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1.
Internalization and degradation of peptides of the bombesin family in Swiss 3T3 cells occurs without ligand-induced receptor down-regulation. 总被引:3,自引:1,他引:2
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The binding of [125I]gastrin releasing peptide ([125I]GRP) to Swiss 3T3 cells at 37 degrees C increases rapidly, reaching a maximum after 30 min and decreasing afterwards. The decrease in cell-associated radioactivity at this temperature is accompanied by extensive degradation of the labelled peptide. At 4 degrees C equilibrium binding is achieved after 6 h and [125I]GRP degradation is markedly inhibited. Extraction of surface-bound ligand at low pH demonstrates that the iodinated peptide is internalized within minutes after addition to 3T3 cells at 37 degrees C. The rate of internalization is strikingly temperature-dependent and is virtually abolished at 4 degrees C. In addition, lysomotropic agents including chloroquine increase the cell-associated radioactivity in cells incubated with [125I]GRP. The binding of [125I]GRP to Swiss 3T3 cells was not affected by pretreatment for up to 24 h with either GRP or bombesin at mitogenic concentrations. Furthermore, pretreatment with GRP did not reduce the affinity labelling of a Mr 75,000-85,000 surface protein recently identified as a putative receptor for bombesin-like peptides. These results demonstrate that while peptides of the bombesin family are rapidly internalized and degraded by Swiss 3T3 cells, the cell surface receptors for these molecules are not down-regulated. 相似文献
2.
Proteinase A, a yeast aspartyl protease that is highly homologous to the mammalian lysosomal aspartyl protease, cathepsin D, was expressed in Xenopus oocytes and its biosynthesis and post-translational modifications were characterized. While 29-45% of the proteinase A was secreted from oocytes, approximately 37% of the cell-associated proteinase A underwent proteolytic cleavage, characteristic of delivery to a lysosomal organelle. Although proteinase A is not targeted to the yeast vacuole by a mannose 6-phosphate receptor-dependent pathway, 2-5% of the proteinase A molecules expressed in oocytes bound to a Man-6-P receptor column. However, analysis of its [2-3H]mannose-labeled oligosaccharides revealed that 14-23% of these units contain phosphomannosyl residues. A hybrid molecule (H6), in which the propiece and first 12 amino acids of proteinase A were changed to the cathepsin D sequence, was also expressed in oocytes. The binding of H6 to the Man-6-P receptor was approximately 12-fold greater than observed for proteinase A. This increased level of receptor binding could be accounted for by three factors: 1) a small increase in the total amount of phosphorylated oligosaccharides, 2) an increase in the number of oligosaccharides which acquire two phosphomonoesters, and 3) the presence of a greater percentage of oligosaccharides with one phosphomonoester which exhibit high affinity binding to the Man-6-P receptor. These results demonstrate that proteinase A is recognized by UDP-GlcNAc:lysosomal enzyme N-acetylglucosaminylphosphotransferase. However, this interaction is altered by the addition of cathepsin D sequences, resulting in the generation of a higher affinity ligand for binding to the Man-6-P receptor. 相似文献
3.
Multivesicular bodies play a key role in vitellogenin endocytosis by Xenopus oocytes 总被引:5,自引:0,他引:5
A combination of electron microscopic tracers and subcellular fractionation has been used to examine the endocytic pathway of the yolk protein precursor, vitellogenin (VG), in Xenopus oocytes. VG was adsorbed to colloidal gold, and the organelles traversed by newly internalized ligand were examined at various time intervals after endocytosis. VG-Au enters oocytes via coated pits and vesicles and then appears rapidly in tubular endosomes and multivesicular bodies (MVBs). MVBs play a central role in VG processing for storage; the large majority of newly internalized VG enters this compartment, remaining there for up to several hours. Condensation of VG into crystalline bodies begins in MVBs, and continues with growth of the crystals until typical platelets are formed. When oocytes are exposed to high [VG], MVBs containing large amounts of internalized VG are morphologically indistinguishable from the primordial yolk platelets described earlier (Dumont, 1978). The use of VG-Au particles of two sizes demonstrates that gold particles in early MVBs were generally associated with the limiting membrane of these organelles, while older MVB compartments have gold particles well separated from the limiting membranes, suggesting that dissociation of VG from its receptor occurs in this compartment. Newly internalized ligand preferentially forms a new MVB, rather than fusing and mixing with previously formed MVBs. Progressive yolk protein condensation gradually transforms MVBs into yolk platelets over a period of several hours. Analysis of 125I-VG-Au behavior after sucrose gradient fractionation of oocytes allowed correlation of biochemical compartments with those observed in the electron microscope. MVBs containing yolk in progressive stages of condensation were found at densities from 1.16 up to 1.21 g/cc. The final, rate-limiting step in VG transport is a shift of ligand from light (1.21 g/cc) to heavy (1.23 g/cc) platelet compartments (Wall and Meleka, 1985). The morphological correlate of this process is movement of VG-Au from small (less than 3-4 microns diameter) to large (greater than 4 microns diameter) platelets. 相似文献
4.
The biosynthesis of the 140-kilodalton fibronectin receptor complex by cultured 3T3 mouse cells was characterized and compared with that of chick embryo fibroblasts. Three murine glycoprotein components of 140-150 kilodaltons (band 1), 125 kilodaltons (band 2), and 105 kilodaltons (band 3) could be immunoprecipitated from metabolically labeled 3T3 cells using polyclonal antibodies. In pulse-chase experiments, bands 1 and 3 of the mouse receptor were labeled to maximal levels immediately after completion of the labeling pulse. However, band 2 was not detected at short chase times, and it reached maximal labeling only after approximately 12 h of chase. The appearance of band 2 occurred at the same rate as the disappearance of band 3. Only bands 1 and 2 could be affinity purified by binding to immobilized fibronectin cell-binding fragment, indicating that they represent mature functional receptor components. When 3T3 cells were incubated with radioactive sugars, band 1 of the receptor labeled well with both [3H]mannose and [3H]glucosamine. However, band 2 labeled well with [3H]glucosamine but contained low amounts of mannose, and band 3 labeled well with [3H]mannose but contained low amounts of glucosamine. Digestion of both bands 2 and 3 with endoglycosidase F yielded similar-sized products of approximately 88,000 daltons, suggesting that post-translational asparagine-linked oligosaccharide processing can account for most of the size difference between these bands. These data suggest that in the mouse fibronectin receptor, band 3 is a biologically inactive precursor of band 2 that does not appear on the cell surface. In contrast, pulse-chase experiments using chicken embryo fibroblasts indicated that the three components of the chicken 140k complex were distinct moieties. Our results demonstrate distinct types of processing for fibronectin receptor complexes from different species. In mammalian cells, this receptor undergoes a surprisingly long (20 h) maturation process involving asparagine-linked oligosaccharides before reaching its final, biologically active form. 相似文献
5.
Isolated hepatocytes from streptozotocin injected rats bound the same amount of [125I]monoiodoinsulin as hepatocytes from control rats. Scatchard analysis confirmed that insulin receptor number and affinity were the same for both groups. Relatively more cell-associated radioactivity was located intracellularly in hepatocytes from streptozotocin injected rats. Pretreatment with chloroquine resulted in a smaller increase in intracellular [125I]monoiodoinsulin in cells isolated from streptozotocin injected rats than for control cells. These results suggest that intracellular insulin processing occurs more slowly in hepatocytes isolated from streptozotocin injected rats than from control rats. 相似文献
6.
Internalization and degradation of receptor-bound interferon-gamma by murine macrophages. Demonstration of receptor recycling 总被引:4,自引:0,他引:4
Although the interferon-gamma (IFN-gamma) receptor on murine and human mononuclear phagocytes has been defined and partially characterized, very little data exists which describes the ultimate fate of receptor-bound ligand. The current studies were specifically designed to define the metabolic processes which act on murine recombinant IFN-gamma following its interaction with murine macrophages at physiologic temperatures. Ligand internalization was demonstrated by comparing binding of [125I]IFN-gamma to macrophages at 4 degrees C and 37 degrees C. When binding was carried out at 4 degrees C, 96% of the cell-associated [125I]IFN-gamma remained accessible at the plasma membrane and could be stripped from the cell by exposure to pronase. In contrast, at 37 degrees C, only 35% of the cell-associated radioactivity was pronase strippable. Macrophages degraded [125I]IFN-gamma into trichloroacetic acid-soluble material at 37 degrees C at a constant rate of 7000 molecules/cell/hr over a 12-hr time period. The amount of IFN-gamma degraded correlated with the amount of IFN-gamma bound to the cell surface. The receptor was neither up- nor down-regulated by ligand or by other agents known to regulate macrophage functional activity such as IFN-alpha, IFN-beta, lipopolysaccharide, or phorbol myristate acetate. The constant uptake of IFN-gamma by macrophages was due to the presence of an intracellular receptor pool (62% of the total receptor number) and to a mechanism of receptor recycling. Evidence for the latter was obtained using lysosomotropic agents which blocked degradation but not binding and internalization of ligand and caused the intracellular accumulation of receptor. By comparing the relationship between receptor occupancy and biologic response induction, two activation mechanisms became apparent. Induction of certain functions, such as H2O2 secretion, appeared to require only a single round of receptor occupancy. However, induction of more complex functions such as nonspecific tumoricidal activity appeared to require three to four rounds of receptor occupancy. These results thus support the concept that IFN-gamma internalization and receptor recycling are essential in the induction of nonspecific tumoricidal activity by macrophages. 相似文献
7.
B M Simmons P D Stahl J H Russell 《Biochemical and biophysical research communications》1987,146(2):849-854
Ricin A chain has previously been shown to intoxicate macrophages in vitro following binding and endocytosis by the macrophage mannose receptor. In this report it is demonstrated that the intravenous injection of ricin A chain in nephrectomized rats leads to a prolonged plasma half-life for [125I]beta-glucuronidase, a ligand for the mannose receptor. Clearance of [125I]asialofetuin, a ligand for the galactose receptor of hepatocytes, was unaffected by injection of A chain. Microscopic examination of the livers of A chain-treated animals revealed a loss of phagocytic cells from the liver sinusoids. These results suggest that ricin A chain may be useful as a toxin specific for mannose receptor bearing cells of the reticuloendothelial system. 相似文献
8.
Analysis of photoaffinity label derivatives to probe thyroid hormone receptor in human fibroblasts, GH1 cells, and soluble receptor preparations 总被引:3,自引:0,他引:3
Z D Horowitz H Sahnoun A Pascual J Casanova H H Samuels 《The Journal of biological chemistry》1988,263(14):6636-6642
The regulation of growth hormone gene expression by thyroid hormone in cultured GH1 cells is mediated by a chromatin-associated receptor. We have previously described a photoaffinity label derivative of 3,5,3'-triiodo-L-thyronine (L-T3) in which the alanine side chain was modified to form N-2-diazo-3,3,3-trifluoropropionyl-L-T3 (L-[125I]T3-PAL). On exposure to 254 nm UV light, L-[125I]T3-PAL generates a carbene which covalently modifies two thyroid hormone receptor forms in intact GH1 cells; an abundant 47,000 Mr species and a less abundant 57,000 Mr form. We have now synthesized similar photoaffinity label derivatives of 3,5,3',5'-tetraiodo-L-thyronine (L-T4) and 3,3',5'-triiodo-L-thyronine (L-rT3). Both compounds identify the same receptor forms in intact cells and in nuclear extracts in vitro as L-[125I]T3-PAL. Labeling by L-[125I]rT3-PAL was low and consistent with the very low occupancy of receptor by L-rT3. Underivatized L-[125I]T3 and L-[125I]T4 labeled the same receptor forms at 254 nm but at a markedly lower efficiency than their PAL derivatives. In contrast, N-bromoacetyl-L-[125I]T3, a chemical affinity labeling agent, did not derivatize either receptor form in vitro. The relative efficiency of coupling to receptor at 254 nm was L-[125I]T4-PAL greater than L-[125I]T3-PAL greater than L-[125I]T4 greater than L-[125I]T3. Although L-[125I]T4-PAL has a lower affinity for receptor than L-[125I]T3-PAL, its coupling efficiency was 5-10-fold higher. This suggests that the alanine side chain of L-[125I]T4-PAL is positioned in the ligand binding region near a residue which is efficiently modified by photoactivation. With L-[125I]T4-PAL we were able to identify three different molecular weight receptor species in human fibroblast nuclei. 相似文献
9.
Chemical cross-linking alters high-density lipoprotein to be recognized by a scavenger receptor in rat peritoneal macrophages. 总被引:1,自引:0,他引:1
A Miyazaki A T Rahim S Araki Y Morino S Horiuchi 《Biochimica et biophysica acta》1991,1082(2):143-151
Rat peritoneal macrophages possess a surface receptor for high-density lipoprotein (HDL). To obtain the functional aspect of the HDL receptor, the present study was undertaken to modify HDL with three different cross-linkers; dimethylsuberimidate, disuccinimidylsuberate and dithiobissuccinimidylpropionate (DSP) and determine their effect on the ligand activity for the HDL receptor. Upon modification at a low reagent concentration, DSP was found to be most effective in cross-linking of HDL apolipoproteins. The ligand activity of DSP-HDL for the HDL receptor was reduced by greater than 60%. Experiments with these macrophages at 37 degrees C showed; (i) the amounts of the cell-associated [125I]DSP-HDL as 3.5-fold higher than [125I]HDL; (ii) the cell-association of [125I]DSP-HDL was effectively (greater than 70%) inhibited by unlabeled DSP-HDL, whereas HDL showed a partial inhibition (30%); (iii) [125I]DSP-HDL underwent chloroquine-sensitive intracellular degradation; and (iv) DSP-HDL induced a 3-fold increase in the incorporation of [14C]oleic acid into cholesteryl oleate when compared with unmodified HDL. Experiments at 0 degrees C showed that the cellular binding of [125I]DSP-HDL was competed by acetylated low-density lipoprotein and dextran sulfate. These findings indicate that DSP-HDL is recognized as a ligand by a scavenger receptor of rat peritoneal macrophages, a notion consistent with HDL modified with tetranitromethane (Kleinherenbrink-Stins, M.F. et al. (1989) J. Lipid Res. 39, 511-520). 相似文献
10.
Vitellogenesis is the process of yolk formation in rapidly growing oocytes of oviparous species. The transport of yolk precursor proteins from the blood plasma into the oocyte is achieved by receptor-mediated endocytosis. Although the Xenopus oocyte is one of the prime experimental systems for expression of foreign genes and their products, the receptor for the main vitellogenic protein, vitellogenin, from this extensively utilized cell has not been identified. Here we have applied ligand and immunoblotting to visualize the Xenopus laevis oocyte receptor for vitellogenin as a protein with an apparent Mr of 115,000 in sodium dodecyl sulfate-polyacrylamide gels under nonreducing conditions. The receptor from the amphibian oocyte also recognizes chicken vitellogenin, and vice versa; furthermore, the two receptor proteins are immunologically related as revealed by Western blotting with anti-chicken vitellogenin receptor antibodies. The receptors from both species bind the lipovitellin moiety of vitellogenin, as revealed by ligand blotting with radiolabeled lipovitellin polypeptides as well as by a novel reverse ligand blotting procedure utilizing nitrocellulose-immobilized ligand. Since vitellogenins of chicken and Xenopus have been shown to be structurally similar and evolutionarily related (Nardelli, D., van het Schip, F. D., Gerber-Huber, S., Haefliger, J.-A., Gruber, M., AB, G., and Wahli, W. (1987) J. Biol. Chem. 262, 15377-15383), it appears that conservation of key structural elements required for efficient vitellogenesis extends from the ligands to their receptors on the oocyte plasma membrane. 相似文献
11.
Trond Berg Terry Ford Grete Kindberg Rune Blomhoff Christian Drevon 《Experimental cell research》1985,156(2):570-574
Isolated rat hepatocytes take up and degrade [125I]tyramine-cellobiose-labelled asialofetuin [( 125I]TC-AF). The labelled degradation products are trapped at the site of degradation. The intracellular transport of [125I]TC-AF was studied by means of cell fractionation in Nycodenz gradients. The labelled ligand was kept in a small, slowly sedimenting vesicle during the first minutes after uptake in the cells, and was then transferred to a larger endosome. Labelled degradation products first appeared in an organelle with the same density distribution as the larger endosome and then in a denser organelle. These observations suggest that two types of lysosome, 'light' lysosomes and 'dense', are sequentially involved in the degradation of the asialoglycoprotein. The bulk of the lysosomal enzymes is associated with the dense lysosome. 相似文献
12.
[125I]NPY bound to a single class of saturable binding sites on bovine hippocampus membranes with a KD of 0.1 mM and Bmax of 165 fmol/mg of protein. The rank order of potency of NPY fragments and other structurally related peptides to inhibit [125I]NPY binding was: PYY greater than or equal to NPY much greater than BPP greater than or equal to APP and NPY greater than NPY-(13-36) greater than NPY-(18-36) greater than or equal to NPY-(20-36) much greater than NPY-(26-36) greater than NPY-(free acid). The identity of the NPY binding site was investigated by affinity labeling. Gel electrophoresis followed by autoradiography revealed a band with a mol mass of 50 kDa. Unlabeled NPY or PYY, but not BPP, HPP and APP, inhibited labeling of [125I]NPY to the 50 kDa protein band. Moreover, labeling was inhibited by NPY greater than NPY-(18-36) greater than or equal to NPY-(13-36) greater than or equal to NPY-(20-36) greater than NPY-(26-36) greater than NPY-(free acid). The binding of [125I]NPY and the intensity of the cross-linked band were reduced in parallel by increasing concentrations of unlabeled NPY (IC50 = 0.7 nM and 0.6 mM, respectively). These studies demonstrate that bovine hippocampal membranes contain a 50 kDa [125I]NPY binding site that has the ligand specificity characteristic of the Y2 receptor subtype. 相似文献
13.
Steven J. Fluharty Lawrence P. Reagan Michael M. White 《Journal of neurochemistry》1991,56(4):1307-1311
Intact Xenopus oocytes contain a homogeneous population of binding sites for the angiotensin II (Ang II) receptor antagonist 125I-[Sarc1,Ile8]-Ang II (125I-SARILE). Binding of 125I-SARILE to intact oocytes was saturable and of high affinity with an apparent KD of 0.7 nM and maximal density of 0.12 fmol/oocyte. Binding of 125I-SARILE to oocytes also was specific for Ang II-related peptides with a rank order potency of: [Sarc1]-Ang II greater than Ang II greater than Ang III much greater than Ang I. However, these endogenous binding sites were present only in follicle-enclosed oocytes and within the follicular layer itself. On the other hand, injection of poly(A)+ RNA isolated from murine N1E-115 neuroblastoma cells into oocytes resulted in the appearance of 125I-SARILE binding sites even in defolliculated oocytes. These expressed receptors exhibited pharmacological properties similar to those endogenously present in the follicular layer, although their levels were much less. Collectively, these results suggest that endogenous Ang II receptors are present on Xenopus oocyte follicle cells, whereas Ang II receptors expressed from exogenous N1E-115 RNA are found on the oocytes themselves. In addition, the high density of Ang II receptors on the follicle cells emphasizes the necessity for care in using Xenopus oocytes for the expression of receptors encoded by exogenous RNAs. 相似文献
14.
15.
Receptor binding and cell-mediated metabolism of [125I]monoiodoglucagon by isolated canine hepatocytes 总被引:3,自引:0,他引:3
We have developed a reverse-phase HPLC method to purify 125I-labeled products resulting from the chloramine-T-based iodination of glucagon and have used the products [(125I)iodoTyr13]glucagon, [(125I)iodoTyr10,13]glucagon, and [(125I)iodoTyr10]glucagon) to study the receptor binding of glucagon and the cell-mediated metabolism of the hormone by isolated canine hepatocytes. The extent of binding of the three labeled glucagons to cell receptors differed at steady state (8.5, 11.9, and 12.6% of the three peptides, respectively, becoming cell-associated), but each of the labeled glucagons approached steady state binding at the same rate. Further, unlabeled glucagon competed for the binding of each of the labeled peptides in parallel under steady state conditions, and each of the peptides showed potent activity in inhibiting [14C]fructose incorporation into glycogen. Gel filtration of the acetic acid-extracted, cell-associated products of radiolabeled glucagon binding revealed 10-20% of the material as a shoulder on the descending limb of the peak of hormone for each of the three labeled peptides. Trypsin digestion of the lower molecular weight peptide derived from [(125I)iodoTyr13]glucagon resulted in a fragment containing residues 13 to 17 as the only detectable radiolabeled product. On the other hand, trypsin digestion of the analogous peptide derived from [(125I)iodoTyr10]glucagon revealed, in addition to the radiolabeled fragment containing residues 1 to 12, a major fragment identified by radiosequence analysis to contain residues 4 to 12 and a minor fragment identified to contain residues 7 to 12. We conclude that (a) notwithstanding apparent differences in affinities exhibited by [(125I)iodoTyr13]glucagon, [(125I)iodoTyr10,13]glucagon, and [(125I)iodoTyr10]glucagon for binding to canine hepatocytes, the interactions of all three peptides with the glucagon receptor are functionally equivalent, and (b) the cell-mediated metabolism of receptor-bound glucagon involves the formation of hormone-derived peptides in which the biologically important NH2-terminal region of the hormone has been modified by limited proteolytic cleavage. 相似文献
16.
Specific proteolysis regulates fusion between endocytic compartments in Xenopus oocytes 总被引:3,自引:0,他引:3
We examined the role of proteolytic ligand modification in endosomal targeting using vitellogenin (VTG) uptake by Xenopus oocytes as a model system. Non-cleavable VTG is internalized, but does not appear in yolk platelets. We identified two inhibitors of VTG processing into the yolk proteins: the ionophore monensin and pepstatin A, a specific inhibitor of cathepsin D. Pepstatin neither affected ligand binding and internalization, nor inhibited the degradation of nonspecifically incorporated proteins, whereas monensin inhibited all of these processes. Inhibiting VTG processing prevented its deposition into yolk platelets by strongly interfering with endosome-yolk platelet fusion. Monensin treatment resulted in morphologically abnormal endosomes, while pepstatin only inhibited VTG cleavage and the subsequent fusion of endosomes with yolk platelets. Since VTG cleavage is initiated prior to its deposition in platelets, we postulate that ligand proteolysis could be necessary for normal endosomal targeting. 相似文献
17.
Cathepsin D Activity in the Vitellogenesis of Xenopus laevis 总被引:3,自引:3,他引:0
An ovarian extract of Xenopus laevis exhibited in SDS-PAGE analyses an activity cleaving vitellogenin to lipovitellins under mildly acidic conditions. This activity was pepstatin-sensitive and inhibited by monospecific anti-rat liver cathepsin D antibody and thus identified as cathepsin D. Immunoblot analysis showed that two proteins of 43 kDa and 36 kDa immunoreacted with the antibody.
Immunocytochemical staining revealed that the enzyme was located in the cortical cytoplasm of stage I and II oocytes and in small yolk platelets and nascent forms of large yolk platelets in the cortical cytoplasm of stage III oocytes. In stage IV and V oocytes, small yolk platelets retained the immuno-staining but large yolk platelets decreased it. No immuno-positive signals were observed in oocytes at stage VI. When examined by immunoelectron microscopy, gold particles indicated that cathepsin D was located on dense lamellar bodies in the cortical cytoplasm of stage I and II oocytes. The particles were located on primordial yolk platelets and on the superficial layer of small yolk platelets in stage III oocytes, while they were sparse or not present at all on large yolk platelets in stage IV and V oocytes. These results indicate that cathepsin D plays a key role in vitellogenesis by cleaving endocytosed vitellogenin to yolk proteins in developing oocytes. 相似文献
Immunocytochemical staining revealed that the enzyme was located in the cortical cytoplasm of stage I and II oocytes and in small yolk platelets and nascent forms of large yolk platelets in the cortical cytoplasm of stage III oocytes. In stage IV and V oocytes, small yolk platelets retained the immuno-staining but large yolk platelets decreased it. No immuno-positive signals were observed in oocytes at stage VI. When examined by immunoelectron microscopy, gold particles indicated that cathepsin D was located on dense lamellar bodies in the cortical cytoplasm of stage I and II oocytes. The particles were located on primordial yolk platelets and on the superficial layer of small yolk platelets in stage III oocytes, while they were sparse or not present at all on large yolk platelets in stage IV and V oocytes. These results indicate that cathepsin D plays a key role in vitellogenesis by cleaving endocytosed vitellogenin to yolk proteins in developing oocytes. 相似文献
18.
Characterization of rat ovarian lutropin receptor. Role of thiol groups in receptor association 总被引:2,自引:0,他引:2
Rat ovarian lutropin receptor occurs predominantly as a monomer of an apparent molecular mass of 70 or 80 kDa determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing and reducing conditions, respectively. The receptor contains 0.4% free cysteine and 1.9% cysteine as cystine, determined by amino acid analysis of the S-carboxymethyl receptor prepared before and after reduction. The presence of free thiol groups was further shown by the specific adsorption of the receptor on p-chloromercuribenzoate-agarose and its susceptibility to 3H labeling with [3H]N-ethylmaleimide or [3H]iodoacetic acid. The receptor readily undergoes association into homo-oligomers. Evidence suggests that the association was caused by the intermolecular oxidation of the free -SH groups to form disulfide bonds. The aggregation could be induced by H2O2 or molecular O2 and was inhibited by sulfhydryl protecting agents such as N-ethylmaleimide, iodoacetic acid, dithiothreitol, cysteine, and Zn(II). The oligomers could be dissociated by reduction into a monomer. 125I-Labeling of the S-carboxymethyl- or N-ethylmaleyl receptor gave a single band of molecular mass 70 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Furthermore, S-alkylation of the receptor did not affect its binding to the ligand. On reduction, however, it lost its ability to bind to the ligand, but the reduced receptor retained its ability to bind to a specific polyclonal rabbit antireceptor antibody indicating the separation of the ligand and antibody binding sites. Endoproteinase Glu-C cleaved the receptor at a single glutamyl residue to give two components, 46 and 36 kDa. The 36-kDa component was extracellularly located since it contained the carbohydrate. On deglycosylation with endoglycosidase F, it yielded two components, 27 and 25 kDa. The deglycosylation of the reduced intact receptor (80 kDa) with endoglycosidase F occurred in two steps giving 73- and 64-kDa polypeptides, indicating the presence of about 20% carbohydrate contained in two or more N-linked chains. 相似文献
19.
Angiotensin II (Ang II) receptor subtypes AT1 and AT2 share 34% overall homology, but the least homology is in their third intracellular loop (3rd ICL). In an attempt to elucidate the role of the 3rd ICL in determining the similarities and differences in the functions of the AT1 and the AT2 receptors, we generated a chimeric receptor in which the 3rd ICL of the AT2 receptor was replaced with that of the AT1 receptor. Ligand-binding properties and signaling properties of this receptor were assayed by expressing this receptor in Xenopus oocytes. Ligand-binding studies using [125I-Sar1-Ile8] Ang II, a peptidic ligand that binds both the AT1 and the AT2 receptor subtypes, and 125I-CGP42112A, a peptidic ligand that is specific for the AT2 receptor, showed that the chimeric receptor has lost affinity to both ligands. However, IP3 levels of the oocytes expressing the chimeric receptor were comparable to the IP3 levels of the oocytes expressing the AT1 receptor, suggesting that the chimeric receptors could couple to phospholipase C pathway in response to Ang II. We have shown previously that the nature of the amino acid present in the position 215 located in the fifth transmembrane domain (TMD) of the AT2 receptor plays an important role in determining its affinity to different ligands. Our results from the ligand-binding studies of the chimeric receptor further support the idea that the structural organization of the region spanning the 5th TMD and the 3rd ICL of the AT2 receptor has an important role in determining the ligand-binding properties of this receptor. 相似文献
20.
To characterize the thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor on baboon platelets the binding of [125I]BOP was studied. [125I]BOP bound to washed baboon platelets in a saturable manner. Scatchard analysis of binding isotherms revealed a Kd of 1.12 +/- 0.08 nM and a binding capacity of 54 +/- 5 fmoles/10(8) platelets (326 sites/platelet). Several TXA2/PGH2 agonists and antagonists displaced [125I]BOP from its baboon platelet binding site with a rank order of potency similar to human platelets: I-BOP greater than SQ29548 greater than U46619 = I-PTA-OH greater than PTA-OH. I-BOP aggregated washed baboon platelets with an EC50 of 10 +/- 4 nM. The results indicate that [125I]BOP binds to the TXA2/PGH2 receptor on baboon platelets and that this receptor is similar to its human counterpart. 相似文献