state by an endogenous reducing system utilizing NADPH and a Mr = 12,000, heat-stable, endogenous, cytosolic protein (Grippo, J. F., Tienrungroj, W., Dahmer, M. K., Housley, P. R., and Pratt, W. B. (1983) J. Biol. Chem. 258, 13658-13664). In this paper we show that NADPH-dependent conversion of the rat liver glucocorticoid receptor from a nonbinding to a steroid-binding form is blocked in an immune-specific manner by antisera raised against purified rat liver thioredoxin reductase or thioredoxin. The inhibition produced by thioredoxin reductase antiserum may be circumvented by dithiothreitol or overcome by addition of purified thioredoxin reductase. These observations prove that the endogenous glucocorticoid receptor-activating factor is thioredoxin and that the enzyme required for generating the steroid-binding conformation of the glucocorticoid receptor by the endogenous receptor-activating system is thioredoxin reductase.     

Serum form of the rat insulin-like growth factor II/mannose 6-phosphate receptor is truncated in the carboxyl-terminal domain

The insulin-like growth factor (IGF)-II/mannose 6-phosphate (Man-6-P) receptor present in mammalian tissues as an apparent molecular mass = 250 kDa glycoprotein has recently been detected in fetal rat serum in a lower molecular mass form (240 kDa). In the present studies the serum receptor was affinity labeled with 125I-IGF-II after its adsorption onto pentamannosyl 6-phosphate-Sepharose, demonstrating that it can also bind both ligands simultaneously. The receptors in both serum and fresh plasma exhibited the lower molecular mass compared to tissue receptors, indicating this form circulates in vivo. In order to probe the structural basis of the serum receptor's lower mass, we raised antipeptide antibodies against cytoplasmic and extracellular domains of the tissue form of the rat receptor deduced from complementary DNA clones (MacDonald, R. G., Pfeffer, S. R., Coussens, L., Tepper, M. A., Brocklebank, C. M., Mole, J. E., Anderson, J. K., Chen, E., Czech, M. P., and Ullrich, A. (1988) Science 239, 1134-1137). Peptide 22C, Glu-Glu-Glu-Thr-Asp-Glu-Asn-Glu-Thr-Glu-Trp-Leu-Met-Glu-Glu-Ile-Gln-Val- Pro-Ala - Pro-Arg, located in the cytoplasmic domain 32 residues carboxyl-terminal to the transmembrane region, and peptide 13D, Tyr-Tyr-Leu-Asn-Val-Cys-Arg-Pro-Leu-Asn-Pro-Val-Pro-Gly-Cys-Asp, located 1476 residues amino-terminal to the transmembrane domain were synthesized and used as immunogens in rabbits. IGF-II/Man-6-P receptors were first immunoprecipitated from either rat serum or a Triton X-100 extract of rat placental plasma membranes using a polyclonal antireceptor antibody. The immunoadsorbed receptors were then reduced, alkylated, electrophoresed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotted onto nitrocellulose, and probed with antipeptide antibodies. Anti-13D revealed the major receptor band in all the membrane and serum samples tested as well as several minor species of lower apparent mass in serum. Fetal and neonatal rat sera contained 3-4 times as much of the receptor as adult serum. In contrast, anti-22C recognized the membrane IGF-II/Man-6-P receptor but failed to recognize any of the serum receptor species. These results indicate that the serum IGF-II/Man-6-P receptor is truncated or altered in its cytoplasmic domain, consistent with the hypothesis that it is derived from cells by proteolytic cleavage.     

The kangaroo cation-independent mannose 6-phosphate receptor binds insulin-like growth factor II with low affinity.

The mammalian cation-independent mannose 6-phosphate receptor (CI-MPR) binds mannose 6-phosphate-bearing glycoproteins and insulin-like growth factor (IGF)-II. However, the CI-MPR from the opossum has been reported to bind bovine IGF-II with low affinity (Dahms, N. M., Brzycki-Wessell, M. A., Ramanujam, K. S., and Seetharam, B. (1993) Endocrinology 133, 440-446). This may reflect the use of a heterologous ligand, or it may represent the intrinsic binding affinity of this receptor. To examine the binding of IGF-II to a marsupial CI-MPR in a homologous system, we have previously purified kangaroo IGF-II (Yandell, C. A., Francis, G. L., Wheldrake, J. F., and Upton, Z. (1998) J. Endocrinol. 156, 195-204), and we now report the purification and characterization of the CI-MPR from kangaroo liver. The interaction of the kangaroo CI-MPR with IGF-II has been examined by ligand blotting, radioreceptor assay, and real-time biomolecular interaction analysis. Using both a heterologous and homologous approach, we have demonstrated that the kangaroo CI-MPR has a lower binding affinity for IGF-II than its eutherian (placental mammal) counterparts. Furthermore, real-time biomolecular interaction analysis revealed that the kangaroo CI-MPR has a higher affinity for kangaroo IGF-II than for human IGF-II. The cDNA sequence of the kangaroo CI-MPR indicates that there is considerable divergence in the area corresponding to the IGF-II binding site of the eutherian receptor. Thus, the acquisition of a high-affinity binding site for regulating IGF-II appears to be a recent event specific to the eutherian lineage.     

A cycloheximide-resistant pool of receptors for asialoglycoproteins and mannose 6-phosphate residues in the Golgi complex of hepatocytes.

Following in vivo administration of cycloheximide (20 mg/kg body weight i.p.) protein synthesis was completely inhibited (99%) in rat liver. No newly synthesized asialoglycoprotein receptor (ASGP-R) could be detected by metabolic labeling. Fluorescence immunocytochemistry of several secretory proteins and plasma membrane proteins, including the receptors for polymeric IgA (IgA-R), demonstrated a rapid loss from the Golgi complex following cycloheximide administration. On the other hand, two membrane proteins, the receptors for ASGP-R and mannose 6-phosphate (MP-R), were not altered in their cellular localization including the Golgi. Using quantitative immunoelectron microscopy with colloidal gold, we found that 2 h and 4 h after cycloheximide administration, the densities of ASGP-R and MP-R in the membranes of the Golgi complex were unaltered compared with control liver. Similarly, there was no significant effect of cycloheximide on the receptor labeling in coated vesicles and compartment of uncoupling receptors and ligands (CURL). These observations are consistent with an involvement of the Golgi and CURL pools of the receptors in intracellular trafficking, endocytosis and receptor recycling.