共查询到20条相似文献,搜索用时 0 毫秒
1.
PG Marron-Terada MA Brzycki-Wessell NM Dahms 《Canadian Metallurgical Quarterly》1998,273(35):22358-22366
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. 相似文献
2.
H Munier-Lehmann F Mauxion U Bauer P Lobel B Hoflack 《Canadian Metallurgical Quarterly》1996,271(25):15166-15174
We have previously generated primary embryonic fibroblasts lacking either the cation-independent mannose 6-phosphate/insulin-like growth factor II receptor (MPR) or the cation-dependent MPR, two trans-membrane proteins that bind the mannose 6-phosphate (Man-6-P) recognition marker on soluble lysosomal enzymes (Ludwig, T., Munier-Lehmann, H., Bauer, U., Hollinshead, M., Ovitt, C., Lobel, P., and Hoflack, B.(1994) EMBO J. 13, 3430-3437). These two cell types partially missort phosphorylated lysosomal enzymes. Using two-dimensional gel electrophoresis, we show here that they secrete, in a large part, different phosphorylated ligands. In order to better understand the sorting function of the MPRs, we have re-expressed each MPR in MPR-negative fibroblasts. We show that the MPRs have similar capacities for transporting the bulk of the newly synthesized lysosomal enzymes and that they target individual ligands with various efficiencies. However, high levels of one MPR do not fully compensate for the absence of the other, demonstrating that the two MPRs have complementary targeting functions, perhaps by recognizing different features on lysosomal enzymes. The analysis of the phosphorylated oligosaccharides shows that the ligands missorted in the absence of the cation-dependent MPR are slightly but significantly depleted in oligosaccharides with two Man-6-P residues, when compared with those missorted in the absence of the cation-independent MPR. While these results could explain some differences between the structure and the sorting function of the two MPRs, they strongly suggest that the reason why cells express two different but related MPRs is to maintain an efficient Man-6-P-dependent targeting process that could be potentially regulated by MPR expression. 相似文献
3.
The 46-kDa mannose 6-phosphate receptor contains multiple binding sites for clathrin adaptors 总被引:1,自引:0,他引:1
S H?ning M Sosa A Hille-Rehfeld K von Figura 《Canadian Metallurgical Quarterly》1997,272(32):19884-19890
The two known mannose 6-phosphate receptors (MPR46 and MPR300) both mediate the transport of Man-6-P-containing lysosomal proteins to lysosomes. However, the MPRs cannot be detected in lysosomes, instead they recycle between the plasma membrane and endosomes and between endosomes and the trans-Golgi network. Both, endocytosis from the plasma membrane and budding of transport vesicles from the trans-Golgi network involves the interaction of the receptor with the clathrin-coated vesicles-associated protein complexes AP1 and AP2. We have analyzed this interaction between the Golgi-restricted AP1 complex and the plasma membrane-restricted AP2 complex with the MPR46 tail in vitro by using a biosensor. AP1 and AP2 both bind to and dissociate from the MPR46 tail with similar kinetics. Using synthetic peptides corresponding to different MPR receptor tail regions in inhibition and binding studies, a common high affinity binding site for AP1 and AP2 and two separate high affinity binding sites for AP1 and AP2, respectively, were identified. 相似文献
4.
SJ York LS Arneson WT Gregory NM Dahms S Kornfeld 《Canadian Metallurgical Quarterly》1999,274(2):1164-1171
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. 相似文献
5.
Neurofilaments, which are exclusively found in nerve cells, are one of the earliest recognizable features of the maturing nervous system. The differential distribution of neurofilament proteins in varying degrees of phosphorylation within a neuron provides the possibility of selectively demonstrating either somata and dendrites or axons. Non-phosphorylated neurofilaments typical of somata and dendrites can be visualized with the aid of monoclonal antibody SMI 311, whereas antibody SMI 312 is directed against highly phosphorylated axonal epitopes of neurofilaments. The maturation of neuronal types, the development of area-specific axonal networks, and the gradients of maturation can thus be demonstrated. Optimal immunostaining with SMI 311 and SMI 312 is achieved when specimens are fixed in a mixture of paraformaldehyde and picric acid for up to 3 days and sections are incubated free-floating. Neurons, with their dendritic domains immunostained by SMI 311 in a Golgi-like manner, can be completely visualized in relatively thick sections. The limitations of Golgi-preparations, such as glia-labeling, artifacts, and the staining of only a small non-representative percentage of existing neurons, are not apparent in SMI preparations, which additionally provide the possibility of selectively staining axonal networks. The results achieved in normal fetal brain provide the basis for studies of developmental disturbances. 相似文献
6.
The bovine cation-dependent mannose 6-phosphate receptor (CD-MPR) is a type 1 transmembrane protein that cycles between the trans-Golgi network, endosomes, and the plasma membrane. When the terminal 40 residues were deleted from the 67-amino acid cytoplasmic tail of the CD-MPR, the half-life of the receptor was drastically decreased and the mutant receptor was recovered in lysosomes. Analysis of additional cytoplasmic tail truncation mutants and alanine-scanning mutants implicated amino acids 34-39 as being critical for avoidance of lysosomal degradation. The cytoplasmic tail of the CD-MPR was partially effective in preventing the lysosomal membrane protein Lamp1 from entering lysosomes. Complete exclusion required both the CD-MPR cytoplasmic tail and transmembrane domain. The transmembrane domain alone had just a minor effect on the distribution of Lamp1. These findings indicate that the cytoplasmic tail of the CD-MPR contains a signal that prevents the receptor from trafficking to lysosomes. The transmembrane domain of the CD-MPR also contributes to this function. 相似文献
7.
The transport of pro-cathepsin D from the trans-Golgi network (TGN) to the endosomal pathway is dependent on binding to the calcium-independent mannose 6-phosphate receptor (ci-M6PR), which is incorporated into TGN-derived clathrin-coated transport vesicles (CCVs). Inhibition of this transport step by wortmannin has led to the proposal that it is dependent upon a phosphoinositide 3-kinase activity necessary for ci-M6PR recruitment into TGN-derived CCVs or in the formation of those vesicles (Brown, W. J., DeWald, D. B., Emr, S. D., Plutner, H., and Balch, W. E. (1995) J. Cell Biol. 130, 781-796; Davidson, H. W. (1995) J. Cell Biol. 130, 797-806). In this study we have addressed the effect of wortmannin on the TGN step of the ci-M6PR cycle. CCVs from K562 cells, pretreated or not with 250 nM wortmannin, were purified on equilibrium density gradients. Quantification of TGN-derived CCVs, assessed by gamma-adaptin content in purified vesicle fractions, showed that the formation of the vesicles was only marginally decreased after 20 min of treatment with the drug, while for the same wortmannin treatment, the amount of ci-M6PR recruited into those vesicles was decreased by 70% compared with control. At a later time point (2 h), a reduction in the amount of gamma-adaptin in CCV fractions was also observed. These findings demonstrate that inhibition of ci-M6PR recruitment into CCVs but not of vesicle formation is the primary reason for the observed defect in cathepsin D transport following wortmannin treatment. 相似文献
8.
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. 相似文献
9.
S Kanamori S Waguri M Shibata K Isahara Y Ohsawa A Konishi S Kametaka T Watanabe S Ebisu E Kominami Y Uchiyama 《Canadian Metallurgical Quarterly》1998,251(1):204-208
Beta1,4-galactosyltransferase (GalTase, EC 2.4.1.38) transfers galactose to the terminal N-acetylglucosamine of complex-type N-glycans, which have great importance for cell-cell interactions during fertilization and early embryogenesis. In this study, the activity of beta1,4-galactosyltransferase in mouse brain during development was measured with the method of reverse HPLC using a fluorescence-labeled biantenary sugar chain, GlcNAcbeta1-2Manalpha1-6(GlcNAcbeta1-2Manalpha1- 3) Manbeta1-4GlcNAcbeta1-4GlcNAc-PA. The level of messenger RNA of this enzyme during the development of mouse brain was also investigated with Northern blot analysis. The results showed that: (1)beta1,4-galactosyltransferase showed similar branch specificity and kinetics for the biantenary substrate during development; (2) GalTase activity in fetal mouse brain was four times higher than that in adult mouse brain and decreased gradually in the course of development; (3) messenger RNA level was highest in fetal mouse and decreased dramatically after birth. However, the contents of mRNA were not parallel to the enzyme activity. 相似文献
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Previous studies indicate that fetal rat heart tissue contains large amounts of insulin-like growth factor (IGF)-II/mannose 6-phosphate (Man 6-P) receptor messenger RNA, with receptor messenger RNA levels falling by 20 days after birth. We examined the amount of IGF receptor protein in developing rat myocardium. To establish a model in which the role of neural, hormonal, and hemodynamic controls of IGF receptor binding could be studied, we compared binding of IGF-I and IGF-II in normally growing rat atria and ventricles with embryonic day 12 (E-12) atria and ventricles maturing in the anterior eye chamber of an adult host rat. In oculo, embryonic myocardium matures without hemodynamic load or exposure to the fetal hormonal milieu. In fetal rat hearts (E-12 to E-19), both IGF-I and IGF-II intensely bound to a protein with a molecular weight corresponding to the IGF-II/Man 6-P receptor. Receptors were identified using sodium dodecyl sulfate/polyacrylamide gel electrophoresis autoradiography and western blot analysis using Ab3637, a specific polyclonal antibody against rat IGF-II/Man 6-P receptor antigens. This antibody competed for binding of both IGF-I and IGF-II to the band with molecular radius corresponding to 260,000 (reduced). In normally growing rat atria, IGF-I binding to the IGF-II/Man 6-P receptor was similar to ventricular tissue; however, there was significantly greater binding of IGF-II than of IGF-I in both atrial and ventricular tissue. High levels of IGF-II binding to the IGF-II/Man 6-P receptor were observed in both fetal rat atrial and ventricular grafts until 6-8 weeks in oculo. As in normally growing heart tissue, there was similar IGF-I binding to the IGF-II/Man 6-P receptor in atrial grafts compared with ventricular grafts from 2-8 weeks after implantation. For the first 2 weeks after grafting, the ventricular grafts had relatively higher IGF-I binding to the IGF-II/Man 6-P receptor compared with later time points examined. The present data indicate that atrial and ventricular binding of IGFs to the IGF-II/Man 6-P receptor decreases with age, suggesting that decreased IGF binding may be independent of postnatal hemodynamic changes. The decrease is similar in in oculo embryonic rat cardiac grafts and normally growing heart tissue.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
12.
The 67-amino acid cytoplasmic tail of the cation-dependent mannose 6-phosphate receptor (CD-MPR) contains a signal(s) that prevents the receptor from entering lysosomes where it would be degraded. To identify the key residues required for proper endosomal sorting, we analyzed the intracellular distribution of mutant forms of the receptor by Percoll density gradients. A receptor with a Trp19 --> Ala substitution in the cytoplasmic tail was highly missorted to lysosomes whereas receptors with either Phe18 --> Ala or Phe13 --> Ala mutations were partially defective in avoiding transport to lysosomes. Analysis of double and triple mutants confirmed the key role of Trp19 for sorting of the CD-MPR in endosomes, with Phe18, Phe13, and several neighboring residues contributing to this function. The addition of the Phe18-Trp19 motif of the CD-MPR to the cytoplasmic tail of the lysosomal membrane protein Lamp1 was sufficient to partially impair its delivery to lysosomes. Replacing Phe18 and Trp19 with other aromatic amino acids did not impair endosomal sorting of the CD-MPR, indicating that two aromatic residues located at these positions are sufficient to prevent the receptor from trafficking to lysosomes. However, alterations in the spacing of the diaromatic amino acid sequence relative to the transmembrane domain resulted in receptor accumulation in lysosomes. These findings indicate that the endosomal sorting of the CD-MPR depends on the correct presentation of a diaromatic amino acid-containing motif in its cytoplasmic tail. Because a diaromatic amino acid sequence is also present in the cytoplasmic tail of other receptors known to be internalized from the plasma membrane, this feature may prove to be a general determinant for endosomal sorting. 相似文献
13.
Insulin-like growth factor II (IGF-II) and phosphomannosylated glycoproteins bind to distinct sites on the same receptor, the IGF-II/mannose 6-phosphate receptor (IGF2R). Analysis of truncated receptors (minireceptors) has been used to map the IGF-II binding site within the receptor's extracytoplasmic domain, which consists of 15 homologous repeats. A minireceptor consisting of repeat 11 contained the minimal elements for binding IGF-II, but with 5- to 10-fold lower relative binding affinity than the full-length receptor. We hypothesized that the complete, high-affinity IGF-II binding site is formed by interaction between the primary site in repeat 11 and a putative affinity-enhancing domain. To determine the minimum portion of the IGF2R's extracytoplasmic domain needed for expression of high-affinity IGF-II binding, a nested set of FLAG epitope-tagged minireceptors encompassing repeats 11 through 15 was prepared and transiently expressed in 293T cells. Minireceptors containing repeats 11-13 or 11-15 exhibited high affinity, comparable to the full-length receptor (IC50 = 1-2 nM), whereas constructs containing repeat 11 only or repeats 11-12 did not (IC50 = 10-20 nM). These data suggested that the affinity-enhancing domain is located within repeat 13, which contains a unique 43-residue insert that has approximately 50% sequence identity to the type II repeat of fibronectin. Although a repeat 13 minireceptor did not bind IGF-II on its own, an 11-13 minireceptor containing a deletion of the 43-residue insert exhibited low IGF-II binding affinity (IC50 = 10-20 nM). Expression of mutant receptors from a full-length IGF2R construct bearing a deletion of the 43-residue insert was very low relative to wild type. Depletion assays using IGF-II-Sepharose showed that the mutant receptor had lower affinity for IGF-II than the wild-type receptor. This study reveals that two independent receptor domains are involved in the formation of a high-affinity binding site for IGF-II, and that a complete repeat 13 is required for high-affinity IGF-II binding. 相似文献
14.
F Blanchard S Raher L Duplomb P Vusio V Pitard JL Taupin JF Moreau B Hoflack S Minvielle Y Jacques A Godard 《Canadian Metallurgical Quarterly》1998,273(33):20886-20893
Comparison of the binding properties of non-glycosylated, glycosylated human leukemia inhibitory factor (LIF) and monoclonal antibodies (mAbs) directed at gp190/LIF-receptor beta subunit showed that most of the low affinity (nanomolar) receptors expressed by a variety of cell lines are not due to gp190. These receptors bind glycosylated LIF produced in Chinese hamster ovary cells (CHO LIF) (Kd = 6.9 nM) but not Escherichia coli-derived LIF or CHO LIF treated with endoglycosidase F. CHO LIF binding to these receptors is neither affected by anti-gp190 mAbs nor by anti-gp130 mAbs and is specifically inhibited by low concentrations of mannose 6-phosphate (Man-6-P) (IC50 = 40 microM), suggesting that they could be related to Man-6-P receptors. The identity of this LIF binding component with the Man-6-P/insulin-like growth factor-II receptor (Man-6-P/IGFII-R) was supported by several findings. (i) It has a molecular mass very similar to that of the Man-6-P/IGFII-R (270 kDa); (ii) the complex of LIF cross-linked to this receptor is immunoprecipitated by a polyclonal anti-Man-6-P/IGFII-R antibody; (iii) this antibody inhibits LIF and IGFII binding to the receptor with comparable efficiencies; (iv) soluble Man-6-P/IGFII-R purified from serum binds glycosylated LIF (Kd = 4.3 nM) but not E. coli LIF. The potential role of Man-6-P/IGFII-R in LIF processing and biological activity is discussed. 相似文献
15.
Different peptide binding specificities of hsp70 family members 总被引:1,自引:0,他引:1
A set of heptapeptides was used to compare the relative peptide affinities of three proteins of the hsp70 family: bacterial DnaK, mammalian cytosolic hsc70, and BiP from mammalian ER. Each hsp displays a characteristic pattern of relative affinities. DnaK and hsc70 are more similar to each other than to BiP. A difference in peptide binding specificity may be an important determinant in adjusting an hsp70 family member to its particular cellular function. 相似文献
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JR Jasper JD Lesnick LK Chang SS Yamanishi TK Chang SA Hsu DA Daunt DW Bonhaus RM Eglen 《Canadian Metallurgical Quarterly》1998,55(7):1035-1043
Alpha-2 adrenergic receptors (alpha2 AR) mediate incorporation of guanosine 5'-O-(gamma-thio)triphosphate ([35S]GTPgammaS) into isolated membranes via receptor-catalyzed exchange of [35S]GTPgammaS for GDP. In the current study, we used [35S]GTPgammaS incorporation to characterize the intrinsic activity and potency of agonists and antagonists at the cloned mouse alpha2a/d and human alpha2a, alpha2b, and alpha2c ARs. Full agonists increased [35S]GTPgammaS binding to membranes by 2- to 3-fold. Antagonists did not increase [35S]GTPgammaS binding but competitively inhibited agonist-stimulated [35S]GTPgammaS binding. Compounds with intrinsic activities less than that of the full agonists norepinephrine (NE) or epinephrine (EPI) were capable of antagonizing agonist-stimulated [35S]GTPgammaS binding. The agonistic properties of a number of alpha2 AR ligands were characterized at each alpha2 AR subtype. The rank order of agonist potency for selected compounds at the human receptors (with intrinsic activity compared with NE, defined as 1.0) was: alpha2a: Dexmedetomidine (0.73) > guanabenz (0.38) > UK-14304 (1.02) > clonidine (0.32) > ST-91 (0.63) > NE (1.00). alpha2b: Dexmedetomidine (1.10) > clonidine (0.18) > guanabenz (0.71) > NE (1.00) > ST-91 (0.44) > UK-14304 (0.59). alpha2c: Dexmedetomidine (1.03) > NE (1.00) > UK-14304 (0.75) > ST-91 (0.32) > or = clonidine (0.23) > guanabenz (0). This report provides a functional characterization of adrenergic receptor ligands at human and mouse alpha2a/d AR. It also illustrates the utility of [35S]GTPgammaS incorporation as a functional marker of receptor activation. 相似文献
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19.
A Nykjaer EI Christensen H Vorum H Hager CM Petersen H R?igaard HY Min F Vilhardt LB M?ller S Kornfeld J Gliemann 《Canadian Metallurgical Quarterly》1998,141(3):815-828
The urokinase-type plasminogen activator receptor (uPAR) plays an important role on the cell surface in mediating extracellular degradative processes and formation of active TGF-beta, and in nonproteolytic events such as cell adhesion, migration, and transmembrane signaling. We have searched for mechanisms that determine the cellular location of uPAR and may participate in its disposal. When using purified receptor preparations, we find that uPAR binds to the cation-independent, mannose 6-phosphate/insulin-like growth factor-II (IGF-II) receptor (CIMPR) with an affinity in the low micromolar range, but not to the 46-kD, cation-dependent, mannose 6-phosphate receptor (CDMPR). The binding is not perturbed by uPA and appears to involve domains DII + DIII of the uPAR protein moiety, but not the glycosylphosphatidylinositol anchor. The binding occurs at site(s) on the CIMPR different from those engaged in binding of mannose 6-phosphate epitopes or IGF-II. To evaluate the significance of the binding, immunofluorescence and immunoelectron microscopy studies were performed in transfected cells, and the results show that wild-type CIMPR, but not CIMPR lacking an intact sorting signal, modulates the subcellular distribution of uPAR and is capable of directing it to lysosomes. We conclude that a site within CIMPR, distinct from its previously known ligand binding sites, binds uPAR and modulates its subcellular distribution. 相似文献
20.
MW Loewus 《Canadian Metallurgical Quarterly》1977,252(20):7221-7223
myo-Inositol-1-phosphate synthase (EC 5.5.1.4) from rat testis, Acer pseudoplatanus L. cell culture and Oryza sativa L. cell culture, converted D-[5-3H]glucose 6-phosphate to myo-[2-3H]inositol 1-phosphate at rates ranging from 0.21 to 0.48 that of unlabeled substrate. D-[3-3H]- and D-[4-3H]glucose 6-phosphate were converted at approximately the same rate as that of unlabeled substrate. In the case of testis enzyme, storage as a frozen solution further lowered the rate with D-[5-3H]glucose 6-phosphate as substrate. When the reaction was run in [3H]water, no 3H appeared in myo-inositol 1-phosphate but a small amount was recovered in substrate isolated from the final reaction mixture. These data support the involvement of carbon 5 of D-glucose 6-phosphate in the mechanism proposed for this conversion. 相似文献