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1.
The internalization signal and the phosphorylation site of transferrin receptor are distinct from the main basolateral sorting information. 总被引:11,自引:3,他引:8
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Wild-type human transferrin receptor (hTfR), like endogenous canine receptor, is expressed almost exclusively (97%) at the basolateral membrane of transfected Madin-Darbey canine kidney (MDCK) cells. We investigated the role of two distinct features of the hTfR cytoplasmic domain, namely the endocytic signal and the unique phosphorylation site, in polarized cell surface delivery. Basolateral location was not altered by point mutation of Ser24-->Ala24, indicating that phosphorylation is not involved in vectorial sorting of hTfR. The steady state distribution of hTfR was partially affected by a deletion of 36 cytoplasmic residues encompassing the internalization sequence. However, 80% of the receptors were still basolateral. As assessed by pulse-chase experiments in combination with biotinylation, newly synthesized wild-type and deletion mutant receptors were directly sorted to the domain of their steady state residency. Although both receptors could bind human transferrin, endocytosis of the deletion mutant was strongly impaired at either surface. These data indicate that the predominant basolateral targeting signal of hTfR is independent of the internalization sequence. 相似文献
2.
Recombinant human adenovirus: targeting to the human transferrin receptor improves gene transfer to brain microcapillary endothelium 总被引:6,自引:0,他引:6
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Some inborn errors of metabolism due to deficiencies of soluble lysosomal enzymes cause global neurodegenerative disease. Representative examples include the infantile and late infantile forms of the ceroid lipofuscinoses (CLN1 or CLN2 deficiency, respectively) and mucopolysaccharidoses type VII (MPS VII), a deficiency of beta-glucuronidase. Treatment of the central nervous system component of these disorders will require widespread protein or enzyme replacement, either through dissemination of the protein or through dissemination of a gene encoding it. We hypothesize that transduction of brain microcapillary endothelium (BME) with recombinant viral vectors, with secretion of enzyme product basolaterally, could allow for widespread enzyme dissemination. To achieve this, viruses should be modified to target the BME. This requires (i) identification of a BME-resident target receptor, (ii) identification of motifs targeted to that molecule, (iii) the construction of modified viruses to allow for binding to the target receptor, and (iv) demonstrated transduction of receptor-expressing cells. In proof of principal experiments, we chose the human transferrin receptor (hTfR), a molecule found at high density on human BME. A nonamer phage display library was panned for motifs which could bind hTfR. Forty-three clones were sequenced, most of which contained an AKxxK/R, KxKxPK/R, or KxK motif. Ten peptides representative of the three motifs were cloned into the HI loop of adenovirus type 5 fiber. All motifs tested retained their ability to trimerize and bind transferrin receptor, and seven allowed for recombinant adenovirus production. Importantly, the fiber-modified viruses facilitated increased gene transfer (2- to 34-fold) to hTfR expressing cell lines and human brain microcapillary endothelia expressing high levels of endogenous receptor. Our data indicate that adenoviruses can be modified in the HI loop for expanded tropism to the hTfR. 相似文献
3.
W V Williams T Kieber-Emmons J VonFeldt M I Greene D B Weiner 《The Journal of biological chemistry》1991,266(8):5182-5190
The variable regions of antibody molecules bind antigens with high affinity and specificity. This binding is imparted largely by the hypervariable portions of the variable region. Hypervariable regions typically fold into reverse turn or loop structures. Peptides derived from antibody hypervariable region sequences can bind antigens with similar specificity, albeit with markedly lower affinity. In this study, cyclic and dimeric peptide analogs of an anti-idiotypic/antireceptor antibody hypervariable region were developed. This antibody (87.92.6) binds to reovirus type 3 receptors on cells as well as to a neutralizing anti-reovirus type 3 monoclonal antibody (9B.G5). The cyclic peptides were utilized to probe the optimal conformation for binding to both the receptor and 9B.G5. By dimerizing or constraining the conformation of these peptides, higher affinity binding was produced. By utilizing several different cyclic peptides, the optimal conformation for binding was established. The conformationally optimized cyclic peptide possessed greater than 40-fold higher affinity for the receptor and the idiotype than the linear analog. This study suggests that conformationally constrained and dimeric peptides derived from antibody hypervariable loop sequences can bind antigens (including receptors) with reasonable affinity. hypervariable loop sequences can bind antigens (including 相似文献
4.
Residues in the apical domain of the feline and canine transferrin receptors control host-specific binding and cell infection of canine and feline parvoviruses
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Canine parvovirus (CPV) and feline panleukopenia virus (FPV) capsids bind to the transferrin receptors (TfRs) of their hosts and use these receptors to infect cells. The binding is partially host specific, as FPV binds only to the feline TfR, while CPV binds to both the canine and feline TfRs. The host-specific binding is controlled by a combination of residues within a raised region of the capsid. To define the TfR structures that interact with the virus, we altered the apical domain of the feline or canine TfR or prepared chimeras of these receptors and tested the altered receptors for binding to FPV or CPV capsids. Most changes in the apical domain of the feline TfR did not affect binding, but replacing Leu221 with Ser or Asp prevented receptor binding to either FPV or CPV capsids, while replacing Leu221 with Lys resulted in a receptor that bound only to CPV but not to FPV. Analysis of recombinants of the feline and canine TfRs showed that sequences controlling CPV-specific binding were within the apical domain and that more than one difference between these receptors determined the CPV-specific binding of the canine TfR. Single changes within the canine TfR which removed a single amino acid insertion or which eliminated a glycosylation site gave that receptor the expanded ability to bind to FPV and CPV. In some cases, binding of capsids to mutant receptors did not result in infection, suggesting a structural role for the receptor in cell infection by the viruses. 相似文献
5.
Fluorescence-activated cell-sorting analysis of fibronectin peptides binding to Trypanosoma cruzi trypomastigotes 总被引:1,自引:0,他引:1
M A Quaissi J P Kusnierz H Gras-Masse H Drobecq P Velge J Cornette A Capron A Tartar 《The Journal of protozoology》1988,35(1):111-114
The binding of synthetic peptides modeled from the sequence of the cell attachment site of fibronectin to T. cruzi trypomastigote surface receptors was investigated by fluorescence-activated cell-sorting analysis using fluorescein-labeled peptides. Peptides with the sequence Arg-Gly-Asp-Ser bound to the parasite surface. A low percentage of fresh parasites recently liberated from infected fibroblasts had the capacity to bind the peptide. In contrast, these parasites showed a time-dependent several-fold increase in their ability to bind the Arg-Gly-Asp-Ser-containing peptides during extracellular incubation. From these observations, it appears that the expression of surface receptors on a particular, mature stage of the parasite parallels its ability to adhere to and infect host cells. 相似文献
6.
M. ALI OUAISSI J. P. KUSNIERZ H. GRAS-MASSE H. DROBECQ P. VELGE J. CORNETTE A. CAPRON A. TARTAR 《The Journal of eukaryotic microbiology》1988,35(1):111-114
ABSTRACT. The binding of synthetic peptides modeled from the sequence of the cell attachment site of fibronectin to T. cruzi trypomastigote surface receptors was investigated by fluorescence-activated cell-sorting analysis using fluorescein-labeled peptides. Peptides with the sequence Arg-Gly-Asp-Ser bound to the parasite surface. A low percentage of fresh parasites recently liberated from infected fibroblasts had the capacity to bind the peptide. In contrast, these parasites showed a time-dependent several-fold increase in their ability to bind the Arg-Gly-Asp-Ser-containing peptides during extracellular incubation. From these observations, it appears that the expression of surface receptors on a particular, mature stage of the parasite parallels its ability to adhere to and infect host cells. 相似文献
7.
A Hirose-Kumagai N Akamatsu 《Biochemical and biophysical research communications》1989,164(3):1105-1112
Partial hepatectomy results in an increase in the ability of the liver cells to bind 125I-labeled transferrin to surface receptors. Scatchard analysis of the binding of transferrin in regenerating rat liver indicates that this increase was due to an increase in the number of transferrin receptors on the cell surface. When we measured total cellular transferrin receptor number, we found that control and regenerating livers had identical amounts of receptor number. The increase in the surface receptor number is apparently due to the translocation of intracellular transferrin receptor to the cell surface. 相似文献
8.
9.
Limin Yu Peggy Shuang Yu Elizabeth Yee Yen Mui Jennifer C. McKelvie Thi Phuong Tu Pham Yan Wen Yap Wan Qing Wong Jiawen Wu Weiqiao Deng Brendan P. Orner 《Bioorganic & medicinal chemistry》2009,17(13):4825-4832
A novel selection approach is presented to screen phage display peptide libraries against sets of receptors that share specificity for the same ligand. This strategy was applied to the discovery of glycomimetic peptides. Through these screens, a number of peptide clones were discovered that bind the lectins used in the screen, in a sugar competitive manner. In addition, the majority of the selected peptides demonstrate sugar type mimicry consistent with lectin specificity. Docking studies were conducted to establish whether the mimetic peptides bind to the lectin ConA at the sugar binding site or to a nearby, alternative site shown to bind to YPY-containing peptides previously discovered from single-target screens. Of the three cyclic peptides subjected to computational docking, CNTPLTSRC had the highest predicted affinity and CSRILTAAC demonstrated specificity for the sugar binding site comparable to the natural ligand itself. 相似文献
10.
Root-Bernstein R 《Journal of theoretical biology》2002,218(1):71-84
Dwyer has suggested that peptide receptors evolved from self-aggregating peptides so that peptide receptors should incorporate regions of high homology with the peptide ligand. If one considers self-aggregation to be a particular manifestation of molecular complementarity in general, then it is possible to extend Dwyer's hypothesis to a broader set of peptides: complementary peptides that bind to each other. In the latter case, one would expect to find homologous copies of the complementary peptide in the receptor. Thirteen peptides, 10 of which are not known to self-aggregate (amylin, ACTH, LHRH, angiotensin II, atrial natriuretic peptide, somatostatin, oxytocin, neurotensin, vasopressin, and substance P), and three that are known to self-aggregate (insulin, glucagon, and gastrin), were chosen. In addition to being self-aggregating, insulin and glucagon are also known to bind to each other, making them a mutually complementary pair. All possible combinations of the 13 peptides and the extracellular regions of their receptors were investigated using bioinformatic tools (a total of 325 combinations). Multiple, statistically significant homologies were found for insulin in the insulin receptor; insulin in the glucagon receptor; glucagon in the glucagon receptor; glucagon in the insulin receptor; and gastrin in gastrin binding protein and its receptor. Most of these homologies are in regions or sequences known to contribute to receptor binding of the respective hormone. These results suggest that the Dwyer hypothesis for receptor evolution may be generalizable beyond self-aggregating to complementary peptides. The evolution of receptors may have been driven by small molecule complementarity augmented by modular evolutionary processes that left a "molecular paleontology" that is still evident in the genome today. This "paleontology" may allow identification of peptide receptor sites. 相似文献
11.
Type I and II insulin-like growth factor receptors on human phytohemagglutinin-activated T lymphocytes 总被引:1,自引:0,他引:1
R W Kozak J F Haskell L A Greenstein M M Rechler T A Waldmann S P Nissley 《Cellular immunology》1987,109(2):318-331
Human T cells activated with mitogens, antigens, or antibodies to the T-cell receptor complex acquire a cascade of new receptors, including the receptors for interleukin-2, transferrin, and insulin. We investigated whether receptors for insulin-like growth factors (IGF) also were expressed on activated T cells. Based on competitive binding studies, immunoprecipitation of labeled cell surface receptors and blocking of radiolabeled peptide binding by a specific monoclonal antibody (alpha IR-3) to the type I IGF receptor, as well as affinity crosslinking of radiolabeled peptides to their receptors, we concluded that both type I and type II IGF receptors are expressed on activated T cells. A specific binding site for IGF-II also was observed on the type I IGF receptor which was not inhibited by alpha IR-3. Receptors for IGF were more numerous on activated T cells than on resting T cells, and their peak expression appeared by the peak of DNA synthesis. Thus, human activated T cells were shown to express both type I and II IGF receptors which could potentially play a role in the regulation of T-cell proliferation, differentiation, and function. 相似文献
12.
Human transferrin, a bilobal protein, with each lobe bearing a single iron-binding site, functions to transport iron into cells. While the N-terminal lobe alone does not measurably bind cellular transferrin receptors or serve as an iron donor for cells, the C-lobe is capable of both functions. We used hydroxyl radical-mediated protein footprinting and mass spectrometry to reveal the conformational changes that occur upon complex formation for the human transferrin C-lobe (residues 334-679) bound to the ectodomain of human transferrin receptor 1 (residues 121-760). Oxidation rates for proteolytic peptides in the C-lobe, the receptor, and their complex have been measured by mass spectrometry; upon formation of the complex, a dramatic decrease in modification rates, indicating protection of specific side chain groups, can be seen in C-lobe sequences corresponding to residues 381-401, 415-433, and 457-470. Peptide sequences experiencing modification rate decreases in the transferrin receptor upon C-lobe binding include residues 232-240, 365-371, 496-508, 580 and 581, 614-623, 634-646, 647-681, and 733-760. In addition, several peptides in the receptor exhibit enhancements in the rate of modification consistent with allosteric effects of complex formation. Using tandem mass spectrometry, the sites of modification with altered reactivity in the complex include Met382, Met389, Trp460, Met464, and Phe427 in the C-lobe and Tyr503, Pro581, Tyr611, Leu619, Met635, Phe650, Trp740, Trp754, and Phe760 within the transferrin receptor. Using available genetic, biochemical, and structural data, we confirm that the conserved RGD sequence (residues 646-648) in the helical domain of the transferrin receptor, including residues from Leu619 to Phe650, is a primary binding site for the transferrin C-lobe. 相似文献
13.
Antibodies to the autophosphorylation sites of the epidermal growth factor receptor protein-tyrosine kinase as probes of structure and function. 总被引:12,自引:1,他引:11
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Antisera were prepared against three synthetic peptides with amino acid sequences identical to those surrounding the three major autophosphorylation sites of the epidermal growth factor (EGF) receptor. The affinity-purified antibodies reacted strongly in an enzyme-linked immunosorbent assay against the immunizing peptide but showed little cross-reaction with the other two phosphorylation site peptides. EGF receptors labelled by autophosphorylation could be specifically precipitated by each of the phosphorylation site antibodies. The antibodies recognised EGF receptors labelled at each of the autophosphorylation sites, indicating that they could bind to the immunizing sequences irrespective of their states of phosphorylation. The antibodies were able to inhibit EGF receptor autophosphorylation without affecting EGF-stimulated tyrosine kinase activity towards exogenous peptide substrates, suggesting that the kinase and autophosphorylation sites were in distinct domains. Immunofluorescent staining of A431 cells showed that the autophosphorylation site sequences resided inside the cell. The autophosphorylation sites were shown to be within a domain of 20 000 mol. wt. which could be cleaved from the receptor through limited proteolysis by the calcium-dependent protease, calpain. The position of cleavage of the EGF receptor by the protease was mapped to lie between residues 996 and 1059. These results are discussed in the context of a model for the structure and function of the human EGF receptor. 相似文献
14.
We applied fluorescence correlation spectroscopy (FCS) to characterize the interaction dynamics of fluorescence-labeled transferrin with transferrin receptor (hTfR) associates isolated from human placenta. The dissociation constant for the equilibrium binding of TMR-labeled ferri-transferrin to hTfR in detergent free solution was determined to be 7 +/- 3 nM. Binding curves were compatible with equal and independent binding sites present on the hTfR associates. Under pseudo-first-order conditions, with respect to transferrin, complex formation is monophasic. From these curves, association and dissociation rate constants for a reversible bimolecular binding reaction were determined, with (1.1 +/- 0.1) x 10(4) M-1 s-1 for the former and (6 +/- 4) x 10(-)4 s-1 for the latter. In dissociation exchange experiments, biphasic curves and concentration-independent reciprocal relaxation times were determined. From isothermal titration calorimetry experiments, we obtained an enthalpy change of -44.4 kJ/mol associated with the reaction. We thus conclude that the reaction is mainly enthalpy driven. 相似文献
15.
H Jüppner A B Abou-Samra S Uneno W X Gu J T Potts G V Segre 《The Journal of biological chemistry》1988,263(18):8557-8560
[Tyr36]human adenylate cyclase stimulating peptide (1-36)-NH2, an amino-terminal analog of a tumor peptide which is associated with hypercalcemia of malignancy, and [Nle8, Nle18, Tyr34]bovine parathyroid hormone (PTH)-(1-34)-NH2 both bind with similar affinities to receptors on rat osteosarcoma cells, ROS 17/2.8, when either of the peptides is used as the radioligand. Pretreatment of the cells with either peptide down-regulates available binding sites for either radioligand and desensitizes the cAMP accumulation stimulated by either peptide. Prior exposure of the cells to dexamethasone increases these responses to both peptides. Photoderivatized radioiodinated [Tyr36]human adenylate cyclase-stimulating peptide (1-36)-NH2 and [Nle8, Nle18, Tyr34]bovine PTH-(1-34)-NH2 both specifically label a Mr = 80,000 membrane protein on ROS 17/2.8 cells. The intensity of labeling this receptor band by either photoprobe is reduced by co-incubation with either peptide over the same dose range. Equivalent dose-dependent down-regulation of receptors which bind both photoprobes is also found when ROS 17/2.8 cells are preincubated with either peptide. Dexamethasone increases the intensity of receptor labeling. Our findings strongly indicate that both peptides recognize the same plasma membrane receptor on ROS 17/2.8 cells. Although the physiological function(s) of human adenylate cyclase-stimulating peptide is unknown, these results could explain why its biological actions on mineral ion metabolism so closely simulate those of PTH and raise interesting questions about the general biological and evolutionary significance of the use of the same receptor by chemically distinct peptides. 相似文献
16.
Ortiz-Zapater E Soriano-Ortega E Marcote MJ Ortiz-Masiá D Aniento F 《The Plant journal : for cell and molecular biology》2006,48(5):757-770
Plant cells possess much of the molecular machinery necessary for receptor-mediated endocytosis (RME), but this process still awaits detailed characterization. In order to identify a reliable and well-characterized marker to investigate RME in plant cells, we have expressed the human transferrin receptor (hTfR) in Arabidopsis protoplasts. We have found that hTfR is mainly found in endosomal (Ara7- and FM4-64-positive) compartments, but also at the plasma membrane, where it mediates binding and internalization of its natural ligand transferrin (Tfn). Cell surface expression of hTfR increases upon treatment with tyrphostin A23, which inhibits the interaction between the YTRF endocytosis signal in the hTfR cytosolic tail and the mu2-subunit of the AP2 complex. Indeed, tyrphostin A23 inhibits Tfn internalization and redistributes most of hTfR to the plasma membrane, suggesting that the endocytosis signal of hTfR is functional in Arabidopsis protoplasts. Co-immunoprecipitation experiments show that hTfR is able to interact with a mu-adaptin subunit from Arabidopsis cytosol, a process that is blocked by tyrphostin A23. In contrast, treatment with brefeldin A, which inhibits recycling from endosomes back to the plasma membrane in plant cells, leads to the accumulation of Tfn and hTfR in larger patches inside the cell, reminiscent of BFA compartments. Therefore, hTfR has the same trafficking properties in Arabidopsis protoplasts as in animal cells, and cycles between the plasma membrane and endosomal compartments. The specific inhibition of Tfn/hTfR internalization and recycling by tyrphostin A23 and BFA, respectively, thus provide valuable molecular tools to characterize RME and the recycling pathway in plant cells. 相似文献
17.
Flexible peptides that fold upon binding to another protein molecule mediate a large number of regulatory interactions in the living cell and may provide highly specific recognition modules. We present Rosetta FlexPepDock ab-initio, a protocol for simultaneous docking and de-novo folding of peptides, starting from an approximate specification of the peptide binding site. Using the Rosetta fragments library and a coarse-grained structural representation of the peptide and the receptor, FlexPepDock ab-initio samples efficiently and simultaneously the space of possible peptide backbone conformations and rigid-body orientations over the receptor surface of a given binding site. The subsequent all-atom refinement of the coarse-grained models includes full side-chain modeling of both the receptor and the peptide, resulting in high-resolution models in which key side-chain interactions are recapitulated. The protocol was applied to a benchmark in which peptides were modeled over receptors in either their bound backbone conformations or in their free, unbound form. Near-native peptide conformations were identified in 18/26 of the bound cases and 7/14 of the unbound cases. The protocol performs well on peptides from various classes of secondary structures, including coiled peptides with unusual turns and kinks. The results presented here significantly extend the scope of state-of-the-art methods for high-resolution peptide modeling, which can now be applied to a wide variety of peptide-protein interactions where no prior information about the peptide backbone conformation is available, enabling detailed structure-based studies and manipulation of those interactions. 相似文献
18.
C R Beddell G C Sheppey T L Blundell K Sasaki S Dockerill P J Goodford 《International journal of peptide and protein research》1977,9(3):161-165
Symmetrical features were observed in the amino acid sequences of some biologically active peptides. It is suggested that this approximate symmetry is reflected in the conformations of the peptides at their respective biological receptors, and has arisen by natural selection as both peptides and receptors evolved to optimise their mutual fit. It follows that the binding site for each peptide at its receptor would share the same symmetry element. This would arise if the peptide binds to two symmetrically related similar or identical submits in the receptor. 相似文献
19.
Novel short peptides isolated from phage display library inhibit vascular endothelial growth factor activity 总被引:1,自引:0,他引:1
Erdag B Balcioglu KB Kumbasar A Celikbicak O Zeder-Lutz G Altschuh D Salih B Baysal K 《Molecular biotechnology》2007,35(1):51-63
Signal transduction through the vascular endothelial growth factor (VEGF)-VEGF receptor (VEGFR) pathway has a pivotal importance
in angiogenesis, and has therefore become a prime target in antitumor therapy. In search for peptides antagonizing VEGF binding
to its receptors, we screened a random heptamer library displayed on phage for peptides that bind the whole VEGF165 molecule and inhibit VEGF dependent human umbilical vein endothelial cell (HUVEC) proliferation. Two selected peptides with
sequences WHLPFKC and WHKPFRF were synthesized. Biacore and matrix-assisted laser desorption/ionization time-of-flight mass
spectrometry analysis indicated that these peptides bind the VEGF homodimer in a concentration-dependent manner, with micromolar
affinity, and with a 2:1 peptide: VEGF stoichiometry. They inhibited HUVEC proliferation in vitro by 77 and 55%, respectively.
Taken together, our results indicate that these peptides could be potent inhibitors of angiogenesis. Furthermore, we show
that the peptide-VEGF binding properties can be quantified, a prerequisite for the further optimization of binders. 相似文献
20.
Acquisition of the functional properties of the transferrin receptor during its biosynthesis 总被引:1,自引:0,他引:1
C A Enns E M Clinton C L Reckhow B J Root S I Do C Cook 《The Journal of biological chemistry》1991,266(20):13272-13277
The properties of the newly synthesized and partially glycosylated forms of the transferrin receptor were examined to determine which co- and post-translational modifications are necessary for the acquisition of transferrin binding activity and transport of the receptor to the cell surface. The nascent transferrin receptor containing core-glycosylated asparagine-linked oligosaccharides does not possess complete intersubunit disulfide bonds, sediments predominantly as a monomer in sucrose density gradients, and shows reduced binding to transferrin-agarose. Within 20-30 min after synthesis, the transferrin receptor acquires the ability to bind to a transferrin-linked affinity column. Intersubunit disulfide bond formation occurs slowly throughout the transit of the receptor to the cell surface. These results indicate that core glycosylation of the receptor may be necessary but is not sufficient for the acquisition of the ability of the receptor to bind transferrin and that intersubunit disulfide bond formation is a post-translational event. Inhibition of complex carbohydrate synthesis by either swainsonine (1 micrograms/ml) or deoxynojirimycin (4 mM) does not inhibit the ability of this receptor to form intersubunit disulfide bonds or to be transported to the cell surface. The partially glycosylated receptor, however, does show an approximately 3-fold reduced affinity for transferrin. 相似文献