scholarly journals The first cysteine-rich domain of the receptor GFRα1 stabilizes the binding of GDNF

2005 ◽  
Vol 387 (3) ◽  
pp. 817-824 ◽  
Author(s):  
Heidi VIRTANEN ◽  
Jianmin YANG ◽  
Maxim M. BESPALOV ◽  
Jukka O. HILTUNEN ◽  
Veli-Matti LEPPÄNEN ◽  
...  
Keyword(s):  
Binding Capacity ◽  
Full Length ◽  
Tyrosine Kinase Receptor ◽  
Soluble Receptors ◽  
Truncated Receptor ◽  
Rich Domain ◽  
Low Concentrations ◽  
Optimal Function ◽  
High Concentrations ◽  
Cysteine Rich Domain

The GDNF (glial cell line-derived neurotrophic factor)-binding receptor GFRα1 (GDNF family receptor α1) is attached to the membrane by a GPI (glycosylphosphatidylinositol) anchor and consists of three cysteine-rich domains. The region corresponding to the second and third domains has been shown previously to participate in ligand binding, and to interact with the transmembrane tyrosine kinase receptor RET. No function has so far been found for the N-terminal, first domain (D1). Here we show that the GPI-anchored full-length receptor binds 125I-GDNF two times more tightly than does a GPI-anchored truncated receptor lacking D1. Scintillation proximity assays with purified receptor proteins also show that the GDNF-binding capacity of the soluble full-length GFRα1 is two times higher than the GDNF-binding capacity of the soluble D1-truncated GFRα1. As RET stabilizes the binding of GDNF equally well to the full-length and truncated receptors, D1 seems not to be involved in the interaction between GFRα1 and RET. Moreover, soluble full-length GFRα1 mediates GDNF-promoted neurite outgrowth in PC6-3 cells more efficiently than the soluble truncated GFRα1 protein. At low concentrations, the soluble fulllength receptor mediates the phosphorylation of RET more efficiently than the soluble truncated receptor. However, when the receptors are overexpressed on the cell surface as GPI-anchored proteins, or added to the growth medium at high concentrations as soluble proteins, full-length and truncated GFRα1 are indistinguishable in GDNF-dependent RET-phosphorylation assays. High levels of the receptors can thus mask a slightly impaired function in the phosphorylation assay. Based on assays with both GPI-anchored and soluble receptors, we therefore conclude that D1 contributes to the optimal function of GFRα1 by stabilizing the interaction between GFRα1 and GDNF.

scholarly journals Involvement of the Zinc-Binding Capacity of Sendai Virus V Protein in Viral Pathogenesis

2000 ◽  
Vol 74 (17) ◽  
pp. 7834-7841 ◽  
Author(s):  
Cheng Huang ◽  
Katsuhiro Kiyotani ◽  
Yutaka Fujii ◽  
Noriko Fukuhara ◽  
Atsushi Kato ◽  
...  
Keyword(s):  
Fusion Proteins ◽  
Sendai Virus ◽  
Binding Capacity ◽  
Viral Pathogenesis ◽  
Growth Patterns ◽  
Zinc Binding ◽  
Wild Type ◽  
V Protein ◽  
Rich Domain ◽  
Cysteine Rich Domain

ABSTRACT The V protein of Sendai virus (SeV) is nonessential to virus replication in cell culture but indispensable to viral pathogenicity in mice. The highly conserved cysteine-rich zinc finger-like domain in its carboxyl terminus is believed to be responsible for this viral pathogenicity. In the present study, we showed that the cysteine-rich domain of the SeV V protein could actually bind zinc by using glutathione-S-transferase fusion proteins. When the seven conserved cysteine residues at positions 337, 341, 353, 355, 358, 362, and 365 were replaced individually, the zinc-binding capacities of the mutant proteins were greatly impaired, ranging from 22 to 68% of that of the wild type. We then recovered two mutant SeVs from cDNA, which have V-C341S and V-C365R mutations and represent maximal and minimal zinc-binding capacities among the corresponding mutant fusion proteins, respectively. The mutant viruses showed viral protein synthesis and growth patterns similar to those of wild-type SeV in cultured cells. However, the mutant viruses were strongly attenuated in mice in a way similar to that of SeV VΔC, which has a truncated V protein lacking the cysteine-rich domain, by exhibiting earlier viral clearance from the mouse lung and less virulence to mice. We therefore conclude that the zinc-binding capacity of the V protein is involved in viral pathogenesis.

scholarly journals Stabilization of the bioactivity of tumor necrosis factor by its soluble receptors.

1992 ◽  
Vol 175 (2) ◽  
pp. 323-329 ◽  
Author(s):  
D Aderka ◽  
H Engelmann ◽  
Y Maor ◽  
C Brakebusch ◽  
D Wallach
Keyword(s):  
Tumor Necrosis Factor ◽  
Tumor Necrosis ◽  
Prolonged Treatment ◽  
Tnf Receptors ◽  
Soluble Receptors ◽  
Low Concentrations ◽  
High Concentrations ◽  
Activity Decay ◽  
Necrosis Factor

The receptors for tumor necrosis factor (TNF) exist in cell-associated as well as soluble forms, both binding specifically to TNF. Since the soluble forms of TNF receptors (sTNF-Rs) can compete with the cell-associated TNF receptors for TNF, it was suggested that they function as inhibitors of TNF activity; at high concentrations, the sTNF-Rs indeed inhibit TNF effects. However, we report here that in the presence of low concentrations of the sTNF-Rs, effects of TNF whose induction depend on prolonged treatment with this cytokine are augmented, reflecting an attenuation by the sTNF-Rs of spontaneous TNF activity decay. Evidence that this stabilization of TNF activity by the sTNF-Rs follows from stabilization of TNF structure within the complexes that TNF forms with the sTNF-Rs is presented here, suggesting that the sTNF-Rs can affect TNF activity not only by interfering with its binding to cells but also by stabilizing its structure and preserving its activity, thus augmenting some of its effects.

scholarly journals The amyloid precursor protein is a conserved Wnt receptor

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Tengyuan Liu ◽  
Tingting Zhang ◽  
Maya Nicolas ◽  
Lydie Boussicault ◽  
Heather Rice ◽  
...  
Keyword(s):  
Amyloid Precursor Protein ◽  
Cortical Neurons ◽  
Neuronal Development ◽  
Full Length ◽  
Precursor Protein ◽  
Dependent Manner ◽  
Protein Levels ◽  
Rich Domain ◽  
Wnt Ligands ◽  
Cysteine Rich Domain

The Amyloid Precursor Protein (APP) and its homologues are transmembrane proteins required for various aspects of neuronal development and activity, whose molecular function is unknown. Specifically, it is unclear whether APP acts as a receptor, and if so what its ligand(s) may be. We show that APP binds the Wnt ligands Wnt3a and Wnt5a and that this binding regulates APP protein levels. Wnt3a binding promotes full-length APP (flAPP) recycling and stability. In contrast, Wnt5a promotes APP targeting to lysosomal compartments and reduces flAPP levels. A conserved Cysteine-Rich Domain (CRD) in the extracellular portion of APP is required for Wnt binding, and deletion of the CRD abrogates the effects of Wnts on flAPP levels and trafficking. Finally, loss of APP results in increased axonal and reduced dendritic growth of mouse embryonic primary cortical neurons. This phenotype can be cell-autonomously rescued by full length, but not CRD-deleted, APP and regulated by Wnt ligands in a CRD-dependent manner.

scholarly journals The Amyloid Precursor Protein is a conserved Wnt receptor

2021 ◽  
Author(s):  
Tengyuan Liu ◽  
Maya Nicolas ◽  
Tingting Zhang ◽  
Heather Rice ◽  
Alessia Soldano ◽  
...  
Keyword(s):  
Amyloid Precursor Protein ◽  
Cortical Neurons ◽  
Dendritic Growth ◽  
Neuronal Development ◽  
Full Length ◽  
Precursor Protein ◽  
Protein Levels ◽  
Rich Domain ◽  
Wnt Ligands ◽  
Cysteine Rich Domain

SUMMARYThe Amyloid Precursor Protein (APP) and its homologues are transmembrane proteins required for various aspects of neuronal development and activity, whose molecular function is unknown. Specifically, it is unclear whether APP acts as a receptor, and if so what its ligand(s) may be. We show that APP binds the Wnt ligands Wnt3a and Wnt5a and that this binding regulates APP protein levels. Wnt3a binding promotes full length APP (flAPP) recycling and stability. In contrast, Wnt5a promotes APP targeting to lysosomal compartments and reduces flAPP levels. A conserved Cysteine Rich Domain (CRD) in the extracellular portion of APP is required for Wnt binding, and deletion of the CRD abrogates the effects of Wnts on flAPP levels and trafficking. Finally, loss of APP results in increased axonal and reduced dendritic growth of mouse embryonic primary cortical neurons. This phenotype can be cell-autonomously rescued by full length, but not CRD-deleted, APP.

Crystal Structure of a Proteolytic Fragment of ADAMTS13 Reveals Alternative Disulfide Pairings in the Cysteine-Rich Domain

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3363-3363
Author(s):  
Christopher G. Skipwith ◽  
Steven Stayrook ◽  
Hanspeter Rottensteiner ◽  
Friedrich Scheiflinger ◽  
X. Long Zheng
Keyword(s):  
Disulfide Bond ◽  
Disulfide Bonds ◽  
Catalytic Domain ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Full Length ◽  
Proteolytic Fragment ◽  
Rich Domain ◽  
The Difference ◽  
Cysteine Rich Domain

Abstract Abstract 3363 Proteolytic cleavage of von Willebrand factor (VWF) by ADAMTS13 depends on its non-catalytic domain (or exosite) interactions. These exosites, particularly the residues between Tyr659 and Tyr665 and their adjacent residues Arg568 and Phe592 in the ADAMTS13 spacer domain, are the major targets of anti-ADAMTS13 autoantibodies in patients with acquired TTP. In an effort to further determine the potential exosite interactions between ADAMTS13 and VWF, we carried out crystal screening of a recombinant full-length ADAMTS13 purified from stably transfected Chinese hamster ovary cells. After over 900 conditions were tested, we obtained multiple well-formed and diffractable crystals. Upon X-ray diffraction, we discovered that only a proteolytic fragment of full-length ADAMTS13 (Met432-Arg670) had been consistently crystallized. Here, we report the structure of this non-catalytic ADAMTS13 fragment at 2.2Å. The fragment encompasses 8 residues from the first TSP-1 repeat, an entire cysteine-rich domain (CA and CB), and a majority of the spacer domain. While recombinant ADAMTS13 in the present study is fully glycosylated, the overall structure agrees very well with the analogous portion of the ADAMTS13-DTCS structure previously reported (RMSD = 0.02Å, 1817 atoms, 232 residues). A region within the CA domain does vary slightly from that in the ADAMTS13-DTCS (RMSD = 0.08Å, 379 atoms, 51 residues). As with the ADAMTS13-DTCS structure, our structure shows 3 intermolecular disulfide bonds in CA, in addition to 2 free cysteine residues. In this region, our structure shows differences in the disulfide bonding pairing, and a slight shift in the α4 helix from Gly479-Cys483. Specifically, our structure reveals an intermolecular disulfide bond between the residues Cys483 and Cys527 and Cys508 and Cys522. These disulfide pair patterns are different from those reported in the ADAMTS13-DTCS structure, in which disulfide bonds are formed between the residues Cys483 and Cys522 (4.6Å apart in our structure) and the residues Cys508 and Cys527 (3.8Å apart in our structure). Our results indicate that, despite the difference in conditions under which crystals of ADAMTS13 fragments are formed, the overall structure of the Cys-rich and spacer domains are quite similar. The difference in the disulfide bond pattering may provide a novel insight into the mechanism regarding the ADAMTS13 exosite interaction with VWF. Our ongoing work is aimed to determine the complex formation between ADAMTS13 and its substrate or autoantibodies. Disclosures: Rottensteiner: Baxter Innovations GmbH: Employment.

Neutrophil Elastase Potentiates Cathepsin G-lnduced Platelet Activation

1992 ◽  
Vol 68 (05) ◽  
pp. 570-576 ◽  
Author(s):  
Mary A Selak
Keyword(s):  
Neutrophil Elastase ◽  
Cell Activation ◽  
Thromboxane A2 ◽  
Creatine Phosphate ◽  
Dense Granule ◽  
Cathepsin G ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Low Concentrations ◽  
High Concentrations

SummaryWe have previously demonstrated that human neutrophil cathepsin G is a strong platelet agonist that binds to a specific receptor. This work describes the effect of neutrophil elastase on cathepsin G-induced platelet responses. While platelets were not activated by high concentrations of neutrophil elastase by itself, elastase enhanced aggregation, secretion and calcium mobilization induced by low concentrations of cathepsin G. Platelet aggregation and secretion were potentiated in a concentration-dependent manner by neutrophil elastase with maximal responses observable at 200 nM. Enhancement was observed when elastase was preincubated with platelets for time intervals of 10–60 s prior to addition of a low concentration of cathepsin G and required catalytically-active elastase since phenylmethanesulphonyl fluoride-inhibited enzyme failed to potentiate cell activation. Neutrophil elastase potentiation of platelet responses induced by low concentrations of cathepsin G was markedly inhibited by creatine phosphate/creatine phosphokinase and/or indomethacin, indicating that the synergism between elastase and cathepsin G required the participation of ADP and thromboxane A2. On the other hand, platelet responses were not attenuated by the PAF antagonist BN 52021, signifying that PAF-acether did not play a role in elastase potentiation. At higher concentrations porcine pancreatic elastase exhibits similar effects to neutrophil elastase, demonstrating that the effect of elastase was not unique to the neutrophil protease. While neutrophil elastase failed to alter the ability of cathepsin G to hydrolyze a synthetic chromogenic substrate, preincubation of platelets with elastase increased the apparent affinity of cathepsin G binding to platelets. In contrast to their effect on cathepsin G-induced platelet responses, neither neutrophil nor pancreatic elasatse potentiated aggregation or dense granule release initiated by ADP, PAF-acether, arachidonic acid or U46619, a thromboxane A2 mimetic. Moreover, unlike its effect on cathepsin G, neutrophil elastase inhibited thrombin-induced responses. The current observations demonstrate that elastase can potentiate platelet responses mediated by low concentrations of cathepsin G, suggesting that both enzymes may function synergistically to activate platelets under conditions where neutrophil degranulation occurs.

In Vitro Effects of Ethanol on Rabbit Platelet Aggregation, Secretion of Granule Contents, and Cyclic AMP Levels in the Presence of Prostacyclin

1989 ◽  
Vol 61 (02) ◽  
pp. 254-258 ◽  
Author(s):  
Margaret L Rand ◽  
Peter L Gross ◽  
Donna M Jakowec ◽  
Marian A Packham ◽  
J Fraser Mustard
Keyword(s):  
Cyclic Amp ◽  
Inhibitory Effect ◽  
Rabbit Platelet ◽  
Inhibitory Effects ◽  
Low Concentrations ◽  
Rabbit Platelets ◽  
High Concentrations ◽  
In Vitro Effects ◽  
Aggregation Of Platelets

SummaryEthanol, at physiologically tolerable concentrations, inhibits platelet responses to low concentrations of collagen or thrombin, but does not inhibit responses of washed rabbit platelets stimulated with high concentrations of ADP, collagen, or thrombin. However, when platelet responses to high concentrations of collagen or thrombin had been partially inhibited by prostacyclin (PGI2), ethanol had additional inhibitory effects on aggregation and secretion. These effects were also observed with aspirin- treated platelets stimulated with thrombin. Ethanol had no further inhibitory effect on aggregation of platelets stimulated with ADP, or the combination of ADP and epinephrine. Thus, the inhibitory effects of ethanol on platelet responses in the presence of PGI2 were very similar to its inhibitory effects in the absence of PGI2, when platelets were stimulated with lower concentrations of collagen or thrombin. Ethanol did not appear to exert its inhibitory effects by increasing cyclic AMP above basal levels and the additional inhibitory effects of ethanol in the presence of PGI2 did not appear to be brought about by further increases in platelet cyclic AMP levels.

Aggregation of Cat Platelets in Vitro

1970 ◽  
Vol 23 (03) ◽  
pp. 601-620 ◽  
Author(s):  
Th. B Tschopp
Keyword(s):  
Adenosine Monophosphate ◽  
Blood Collection ◽  
Base Line ◽  
Reversible Aggregation ◽  
Low Concentrations ◽  
Irreversible Aggregation ◽  
High Concentrations ◽  
Two Phases ◽  
Improved Technique

SummaryAggregation of cat platelets in the citrated plasma is examined by means of Born’s absorptiometer. A marked tendency of the platelets of this species to spontaneous aggregation necessitated first of all the development of an improved technique of blood collection.A hypothesis according to which 5-HT is released from the platelets, explains the absence of oscillations on the base line of the absorptiometer, the absence of platelet swelling, when ADP is added, and the effect of stirring on the aggregation curves in cat PRP. The average volume of cat platelets amounts to 10.46 μ3 when directly fixed in the blood, when fixed from PRP to 12.17 μ3, when fixed from stirred PRP to 13.51 μ3.In low concentrations (0.3-2 μM) ADP produce reversible aggregation; in narrowly restricted, individually dissimilar mean concentrations irreversible aggregation in two phases and in high concentrations, irreversible aggregation in one phase. Like ADP serotonin produces 2 phase irreversible aggregation in concentrations of 3-10 μM, but unlike ADP, the aggregation velocity decreases again with high 5-HT concentrations (>100 μM). Adrenaline does not produce aggregation and it is likely that adenosine and adenosine monophosphate inhibit the aggregation by serotonin but not by ADP. Species differences in the aggregation of human, rabbit and cat platelets are discussed.

The Action of an Aniline Derivative (AN 162) on Blood Coagulation and on Platelets

1971 ◽  
Vol 26 (01) ◽  
pp. 145-166
Author(s):  
E Deutsch ◽  
K Lechner ◽  
K Moser ◽  
L Stockinger
Keyword(s):  
Blood Coagulation ◽  
Citric Acid Cycle ◽  
Second Phase ◽  
Aniline Derivative ◽  
Acid Cycle ◽  
Enhancing Effect ◽  
Low Concentrations ◽  
Dual Action ◽  
High Concentrations ◽  
Induced Aggregation

Summary1. The aniline derivative AN 162, Donau Pharmazie, Linz, Austria, has a dual action on the blood coagulation: an anticoagulant and an coagulation enhancing effect.2. The anticoagulant action may only be demonstrated with high concentrations (over 1 X 10”3 M related to plasma) preferentially in PPP. It is partially caused by an inhibition of the endogenous way of generation of the prothrombin converting principle. In addition it is suggested that it interferes with the fibrinogen-fibrin reaction in a manner not yet understood.3. The coagulant action is caused by a greater availability of platelet constituents at low concentrations of AN 162 (over 1 × 10-4 M) and by the induction of a release reaction at higher concentrations. The platelet factors 3 and 4, serotonin, adenine, and acid phosphatase are released.4. AN 162 inhibits platelet aggregation. This inhibition can be demonstrated by the PAT of Breddin and in the stirred aggregation test of Born. It is more effective to inhibit the collagen-induced and the second phase of the adrenaline-induced aggregation than the ADP induced one. The platelet retention (test of Hellem) is also reduced.5. The action of AN 162 on the platelets is caused by a damage of the platelet membrane which becomes permeabel for both, soluble platelet constitutents and granula.6. AN 162 interferes with the energy metabolism of the platelets. It causes a loss of ATP, and inhibits the key-enzymes of glycolysis, citric acid cycle, fatty acid oxydation and glutathione reduction.7. AN 162 inhibits the growth of fibroblasts without influence on mitosis.

Functional Involvement of Thrombospondin in Platelet Aggregation Induced by Low Versus High Concentrations of Thrombin

1986 ◽  
Vol 55 (01) ◽  
pp. 136-142 ◽  
Author(s):  
K J Kao ◽  
David M Shaut ◽  
Paul A Klein
Keyword(s):  
Platelet Aggregation ◽  
Binding Sites ◽  
Inhibitory Effect ◽  
Activated Platelets ◽  
Low Concentrations ◽  
Platelet Binding ◽  
Functional Involvement ◽  
Thrombin Activation ◽  
Platelet Aggregates ◽  
High Concentrations

SummaryThrombospondin (TSP) is a major platelet secretory glycoprotein. Earlier studies of various investigators demonstrated that TSP is the endogenous platelet lectin and is responsible for the hemagglutinating activity expressed on formaldehyde-fixed thrombin-treated platelets. The direct effect of highly purified TSP on thrombin-induced platelet aggregation was studied. It was observed that aggregation of gel-filtered platelets induced by low concentrations of thrombin (≤0.05 U/ml) was progressively inhibited by increasing concentrations of exogenous TSP (≥60 μg/ml). However, inhibition of platelet aggregation by TSP was not observed when higher than 0.1 U/ml thrombin was used to activate platelets. To exclude the possibility that TSP inhibits platelet aggregation by affecting thrombin activation of platelets, three different approaches were utilized. First, by using a chromogenic substrate assay it was shown that TSP does not inhibit the proteolytic activity of thrombin. Second, thromboxane B2 synthesis by thrombin-stimulated platelets was not affected by exogenous TSP. Finally, electron microscopy of thrombin-induced platelet aggregates showed that platelets were activated by thrombin regardless of the presence or absence of exogenous TSP. The results indicate that high concentrations of exogenous TSP (≥60 μg/ml) directly interfere with interplatelet recognition among thrombin-activated platelets. This inhibitory effect of TSP can be neutralized by anti-TSP Fab. In addition, anti-TSP Fab directly inhibits platelet aggregation induced by a low (0.02 U/ml) but not by a high (0.1 U/ml) concentration of thrombin. In conclusion, our findings demonstrate that TSP is functionally important for platelet aggregation induced by low (≤0.05 U/ml) but not high (≥0.1 U/ml) concentrations of thrombin. High concentrations of exogenous TSP may univalently saturate all its platelet binding sites consequently interfering with TSP-crosslinking of thrombin-activated platelets.

Sign in / Sign up

Export Citation Format

Share Document