共查询到20条相似文献,搜索用时 31 毫秒
1.
Robin J. Marjoram Bryan Voss Yumei Pan S. Kent Dickeson Mary M. Zutter Heidi E. Hamm Samuel A. Santoro 《The Journal of biological chemistry》2009,284(50):34640-34647
Thrombin and fibrillar collagen are potent activators of platelets at sites of vascular injury. Both agonists cause platelet shape change, granule secretion, and aggregation to form the primary hemostatic plug. Human platelets express two thrombin receptors, protease-activated receptors 1 and 4 (PAR1 and PAR4) and two collagen receptors, the α2β1 integrin (α2β1) and the glycoprotein VI (GPVI)/FcRγ chain complex. Although these receptors and their signaling mechanisms have been intensely studied, it is not known whether and how these receptors cooperate in the hemostatic function of platelets. This study examined cooperation between the thrombin and collagen receptors in platelet adhesion by utilizing a collagen-related peptide (α2-CRP) containing the α2β1-specific binding motif, GFOGER, in conjunction with PAR-activating peptides. We demonstrate that platelet adhesion to α2-CRP is substantially enhanced by suboptimal PAR activation (agonist concentrations that do not stimulate platelet aggregation) using the PAR4 agonist peptide and thrombin. The enhanced adhesion induced by suboptimal PAR4 activation was α2β1-dependent and GPVI/FcRγ-independent as revealed in experiments with α2β1- or FcRγ-deficient mouse platelets. We further show that suboptimal activation of other platelet Gq-linked G protein-coupled receptors (GPCRs) produces enhanced platelet adhesion to α2-CRP. The enhanced α2β1-mediated platelet adhesion is controlled by phospholipase C (PLC), but is not dependent on granule secretion, activation of αIIbβ3 integrin, or on phosphoinositol-3 kinase (PI3K) activity. In conclusion, we demonstrate a platelet priming mechanism initiated by suboptimal activation of PAR4 or other platelet Gq-linked GPCRs through a PLC-dependent signaling cascade that promotes enhanced α2β1 binding to collagens containing GFOGER sites. 相似文献
2.
Tijs van Wieringen Sebastian Kalamajski ?sa Lidén Dominique Bihan Bengt Guss Dick Heineg?rd Richard W. Farndale Kristofer Rubin 《The Journal of biological chemistry》2010,285(46):35803-35813
Collagen fibers expose distinct domains allowing for specific interactions with other extracellular matrix proteins and cells. To investigate putative collagen domains that govern integrin αVβ3-mediated cellular interactions with native collagen fibers we took advantage of the streptococcal protein CNE that bound native fibrillar collagens. CNE specifically inhibited αVβ3-dependent cell-mediated collagen gel contraction, PDGF BB-induced and αVβ3-mediated adhesion of cells, and binding of fibronectin to native collagen. Using a Toolkit composed of overlapping, 27-residue triple helical segments of collagen type II, two CNE-binding sites present in peptides II-1 and II-44 were identified. These peptides lack the major binding site for collagen-binding β1 integrins, defined by the peptide GFOGER. Peptide II-44 corresponds to a region of collagen known to bind collagenases, discoidin domain receptor 2, SPARC (osteonectin), and fibronectin. In addition to binding fibronectin, peptide II-44 but not II-1 inhibited αVβ3-mediated collagen gel contraction and, when immobilized on plastic, supported adhesion of cells. Reduction of fibronectin expression by siRNA reduced PDGF BB-induced αVβ3-mediated contraction. Reconstitution of collagen types I and II gels in the presence of CNE reduced collagen fibril diameters and fibril melting temperatures. Our data indicate that contraction proceeded through an indirect mechanism involving binding of cell-produced fibronectin to the collagen fibers. Furthermore, our data show that cell-mediated collagen gel contraction does not directly depend on the process of fibril formation. 相似文献
3.
Philippe Ohlmann Anna Lecchi Ali El-Tayeb Christa E. Müller Marco Cattaneo Christian Gachet 《Purinergic signalling》2013,9(1):59-66
Various radioligands have been used to characterize and quantify the platelet P2Y12 receptor, which share several weaknesses: (a) they are metabolically unstable and substrates for ectoenzymes, (b) they are agonists, and (c) they do not discriminate between P2Y1 and P2Y12. We used the [3H]PSB-0413 selective P2Y12 receptor antagonist radioligand to reevaluate the number of P2Y12 receptors in intact platelets and in membrane preparations. Studies in humans showed that: (1) [3H]PSB-0413 bound to 425 ± 50 sites/platelet (KD = 3.3 ± 0.6 nM), (2) 0.5 ± 0.2 pmol [3H]PSB-0413 bound to 1 mg protein of platelet membranes (KD = 6.5 ± 3.6 nM), and (3) competition studies confirmed the known features of P2Y12, with the expected rank order of potency: AR-C69931MX > 2MeSADP ≫ ADPβS > ADP, while the P2Y1 ligand MRS2179 and the P2X1 ligand α,β-Met-ATP did not displace [3H]PSB-0413 binding. Patients with severe P2Y12 deficiency displayed virtually no binding of [3H]PSB-0413 to intact platelets, while a patient with a dysfunctional P2Y12 receptor had normal binding. Studies in mice showed that: (1) [3H]PSB-0413 bound to 634 ± 87 sites/platelet (KD = 14 ± 4.5 nM) and (2) 0.7 pmol ± 0.3 [3H]PSB-0413 bound to 1 mg protein of platelet membranes (KD = 9.1 ± 5.3 nM). Clopidogrel and other thiol reagents like pCMBS or DTT abolished the binding both to intact platelets and membrane preparations. Therefore, [3H]PSB-0413 is an accurate and selective tool for radioligand binding studies aimed at quantifying P2Y12 receptors, to identify patients with P2Y12 deficiencies or quantify the effect of P2Y12 targeting drugs. 相似文献
4.
Background
We have shown that 1,2,3,4,6-penta-O-galloyl-α-D-glucopyranose (α-PGG), an orally effective hypoglycemic small molecule, binds to insulin receptors and activates insulin-mediated glucose transport. Insulin has been shown to bind to its receptors on platelets and inhibit platelet activation. In this study we tested our hypothesis that if insulin possesses anti-platelet properties then insulin mimetic small molecules should mimic antiplatelet actions of insulin.Principal Findings
Incubation of human platelets with insulin or α-PGG induced phosphorylation of insulin receptors and IRS-1 and blocked ADP or collagen induced aggregation. Pre-treatment of platelets with α-PGG inhibited thrombin-induced release of P-selectin, secretion of ATP and aggregation. Addition of ADP or thrombin to platelets significantly decreased the basal cyclic AMP levels. Pre-incubation of platelets with α-PGG blocked ADP or thrombin induced decrease in platelet cyclic AMP levels but did not alter the basal or PGE1 induced increase in cAMP levels. Addition of α-PGG to platelets blocked agonist induced rise in platelet cytosolic calcium and phosphorylation of Akt. Administration of α-PGG (20 mg kg−1) to wild type mice blocked ex vivo platelet aggregation induced by ADP or collagen.Conclusions
These data suggest that α-PGG inhibits platelet activation, at least in part, by inducing phosphorylation of insulin receptors leading to inhibition of agonist induced: (a) decrease in cyclic AMP; (b) rise in cytosolic calcium; and (c) phosphorylation of Akt. These findings taken together with our earlier reports that α-PGG mimics insulin signaling suggest that inhibition of platelet activation by α-PGG mimics antiplatelet actions of insulin. 相似文献5.
Nataly P. Podolnikova Sergiy Yakovlev Valentin P. Yakubenko Xu Wang Oleg V. Gorkun Tatiana P. Ugarova 《The Journal of biological chemistry》2014,289(4):2371-2383
The currently available antithrombotic agents target the interaction of platelet integrin αIIbβ3 (GPIIb-IIIa) with fibrinogen during platelet aggregation. Platelets also bind fibrin formed early during thrombus growth. It was proposed that inhibition of platelet-fibrin interactions may be a necessary and important property of αIIbβ3 antagonists; however, the mechanisms by which αIIbβ3 binds fibrin are uncertain. We have previously identified the γ370–381 sequence (P3) in the γC domain of fibrinogen as the fibrin-specific binding site for αIIbβ3 involved in platelet adhesion and platelet-mediated fibrin clot retraction. In the present study, we have demonstrated that P3 can bind to several discontinuous segments within the αIIb β-propeller domain of αIIbβ3 enriched with negatively charged and aromatic residues. By screening peptide libraries spanning the sequence of the αIIb β-propeller, several sequences were identified as candidate contact sites for P3. Synthetic peptides duplicating these segments inhibited platelet adhesion and clot retraction but not platelet aggregation, supporting the role of these regions in fibrin recognition. Mutant αIIbβ3 receptors in which residues identified as critical for P3 binding were substituted for homologous residues in the I-less integrin αMβ2 exhibited reduced cell adhesion and clot retraction. These residues are different from those that are involved in the coordination of the fibrinogen γ404–411 sequence and from auxiliary sites implicated in binding of soluble fibrinogen. These results map the binding of fibrin to multiple sites in the αIIb β-propeller and further indicate that recognition specificity of αIIbβ3 for fibrin differs from that for soluble fibrinogen. 相似文献
6.
Lahti M Bligt E Niskanen H Parkash V Brandt AM Jokinen J Patrikainen P Käpylä J Heino J Salminen TA 《The Journal of biological chemistry》2011,286(50):43343-43351
We have analyzed the structure and function of the integrin α1I domain harboring a gain-of-function mutation E317A. To promote protein crystallization, a double variant with an additional C139S mutation was used. In cell adhesion assays, the E317A mutation promoted binding to collagen. Similarly, the double mutation C139S/E317A increased adhesion compared with C139S alone. Furthermore, soluble α1I C139S/E317A was a higher avidity collagen binder than α1I C139S, indicating that the double variant represents an activated form. The crystal structure of the activated variant of α1I was solved at 1.9 Å resolution. The E317A mutation results in the unwinding of the αC helix, but the metal ion has moved toward loop 1, instead of loop 2 in the open α2I. Furthermore, unlike in the closed αI domains, the metal ion is pentacoordinated and, thus, prepared for ligand binding. Helix 7, which has moved downward in the open α2I structure, has not changed its position in the activated α1I variant. During the integrin activation, Glu335 on helix 7 binds to the metal ion at the metal ion-dependent adhesion site (MIDAS) of the β1 subunit. Interestingly, in our cell adhesion assays E317A could activate collagen binding even after mutating Glu335. This indicates that the stabilization of helix 7 into its downward position is not required if the α1 MIDAS is already open. To conclude, the activated α1I domain represents a novel conformation of the αI domain, mimicking the structural state where the Arg287-Glu317 ion pair has just broken during the integrin activation. 相似文献
7.
8.
Simone M. Schoenwaelder Akiko Ono Warwick S. Nesbitt Joanna Lim Kate Jarman Shaun P. Jackson 《The Journal of biological chemistry》2010,285(4):2886-2896
Phosphoinositide (PI) 3-kinase (PI3K) signaling processes play an important role in regulating the adhesive function of integrin αIIbβ3, necessary for platelet spreading and sustained platelet aggregation. PI3K inhibitors are effective at reducing platelet aggregation and thrombus formation in vivo and as a consequence are currently being evaluated as novel antithrombotic agents. PI3K regulation of integrin αIIbβ3 activation (affinity modulation) primarily occurs downstream of Gi-coupled and tyrosine kinase-linked receptors linked to the activation of Rap1b, AKT, and phospholipase C. In the present study, we demonstrate an important role for PI3Ks in regulating the avidity (strength of adhesion) of high affinity integrin αIIbβ3 bonds, necessary for the cellular transmission of contractile forces. Using knock-out mouse models and isoform-selective PI3K inhibitors, we demonstrate that the Type Ia p110β isoform plays a major role in regulating thrombin-stimulated fibrin clot retraction in vitro. Reduced clot retraction induced by PI3K inhibitors was not associated with defects in integrin αIIbβ3 activation, actin polymerization, or actomyosin contractility but was associated with a defect in integrin αIIbβ3 association with the contractile cytoskeleton. Analysis of integrin αIIbβ3 adhesion contacts using total internal reflection fluorescence microscopy revealed an important role for PI3Ks in regulating the stability of high affinity integrin αIIbβ3 bonds. These studies demonstrate an important role for PI3K p110β in regulating the avidity of high affinity integrin αIIbβ3 receptors, necessary for the cellular transmission of contractile forces. These findings may provide new insight into the potential antithrombotic properties of PI3K p110β inhibitors. 相似文献
9.
Madhavika N. Serasinghe A. M. P. B. Seneviratne Alan V. Smrcka Yisang Yoon 《The Journal of biological chemistry》2010,285(1):620-630
Mammalian mitochondrial fission requires at least two proteins, hFis1 and the dynamin-like GTPase DLP1/Drp1. The mitochondrial protein hFis1 is anchored at the outer membrane by a C-terminal transmembrane domain. The cytosolic domain of hFis1 contains six α helices [α1–α6] out of which [α2–α5] form tetratricopeptide repeat (TPR)-like motifs. DLP1 and possibly other proteins are thought to interact with the hFis1 TPR region during the fission process. It has also been suggested that the α1-helix regulates protein-protein interactions at the TPR. We performed random peptide phage display screening using the hFis1[α2–α6] as the target and identified ten different peptide sequences. Phage ELISA using mutant hFis1 indicates that the peptide binding requires the α2 and α3 helices and the intact TPR structure. Competition experiments and surface plasmon resonance analyses confirmed that a subset of free peptides enriched with proline residues directly bind to the target. Two of these peptides bind to the α1-containing intact cytosolic domain of hFis1 with decreased affinity. Peptide microinjection into cells abolished the mitochondrial swelling induced by overexpression of α1-deleted hFis1, and significantly decreased cytochrome c release from mitochondria upon apoptotic induction. Our data demonstrate that hFis1 can bind to multiple amino acid sequences selectively, and that the TPR constitutes the main binding region of hFis1, providing a first insight into the hFis1 TPR as a potential therapeutic target. 相似文献
10.
Sergey Ryzhov Bong Hwan Sung Qinkun Zhang Alissa Weaver Richard J. Gumina Italo Biaggioni Igor Feoktistov 《Purinergic signalling》2014,10(3):477-486
Adenosine levels increase in ischemic hearts and contribute to the modulation of that pathological environment. We previously showed that A2B adenosine receptors on mouse cardiac Sca1+CD31− mesenchymal stromal cells upregulate secretion of paracrine factors that may contribute to the improvement in cardiac recovery seen when these cells are transplanted in infarcted hearts. In this study, we tested the hypothesis that A2B receptor signaling regulates the transition of Sca1+CD31− cells, which occurs after myocardial injury, into a myofibroblast phenotype that promotes myocardial repair and remodeling. In vitro, TGFβ1 induced the expression of the myofibroblast marker α-smooth muscle actin (αSMA) and increased collagen I generation in Sca1+CD31− cells. Stimulation of A2B receptors attenuated TGFβ1-induced collagen I secretion but had no effect on αSMA expression. In vivo, myocardial infarction resulted in a rapid increase in the numbers of αSMA-positive cardiac stromal cells by day 5 followed by a gradual decline. Genetic deletion of A2B receptors had no effect on the initial accumulation of αSMA-expressing stromal cells but hastened their subsequent decline; the numbers of αSMA-positive cells including Sca1+CD31− cells remained significantly higher in wild type compared with A2B knockout hearts. Thus, our study revealed a significant contribution of cardiac Sca1+CD31− cells to the accumulation of αSMA-expressing cells after infarction and implicated A2B receptor signaling in regulation of myocardial repair and remodeling by delaying deactivation of these cells. It is plausible that this phenomenon may contribute to the beneficial effects of transplantation of these cells to the injured heart.
Electronic supplementary material
The online version of this article (doi:10.1007/s11302-014-9410-y) contains supplementary material, which is available to authorized users. 相似文献11.
Background
The Ecballium elaterium trypsin inhibitor (EETI-II), a 28-amino acid member of the knottin family of peptides, contains three interwoven disulfide bonds that form multiple solvent-exposed loops. Previously, the trypsin binding loop of EETI-II has been engineered to confer binding to several alternative molecular targets. Here, EETI-II was further explored as a molecular scaffold for polypeptide engineering by evaluating the ability to mutate two of its structurally adjacent loops.Methodology/Principal Findings
Yeast surface display was used to engineer an EETI-II mutant containing two separate integrin binding epitopes. The resulting knottin peptide was comprised of 38 amino acids, and contained 11- and 10-residue loops compared to wild-type EETI-II, which naturally contains 6- and 5-residue loops, respectively. This knottin peptide bound to αvβ3 and αvβ5 integrins with affinities in the low nanomolar range, but bound weakly to the related integrins α5β1 and αiibβ3. In addition, the engineered knottin peptide inhibited tumor cell adhesion to vitronectin, an extracellular matrix protein that binds to αvβ3 and αvβ5 integrins. A 64Cu radiolabeled version of this knottin peptide demonstrated moderate serum stability and excellent tumor-to-muscle and tumor-to-blood ratios by positron emission tomography imaging in human tumor xenograft models. Tumor uptake was ∼3–5% injected dose per gram (%ID/g) at one hour post injection, with rapid clearance of probe through the kidneys.Conclusions/Significance
We demonstrated that multiple loops of EETI-II can be mutated to bind with high affinity to tumor-associated integrin receptors. The resulting knottin peptide contained 21 (>50%) non-native amino acids within two mutated loops, indicating that extended loop lengths and sequence diversity were well tolerated within the EETI-II scaffold. A radiolabeled version of this knottin peptide showed promise for non-invasive imaging of integrin expression in living subjects. However, reduced serum and metabolic stability were observed compared to an engineered integrin-binding EETI-II knottin peptide containing only one mutated loop. 相似文献12.
13.
Karen Vanhoorelbeke Simon F. De Meyer Inge Pareyn Chantal Melchior Sebastien Plan?on Christiane Margue Olivier Pradier Pierre Fondu Nelly Kieffer Timothy A. Springer Hans Deckmyn 《The Journal of biological chemistry》2009,284(22):14914-14920
Three heterozygous mutations were identified in the genes encoding platelet
integrin receptor αIIbβ3 in a patient with an ill defined platelet
disorder: one in the β3 gene (S527F) and two in the αIIb gene
(R512W and L841M). Five stable Chinese hamster ovary cell lines were
constructed expressing recombinant αIIbβ3 receptors bearing the
individual R512W, L841M, or S527F mutation; both the R512W and L841M
mutations; or all three mutations. All receptors were expressed on the cell
surface, and mutations R512W and L841M had no effect on integrin function.
Interestingly, the β3 S527F mutation produced a constitutively active
receptor. Indeed, both fibrinogen and the ligand-mimetic antibody PAC-1 bound
to non-activated αIIbβ3 receptors carrying the S527F mutation,
indicating that the conformation of this receptor was altered and corresponded
to the high affinity ligand binding state. In addition, the conformational
change induced by S527F was evident from basal anti-ligand-induced binding
site antibody binding to the receptor. A molecular model bearing this mutation
was constructed based on the crystal structure of αIIbβ3 and
revealed that the S527F mutation, situated in the third integrin epidermal
growth factor-like (I-EGF3) domain, hindered the αIIbβ3 receptor
from adopting a wild type-like bent conformation. Movement of I-EGF3 into a
cleft in the bent conformation may be hampered both by steric hindrance
between Phe527 in β3 and the calf-1 domain in αIIb and
by decreased flexibility between I-EGF2 and I-EGF3.The platelet receptor αIIbβ3 belongs to the family of integrin
receptors that consist of noncovalently linked α/β-heterodimers.
They are cell-surface receptors that play a role in cell-cell and cell-matrix
interactions. Under resting conditions, integrin receptors adopt the low
affinity conformation and do not interact with their ligands. Inside-out
signaling turns the receptor into a high affinity conformation capable of
ligand binding. Ligand binding itself induces additional conformational
changes resulting in exposure of neoantigenic sites called ligand-induced
binding sites (LIBS)3
and generates in turn outside-in signaling, which triggers a range of
downstream signals (1,
2).Integrin αIIbβ3 is expressed on platelets and megakaryocytes. In
flowing blood under resting conditions, αIIbβ3 does not interact
with its ligand fibrinogen. When a blood vessel is damaged, platelets adhere
at sites of vascular injury and become activated. As a consequence,
αIIbβ3 adopts the high affinity conformation and binds fibrinogen.
This results in platelet aggregation and thrombus formation, which eventually
will stop the bleeding (3).The topology of integrins comprises an extracellular, globular, N-terminal
ligand-binding head domain (the β-propeller domain in the αIIb
chain and the βI domain in the β3 chain) standing on two long legs
or stalks (consisting of thigh, calf-1, and calf-2 domains in the αIIb
chain and hybrid, plexin/semaphorin/integrin (PSI), four integrin endothelial
growth factor-like (I-EGF), and β-tail domains in the β3 chain),
followed by transmembrane and cytoplasmic domains
(1,
2). X-ray crystal structures of
the extracellular domain of non-activated αVβ3 revealed that the
legs are severely bent, putting the head domain next to the membrane-proximal
portions of the legs (4,
5). The bending occurs between
I-EGF1 and I-EGF2 in the β-subunit and between the thigh and calf-1
domains in the α-subunit. This bent conformation represents the low
affinity state of the receptor. The high affinity state of the receptor is
induced by activation and is associated with a large-scale conformational
rearrangement in which the integrin extends with a switchblade-like motion
(2). Recently, the crystal
structure of the entire extracellular domain of αIIbβ3 in its low
affinity conformation was resolved and revealed that this integrin also adopts
the bent conformation under resting conditions
(6). Structural rearrangements
in αIIbβ3 between the bent and extended conformations are similar
to what has been reported for other integrins
(7).We report here that the S527F mutation in the I-EGF3 region of the β3
polypeptide chain of the αIIbβ3 receptor induces a constitutively
active receptor adopting an extended high affinity conformation. This was
evidenced by spontaneous PAC-1, fibrinogen, and anti-LIBS antibody binding.
These data were further corroborated by modeling the replacement of
Ser527 with Phe in the crystal structure of the extracellular
domain of αIIbβ3. In this model, the S527F mutation decreases the
flexibility of I-EGF3 and appears to prevent movement of the lower β-leg
into the cleft between the upper β-leg and the lower α-leg. As a
consequence, formation of the bent conformation of the non-activated receptor
is hampered. 相似文献
14.
Classical benzodiazepines, such as diazepam, interact with αxβ2γ2 GABAA receptors, x = 1, 2, 3, 5 and modulate their function. Modulation of different receptor isoforms probably results in selective behavioural effects as sedation and anxiolysis. Knowledge of differences in the structure of the binding pocket in different receptor isoforms is of interest for the generation of isoform-specific ligands. We studied here the interaction of the covalently reacting diazepam analogue 3-NCS with α1S204Cβ2γ2, α1S205Cβ2γ2 and α1T206Cβ2γ2 and with receptors containing the homologous mutations in α2β2γ2, α3β2γ2, α5β1/2γ2 and α6β2γ2. The interaction was studied using radioactive ligand binding and at the functional level using electrophysiological techniques. Both strategies gave overlapping results. Our data allow conclusions about the relative apposition of α1S204Cβ2γ2, α1S205Cβ2γ2 and α1T206Cβ2γ2 and homologous positions in α2, α3, α5 and α6 with C-atom adjacent to the keto-group in diazepam. Together with similar data on the C-atom carrying Cl in diazepam, they indicate that the architecture of the binding site for benzodiazepines differs in each GABAA receptor isoform α1β2γ2, α2β2γ2, α3β2γ2, α5β1/2γ2 and α6β2γ2. 相似文献
15.
Guo-Dong Li David C. Chiara Jonathan B. Cohen Richard W. Olsen 《The Journal of biological chemistry》2009,284(18):11771-11775
Photoaffinity labeling of γ-aminobutyric acid type A
(GABAA)-receptors (GABAAR) with an etomidate analog and
mutational analyses of direct activation of GABAAR by neurosteroids
have each led to the proposal that these structurally distinct general
anesthetics bind to sites in GABAARs in the transmembrane domain at
the interface between the β and α subunits. We tested whether the
two ligand binding sites might overlap by examining whether neuroactive
steroids inhibited etomidate analog photolabeling. We previously identified
(Li, G. D., Chiara, D. C., Sawyer, G. W., Husain, S. S., Olsen, R. W., and
Cohen, J. B. (2006) J. Neurosci. 26, 11599–11605) azietomidate
photolabeling of GABAAR α1Met-236 and βMet-286 (in
αM1 and βM3). Positioning these two photolabeled amino acids in a
single type of binding site at the interface of β and α subunits
(two copies per pentamer) is consistent with a GABAAR homology
model based upon the structure of the nicotinic acetylcholine receptor and
with recent αM1 to βM3 cross-linking data. Biologically active
neurosteroids enhance rather than inhibit azietomidate photolabeling, as
assayed at the level of GABAAR subunits on analytical SDS-PAGE, and
protein microsequencing establishes that the GABAAR-modulating
neurosteroids do not inhibit photolabeling of GABAAR
α1Met-236 or βMet-286 but enhance labeling of α1Met-236. Thus
modulatory steroids do not bind at the same site as etomidate, and neither of
the amino acids identified as neurosteroid activation determinants (Hosie, A.
M., Wilkins, M. E., da Silva, H. M., and Smart, T. G. (2006) Nature
444, 486–489) are located at the subunit interface defined by our
etomidate site model.GABAA3
receptors (GABAAR) are major mediators of brain inhibitory
neurotransmission and participate in most circuits and behavioral pathways
relevant to normal and pathological function
(1). GABAAR are
subject to modulation by endogenous neurosteroids, as well as myriad
clinically important central nervous system drugs including general
anesthetics, benzodiazepines, and possibly ethanol
(1,
2). The mechanism of
GABAAR modulation by these different classes of drugs is of major
interest, including identification of the receptor amino acid residues
involved in binding and action of the drugs.In the absence of high resolution crystal structures of drug-receptor
complexes, the locations of anesthetic binding sites in GABAARs
have been predicted based upon analyses of functional properties of point
mutant receptors, which identified residues in the α and β subunit
M1–M4 transmembrane helices important for modulation by volatile
anesthetics (primarily α subunit) and by intravenous agents, including
etomidate and propofol (β subunit)
(3–5).
Position βM2–15, numbered relative to the N terminus of the helix,
functions as a major determinant of etomidate and propofol potency as GABA
modulators in vitro and in vivo
(6–8).
By contrast, this residue is not implicated for modulation by the
neurosteroids, potent endogenous modulators of GABAAR
(9).Photoaffinity labeling, which allows the identification of residues in
proximity to drug binding sites
(10,
11), has been used to identify
two GABAAR amino acids covalently modified by the etomidate analog
[3H]azietomidate
(12): α1Met-236 within
αM1 and βMet-286 within βM3. Photolabeling of these residues
was inhibited equally by nonradioactive etomidate and enhanced proportionately
by GABA present in the assay, consistent with the presence of these two
residues in a common drug binding pocket that would be located at the
interface between the β and α subunits in the transmembrane domain
(12). Mutational analyses
identify these positions as etomidate and propofol sensitivity determinants
(13–15).A recent mutagenesis study
(16) identified two other
residues in GABAAR αM1 and βM3 as critical for direct
activation by neurosteroids, αThr-236 (rat numbering, corresponding to
α1Thr-237, bovine numbering used here and by Li et al.
(12))4
and βTyr-284. These residues were also proposed to contribute to a
neurosteroid binding pocket in the transmembrane domain at the interface
between β and α subunits, based upon their location in an
alternative GABAAR structural model that positioned those amino
acids, and not α1Met-236 or βMet-286, at the subunit interface. For
GABAARs and other members of the Cys-loop superfamily of
neurotransmitter-gated ion channels, the transmembrane domain of each subunit
is made up of a loose bundle of four α helices (M1–M4), with M2
from each subunit contributing to the lumen of the ion channel and M4
positioned peripherally in greatest contact with lipid, as seen in the
structures of the Torpedo nicotinic acetylcholine receptor (nAChR)
(17) and in distantly related
prokaryote homologs (18).
However, uncertainties in the alignment of GABAAR subunit sequences
relative to those of the nAChR result in alternative GABAAR
homology models (12,
19,
20) that differ in the
location of amino acids in the M3 and M4 membrane-spanning helices and in the
M1 helix in some models (16,
21).If etomidate and neurosteroids both bind at the same β/α
interface in the GABAAR transmembrane domain, the limited space
available for ligand binding suggests that their binding pockets might overlap
and that ligand binding would be mutually exclusive. To address this question,
we photolabeled purified bovine brain GABAAR with
[3H]azietomidate in the presence of different neuroactive steroids
and determined by protein microsequencing whether active neurosteroids
inhibited labeling of α1Met-236 and βMet-286, as expected for
mutually exclusive binding, or resulted in [3H]azietomidate
photolabeling of other amino acids, a possible consequence of allosteric
interactions. Active steroids failed to inhibit labeling and enhanced labeling
of α1Met-236, clearly indicating that the neurosteroid and the etomidate
sites are distinct. Our GABAAR homology model that positions
α1Met-236 and βMet-286 at the β/α interface, but not
that of Hosie et al.
(16), is also consistent with
cysteine substitution cross-linking studies
(20,
22), which define the
proximity relations between amino acids in the αM1, αM2,
αM3, and βM3 helices, and these results support the interpretation
that the two residues photolabeled by [3H]azietomidate are part of
a single subunit interface binding pocket, whereas the steroid sensitivity
determinants identified by mutagenesis neither are at the β/α
subunit interface nor are contributors to a common binding pocket. 相似文献
16.
Karen Gilio Imke C. A. Munnix Pierre Mangin Judith M. E. M. Cosemans Marion A. H. Feijge Paola E. J. van der Meijden Serv�� Olieslagers Magdalena B. Chrzanowska-Wodnicka Rivka Lillian Simone Schoenwaelder Shigeo Koyasu Stewart O. Sage Shaun P. Jackson Johan W. M. Heemskerk 《The Journal of biological chemistry》2009,284(49):33750-33762
Platelets are activated by adhesion to vascular collagen via the immunoglobulin receptor, glycoprotein VI (GPVI). This causes potent signaling toward activation of phospholipase Cγ2, which bears similarity to the signaling pathway evoked by T- and B-cell receptors. Phosphoinositide 3-kinase (PI3K) plays an important role in collagen-induced platelet activation, because this activity modulates the autocrine effects of secreted ADP. Here, we identified the PI3K isoforms directly downstream of GPVI in human and mouse platelets and determined their role in GPVI-dependent thrombus formation. The targeting of platelet PI3Kα or -β strongly and selectively suppressed GPVI-induced Ca2+ mobilization and inositol 1,4,5-triphosphate production, thus demonstrating enhancement of phospholipase Cγ2 by PI3Kα/β. That PI3Kα and -β have a non-redundant function in GPVI-induced platelet activation and thrombus formation was concluded from measurements of: (i) serine phosphorylation of Akt, (ii) dense granule secretion, (iii) intracellular Ca2+ increases and surface expression of phosphatidylserine under flow, and (iv) thrombus formation, under conditions where PI3Kα/β was blocked or p85α was deficient. In contrast, GPVI-induced platelet activation was insensitive to inhibition or deficiency of PI3Kδ or -γ. Furthermore, PI3Kα/β, but not PI3Kγ, contributed to GPVI-induced Rap1b activation and, surprisingly, also to Rap1b-independent platelet activation via GPVI. Together, these findings demonstrate that both PI3Kα and -β isoforms are required for full GPVI-dependent platelet Ca2+ signaling and thrombus formation, partly independently of Rap1b. This provides a new mechanistic explanation for the anti-thrombotic effect of PI3K inhibition and makes PI3Kα an interesting new target for anti-platelet therapy. 相似文献
17.
Mary Ann Weis David M. Hudson Lammy Kim Melissa Scott Jiann-Jiu Wu David R. Eyre 《The Journal of biological chemistry》2010,285(4):2580-2590
Collagen triple helices are stabilized by 4-hydroxyproline residues. No function is known for the much less common 3-hydroxyproline (3Hyp), although genetic defects inhibiting its formation cause recessive osteogenesis imperfecta. To help understand the pathogenesis, we used mass spectrometry to identify the sites and local sequence motifs of 3Hyp residues in fibril-forming collagens from normal human and bovine tissues. The results confirm a single, essentially fully occupied 3Hyp site (A1) at Pro986 in A-clade chains α1(I), α1(II), and α2(V). Two partially modified sites (A2 and A3) were found at Pro944 in α1(II) and α2(V) and Pro707 in α2(I) and α2(V), which differed from A1 in sequence motif. Significantly, the distance between sites 2 and 3, 237 residues, is close to the collagen D-period (234 residues). A search for additional D-periodic 3Hyp sites revealed a fourth site (A4) at Pro470 in α2(V), 237 residues N-terminal to site 3. In contrast, human and bovine type III collagen contained no 3Hyp at any site, despite a candidate proline residue and recognizable A1 sequence motif. A conserved histidine in mammalian α1(III) at A1 may have prevented 3-hydroxylation because this site in chicken type III was fully hydroxylated, and tyrosine replaced histidine. All three B-clade type V/XI collagen chains revealed the same three sites of 3Hyp but at different loci and sequence contexts from those in A-clade collagen chains. Two of these B-clade sites were spaced apart by 231 residues. From these and other observations we propose a fundamental role for 3Hyp residues in the ordered self-assembly of collagen supramolecular structures. 相似文献
18.
Sarto-Jackson I Milenkovic I Smalla KH Gundelfinger ED Kaehne T Herrera-Molina R Thomas S Kiebler MA Sieghart W 《The Journal of biological chemistry》2012,287(17):14201-14214
γ-Aminobutyric acid type A (GABAA) receptors are pentameric ligand-gated ion channels that mediate fast inhibition in the central nervous system. Depending on their subunit composition, these receptors exhibit distinct pharmacological properties and differ in their ability to interact with proteins involved in receptor anchoring at synaptic or extra-synaptic sites. Whereas GABAA receptors containing α1, α2, or α3 subunits are mainly located synaptically where they interact with the submembranous scaffolding protein gephyrin, receptors containing α5 subunits are predominantly found extra-synaptically and seem to interact with radixin for anchorage. Neuroplastin is a cell adhesion molecule of the immunoglobulin superfamily that is involved in hippocampal synaptic plasticity. Our results reveal that neuroplastin and GABAA receptors can be co-purified from rat brain and exhibit a direct physical interaction as demonstrated by co-precipitation and Förster resonance energy transfer (FRET) analysis in a heterologous expression system. The brain-specific isoform neuroplastin-65 co-localizes with GABAA receptors as shown in brain sections as well as in neuronal cultures, and such complexes can either contain gephyrin or be devoid of gephyrin. Neuroplastin-65 specifically co-localizes with α1 or α2 but not with α3 subunits at GABAergic synapses. In addition, neuroplastin-65 also co-localizes with GABAA receptor α5 subunits at extra-synaptic sites. Down-regulation of neuroplastin-65 by shRNA causes a loss of GABAA receptor α2 subunits at GABAergic synapses. These results suggest that neuroplastin-65 can co-localize with a subset of GABAA receptor subtypes and might contribute to anchoring and/or confining GABAA receptors to particular synaptic or extra-synaptic sites, thus affecting receptor mobility and synaptic strength. 相似文献
19.
Jeppe A. Olsen Jette S. Kastrup Dan Peters Michael Gajhede Thomas Balle Philip K. Ahring 《The Journal of biological chemistry》2013,288(50):35997-36006
Positive allosteric modulators (PAMs) of α4β2 nicotinic acetylcholine receptors have the potential to improve cognitive function and alleviate pain. However, only a few selective PAMs of α4β2 receptors have been described limiting both pharmacological understanding and drug-discovery efforts. Here, we describe a novel selective PAM of α4β2 receptors, NS206, and compare with a previously reported PAM, NS9283. Using two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes, NS206 was observed to positively modulate acetylcholine (ACh)-evoked currents at both known α4β2 stoichiometries (2α:3β and 3α:2β). In the presence of NS206, peak current amplitudes surpassed those of maximal efficacious ACh stimulations (Emax(ACh)) with no or limited effects at potencies and current waveforms (as inspected visually). This pharmacological action contrasted with that of NS9283, which only modulated the 3α:2β receptor and acted by left shifting the ACh concentration-response relationship. Interestingly, the two modulators can act simultaneously in an additive manner at 3α:2β receptors, which results in current levels exceeding Emax(ACh) and a left-shifted ACh concentration-response relationship. Through use of chimeric and point-mutated receptors, the binding site of NS206 was linked to the α4-subunit transmembrane domain, whereas binding of NS9283 was shown to be associated with the αα-interface in 3α:2β receptors. Collectively, these data demonstrate the existence of two distinct modulatory sites in α4β2 receptors with unique pharmacological attributes that can act additively. Several allosteric sites have been identified within the family of Cys-loop receptors and with the present data, a detailed picture of allosteric modulatory mechanisms of these important receptors is emerging. 相似文献
20.
Maciej J. Stawikowski Beatrix Aukszi Roma Stawikowska Mare Cudic Gregg B. Fields 《The Journal of biological chemistry》2014,289(31):21591-21604
Although type IV collagen is heavily glycosylated, the influence of this post-translational modification on integrin binding has not been investigated. In the present study, galactosylated and nongalactosylated triple-helical peptides have been constructed containing the α1(IV)382–393 and α1(IV)531–543 sequences, which are binding sites for the α2β1 and α3β1 integrins, respectively. All peptides had triple-helical stabilities of 37 °C or greater. The galactosylation of Hyl393 in α1(IV)382–393 and Hyl540 and Hyl543 in α1(IV)531–543 had a dose-dependent influence on melanoma cell adhesion that was much more pronounced in the case of α3β1 integrin binding. Molecular modeling indicated that galactosylation occurred on the periphery of α2β1 integrin interaction with α1(IV)382–393 but right in the middle of α3β1 integrin interaction with α1(IV)531–543. The possibility of extracellular deglycosylation of type IV collagen was investigated, but no β-galactosidase-like activity capable of collagen modification was found. Thus, glycosylation of collagen can modulate integrin binding, and levels of glycosylation could be altered by reduction in expression of glycosylation enzymes but most likely not by extracellular deglycosylation activity. 相似文献