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The functional characteristics of a nonacidic, inositol 1,4,5-trisphosphate– and thapsigargin-insensitive Ca2+ pool have been characterized in mammalian cells derived from the rat pituitary gland (GH3, GC, and GH3B6), the adrenal tissue (PC12), and mast cells (RBL-1). This Ca2+ pool is released into the cytoplasm by the Ca2+ ionophores ionomycin or A23187 after the discharge of the inositol 1,4,5-trisphosphate–sensitive store with an agonist coupled to phospholipase C activation and/or thapsigargin. The amount of Ca2+ trapped within this pool increased significantly after a prolonged elevation of intracellular Ca2+ concentration elicited by activation of Ca2+ influx. This pool was affected neither by caffeine-ryanodine nor by mitochondrial uncouplers. Probing mitochondrial Ca2+ with recombinant aequorin confirmed that this pool did not coincide with mitochondria, whereas its homogeneous distribution across the cytosol, as revealed by confocal microscopy, and its insensitivity to brefeldin A make localization within the Golgi complex unlikely. A proton gradient as the driving mechanism for Ca2+ uptake was excluded since ionomycin is inefficient in releasing Ca2+ from acidic pools and Ca2+ accumulation/release in/from this store was unaffected by monensin or NH4Cl, drugs known to collapse organelle acidic pH gradients. Ca2+ sequestration inside this pool, thus, may occur through a low-affinity, high-capacity Ca2+–ATPase system, which is, however, distinct from classical endosarcoplasmic reticulum Ca2+–ATPases. The cytological nature and functional role of this Ca2+ storage compartment are discussed.The cytosolic free Ca2+ concentration ([Ca2+]i)1 of eukaryotic cells rests in the range of 50–200 nM, i.e., at a very low level, if compared to the Ca2+ concentration of physiological media (2 mM). However, the total cellular Ca2+ content is closer to this latter value (1–3 mmol/l of cell water). In other words, eukaryotic cells sequester large amounts of Ca2+ mainly by uptake inside intracellular Ca2+ stores (∼90%) (for reviews see Pozzan et al., 1994; Clapham, 1995).The complexity of intracellular Ca2+ stores has been intensively investigated in recent years (for reviews see Meldolesi et al., 1990; Pozzan et al., 1994; Simpson et al., 1995). Attention has been focused mainly on Ca2+ stores that are highly dynamic because of their ability to rapidly take up and release Ca2+. Ca2+ sequestration into these pools depends on Ca2+–ATPases, known as sarco/endoplasmic reticulum Ca2+–ATPases (SERCAs) (Burk et al., 1989; Bobe et al., 1994; Wuytack et al., 1994). All the SERCA isoforms share the property of being selectively inhibited by thapsigargin (Tg), a tumor-promoting sesquiterpene lactone (Lytton et al., 1991). Tg acts with both high affinity, at nanomolar concentrations, and high specificity, with virtually no effect on the Ca2+– or Na+/K+– ATPase of the plasmalemma.Other drugs, such as 2,5-di(tert-butyl)-1,4-benzohydroquinone (tBHQ) and cyclopiazonic acid (CA), also block SERCAs, albeit with a significantly lower affinity (Mason et al., 1991). Ca2+ release, on the other hand, depends mainly on two types of Ca2+ release channels named inositol 1,4,5-trisphosphate (InsP3) and ryanodine receptors (for reviews see Mikoshiba, 1993; Sorrentino and Volpe, 1993; Ehrlich, 1995). These channels are expressed in variable proportions in different cell types and couple extracellular stimuli to the release of Ca2+, with possible ensuing generation of Ca2+ waves and spikes (for reviews see Amundson and Clapham, 1993; Taylor, 1994; Bootman and Berridge, 1995). The relationship between these types of Ca2+-release channels is still largely debated. The ryanodine-sensitive channel is also activated by caffeine, and ryanodine- and caffeine-sensitive stores are generally regarded to comprise the same pool (Zacchetti et al., 1991; Barry and Cheek, 1994; but also see Giannini et al., 1992; McNulty and Taylor, 1993).In the vast majority of cell types so far investigated, the InsP3- (and/or the ryanodine-) sensitive stores almost completely overlap with those sensitive to Tg (Zacchetti et al., 1991; Gamberucci et al., 1995) and are thus referred to also as Tg-sensitive Ca2+ pools. From the cytological point of view, the InsP3-/Tg-sensitive Ca2+ pool is identified with the ER or with a subfraction of it (Hashimoto et al., 1988).The complexity of the relationships between the InsP3- and ryanodine/caffeine-sensitive stores does not cover the entire issue of intracellular Ca2+ pool heterogeneity. Other types of Ca2+ pools are known to exist, the size of which varies considerably among different cell types. These latter Ca2+ stores account for roughly half of all sequestered Ca2+ (Chandra et al., 1991; Fasolato et al., 1991; Shorte et al., 1991; Bastianutto et al., 1995; Mery et al., 1996). They have been identified through the increase in [Ca2+]i upon application of Ca2+ ionophores, after depletion of the Tgsensitive pool with a combination, or a sequence, of InsP3generating agonists, Tg, and caffeine. These residual Tginsensitive pools appear rather heterogeneous in terms of cytological identity and pharmacological sensitivity. Part of these pools shows an acidic lumenal pH and is discharged only by a combination of a Ca2+ ionophore and of agents that collapse internal acidic pH gradients (such as monensin and NH4Cl). 45Ca2+ labeling of Tg-insensitive pools is slower than that of the Tg-sensitive store, and, for this reason, they have been generally indicated as slowly exchanging Ca2+ pools (Fasolato et al., 1991). As far as their identification is concerned, the acidic pool seems largely identifiable with secretory compartments and lysosomes, while very little is known yet about the rest of the Tg-insensitive store.Here we demonstrate that a nonacidic, InsP3- and Tg- insensitive Ca2+ pool rapidly accumulates large amounts of Ca2+ when high and sustained increases of [Ca2+]i are induced by opening of voltage- or store-operated Ca2+ channels. This Ca2+ storage compartment is insensitive to mitochondrial uncouplers and appears diffusely distributed in the cell cytosol. The possibility is discussed that this low-affinity, high-capacity Ca2+ pool represents a previously unidentified subcompartment of the ER expressing a Tg-insensitive Ca2+–ATPase. 相似文献
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Bezprozvanny I 《Neurochemical research》2011,36(7):1186-1197
Huntington’s disease (HD) and spinocerebellar ataxias (SCAs) are autosomal-dominant neurodegenerative disorders. HD is caused
by polyglutamine (polyQ) expansion in the amino-terminal region of a protein huntingtin (Htt) and primarily affects medium
spiny striatal neurons (MSN). Many SCAs are caused by polyQ-expansion in ataxin proteins and primarily affect cerebellar Purkinje
cells. The reasons for neuronal dysfunction and death in HD and SCAs remain poorly understood and no cure is available for
the patients. Our laboratory discovered that mutant huntingtin, ataxin-2 and ataxin-3 proteins specifically bind to the carboxy-terminal
region of the type 1 inositol 1,4,5-trisphosphate receptor (IP3R1), an intracellular Ca2+ release channel. Moreover, we found that association of mutant huntingtin or ataxins with IP3R1 causes sensitization of IP3R1 to activation by IP3 in planar lipid bilayers and in neuronal cells. These results suggested that deranged neuronal Ca2+ signaling might play an important role in pathogenesis of HD, SCA2 and SCA3. In support of this idea, we demonstrated a connection
between abnormal Ca2+ signaling and neuronal cell death in experiments with HD, SCA2 and SCA3 transgenic mouse models. Additional data in the literature
indicate that abnormal neuronal Ca2+ signaling may also play an important role in pathogenesis of SCAl, SCA5, SCA6, SCA14 and SCA15/16. Based on these results
I propose that IP3R and other Ca2+ signaling proteins should be considered as potential therapeutic targets for treatment of HD and SCAs. 相似文献
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Usha Padmanabhan D. Eric Dollins Peter C. Fridy John D. York C. Peter Downes 《The Journal of biological chemistry》2009,284(16):10571-10582
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The landmark paper by Hirose et al. (Hirose, K., Kadowaki, S., Tanabe, M., Takeshima, H., Iino, M., Science 284:1527–1530,
1999) presented experimental investigations to show that not only can calcium upregulate IP3, but that it can also have an inhibitory effect on IP3. In this paper, we present a preliminary model, which is consistent with these experiments. This model includes positive
and negative feedback between calcium and IP3 and is able to reproduce more precisely the data presented in Hirose et al. (Hirose, K., Kadowaki, S., Tanabe, M., Takeshima,
H., Iino, M., Science 284:1527–1530, 1999). In the second part of the paper, the intracellular and intercellular calcium movement
in Madin–Darby canine kidney epithelial cells is investigated. With the aid of the model we are able to identify the aspects
of IP3 and calcium signalling, which should be studied further experimentally before refining the model. 相似文献
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Aluminum Inhibition of the Inositol 1,4,5-Trisphosphate Signal Transduction Pathway in Wheat Roots: A Role in Aluminum Toxicity? 总被引:11,自引:1,他引:10 下载免费PDF全文
In crop plants, aluminum (Al) rhizotoxicity is a major problem worldwide; however, the cause of Al toxicity remains elusive. The effects of Al on the inositol 1,4,5-trisphosphate (Ins[1,4,5]P3)-mediated signal transduction pathway were investigated in wheat roots. Exogenously applied Al (50 [mu]M) rapidly inhibited root growth (<2 hr) but did not affect general root metabolism. An Ins(1,4,5)P3 transient was generated in root tips, either before or after exposure to Al for 1 hr, by treating the roots with H2O2 (10 mM). Background (unstimulated) levels of Ins(1,4,5)P3 were similar in both Al-treated and Al-untreated root apices. However, H2O2-stimulated levels of Ins(1,4,5)P3 in root apices showed a significant (>50%) reduction after Al exposure in comparison with untreated controls, indicating that Al may be interfering with the phosphoinositide signaling pathway. When phospholipase C (PLC) was assayed directly in the presence of Al or other metal cations in microsomal membranes, AlCl3 and Al-citrate specifically inhibited PLC action in a dose-dependent manner and at physiologically relevant Al levels. Al exposure had no effect on inositol trisphosphate dephosphorylation or on a range of enzymes isolated from wheat roots, suggesting that Al exposure may specifically target PLC. Possible mechanisms of PLC inhibition by Al and the role of Ins(1,4,5)P3 in Al toxicity and growth are discussed. This study provides compelling evidence that the phytotoxic metal cation Al has an intracellular target site that may be integrally involved in root growth. 相似文献
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Cell death can be divided into the anti-inflammatory process of apoptosis and the
pro-inflammatory process of necrosis. Necrosis, as apoptosis, is a regulated form of cell
death, and Poly-(ADP-Ribose) Polymerase-1 (PARP-1) and Receptor-Interacting Protein (RIP)
1/3 are major mediators. We previously showed that absence or inhibition of PARP-1
protects mice from nephritis, however only the male mice. We therefore hypothesized that
there is an inherent difference in the cell death program between the sexes. We show here
that in an immune-mediated nephritis model, female mice show increased apoptosis compared
to male mice. Treatment of the male mice with estrogens induced apoptosis to levels
similar to that in female mice and inhibited necrosis. Although PARP-1 was activated in
both male and female mice, PARP-1 inhibition reduced necrosis only in the male mice. We
also show that deletion of RIP-3 did not have a sex bias. We demonstrate here that male
and female mice are prone to different types of cell death. Our data also suggest that
estrogens and PARP-1 are two of the mediators of the sex-bias in cell death. We therefore
propose that targeting cell death based on sex will lead to tailored and better treatments
for each gender. 相似文献
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Orwah Saleh Bertolt Gust Bj?rn Boll Hans-Peter Fiedler Lutz Heide 《The Journal of biological chemistry》2009,284(21):14439-14447
The bacterium Streptomyces anulatus 9663, isolated from the
intestine of different arthropods, produces prenylated derivatives of
phenazine 1-carboxylic acid. From this organism, we have identified the
prenyltransferase gene ppzP. ppzP resides in a gene cluster
containing orthologs of all genes known to be involved in phenazine
1-carboxylic acid biosynthesis in Pseudomonas strains as well as
genes for the six enzymes required to generate dimethylallyl diphosphate via
the mevalonate pathway. This is the first complete gene cluster of a phenazine
natural compound from streptomycetes. Heterologous expression of this cluster
in Streptomyces coelicolor M512 resulted in the formation of
prenylated derivatives of phenazine 1-carboxylic acid. After inactivation of
ppzP, only nonprenylated phenazine 1-carboxylic acid was formed.
Cloning, overexpression, and purification of PpzP resulted in a 37-kDa soluble
protein, which was identified as a 5,10-dihydrophenazine 1-carboxylate
dimethylallyltransferase, forming a C–C bond between C-1 of the
isoprenoid substrate and C-9 of the aromatic substrate. In contrast to many
other prenyltransferases, the reaction of PpzP is independent of the presence
of magnesium or other divalent cations. The Km value for
dimethylallyl diphosphate was determined as 116 μm. For
dihydro-PCA, half-maximal velocity was observed at 35 μm.
Kcat was calculated as 0.435 s-1. PpzP shows
obvious sequence similarity to a recently discovered family of
prenyltransferases with aromatic substrates, the ABBA prenyltransferases. The
present finding extends the substrate range of this family, previously limited
to phenolic compounds, to include also phenazine derivatives.The transfer of isoprenyl moieties to aromatic acceptor molecules gives
rise to an astounding diversity of secondary metabolites in bacteria, fungi,
and plants, including many compounds that are important in pharmacotherapy.
However, surprisingly little biochemical and genetic data are available on the
enzymes catalyzing the C-prenylation of aromatic substrates. Recently, a new
family of aromatic prenyltransferases was discovered in streptomycetes
(1), Gram-positive soil
bacteria that are prolific producers of antibiotics and other biologically
active compounds (2). The
members of this enzyme family show a new type of protein fold with a unique
α-β-β-α architecture
(3) and were therefore termed
ABBA prenyltransferases (1).
Only 13 members of this family can be identified by sequence similarity
searches in the data base at present, and only four of them have been
investigated biochemically
(3–6).
Up to now, only phenolic compounds have been identified as aromatic substrates
of ABBA prenyltransferases. We now report the discovery of a new member of the
ABBA prenyltransferase family, catalyzing the transfer of a dimethylallyl
moiety to C-9 of 5,10-dihydrophenazine 1-carboxylate
(dihydro-PCA).2
Streptomyces strains produce many of prenylated phenazines as natural
products. For the first time, the present paper reports the identification of
a prenyltransferase involved in their biosynthesis.Streptomyces anulatus 9663, isolated from the intestine of
different arthropods, produces several prenylated phenazines, among them
endophenazine A and B (Fig.
1A) (7).
We wanted to investigate which type of prenyltransferase might catalyze the
prenylation reaction in endophenazine biosynthesis. In streptomycetes and
other microorganisms, genes involved in the biosynthesis of a secondary
metabolite are nearly always clustered in a contiguous DNA region. Therefore,
the prenyltransferase of endophenazine biosynthesis was expected to be
localized in the vicinity of the genes for the biosynthesis of the phenazine
core (i.e. of PCA).Open in a separate windowFIGURE 1.A, prenylated phenazines from S. anulatus 9663.
B, biosynthetic gene cluster of endophenazine A.In Pseudomonas, an operon of seven genes named phzABCDEFG
is responsible for the biosynthesis of PCA
(8). The enzyme PhzC catalyzes
the condensation of phosphoenolpyruvate and erythrose-4-phosphate
(i.e. the first step of the shikimate pathway), and further enzymes
of this pathway lead to the intermediate chorismate. PhzD and PhzE catalyze
the conversion of chorismate to 2-amino-2-deoxyisochorismate and the
subsequent conversion to 2,3-dihydro-3-hydroxyanthranilic acid, respectively.
These reactions are well established biochemically. Fewer data are available
about the following steps (i.e. dimerization of
2,3-dihydro-3-hydroxyanthranilic acid, several oxidation reactions, and a
decarboxylation, ultimately leading to PCA via several instable
intermediates). From Pseudomonas, experimental data on the role of
PhzF and PhzA/B have been published
(8,
9), whereas the role of PhzG is
yet unclear. Surprisingly, the only gene cluster for phenazine biosynthesis
described so far from streptomycetes
(10) was found not to contain
a phzF orthologue, raising the question of whether there may be
differences in the biosynthesis of phenazines between Pseudomonas and
Streptomyces.Screening of a genomic library of the endophenazine producer strain S.
anulatus now allowed the identification of the first complete gene
cluster of a prenylated phenazine, including the structural gene of
dihydro-PCA dimethylallyltransferase. 相似文献
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细胞内质网上的肌醇1,4,5-三磷酸受体(inositol 1,4,5-trisphosphate receptors, IP3Rs)是调节Ca~(2+)释放的重要离子通道。Ca~(2+)稳态是维持机体细胞生理功能的重要基础,Ca~(2+)信号参与酶激活、囊泡释放和细胞凋亡等多种细胞过程。研究表明,Ca~(2+)信号异常与阿尔茨海默病(Alzheimer's disease, AD)密切相关,神经元中钙信号异常可以导致细胞稳态失衡、突触功能丧失,甚至细胞死亡。现对IP3Rs的生物特性及其介导的Ca~(2+)释放在阿尔茨海默病发生发展过程中的作用进行综述。 相似文献
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G. M. Visser R. Keemink Cecile Schattenkerk B. Kraal J. H. Van Boom 《Nucleosides, nucleotides & nucleic acids》2013,32(3):277-286
Abstract Phosphorylation of 2′-0-acetyl-3′-trifluoroacetamido-3′-deoxy-N2-palmitoylguanosine with N-morpholino-O, O-bis(1-benzotriazolyl)phos-phate gives a 5′-phosphotriester. Removal of the benzotriazolyl group and addition of pyrophosphoric acid gave, after deblocking all protecting groups, GTP(3′NH2). 相似文献
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Roujian Lu Yong Li Youwen Zhang Yunjia Chen Angela D. Shields Danny G. Winder Timothy Angelotti Kai Jiao Lee E. Limbird Yi Zhou Qin Wang 《The Journal of biological chemistry》2009,284(19):13233-13243
Although ligand-selective regulation of G protein-coupled receptor-mediated
signaling and trafficking are well documented, little is known about whether
ligand-selective effects occur on endogenous receptors or whether such effects
modify the signaling response in physiologically relevant cells. Using a gene
targeting approach, we generated a knock-in mouse line, in which N-terminal
hemagglutinin epitope-tagged α2A-adrenergic receptor (AR)
expression was driven by the endogenous mouse α2AAR gene
locus. Exploiting this mouse line, we evaluated α2AAR
trafficking and α2AAR-mediated inhibition of Ca2+
currents in native sympathetic neurons in response to clonidine and
guanfacine, two drugs used for treatment of hypertension, attention deficit
and hyperactivity disorder, and enhancement of analgesia through actions on
the α2AAR subtype. We discovered a more rapid desensitization
of Ca2+ current suppression by clonidine than guanfacine, which
paralleled a more marked receptor phosphorylation and endocytosis of
α2AAR evoked by clonidine than by guanfacine.
Clonidine-induced α2AAR desensitization, but not receptor
phosphorylation, was attenuated by blockade of endocytosis with concanavalin
A, indicating a critical role for internalization of α2AAR in
desensitization to this ligand. Our data on endogenous receptor-mediated
signaling and trafficking in native cells reveal not only differential
regulation of G protein-coupled receptor endocytosis by different ligands, but
also a differential contribution of receptor endocytosis to signaling
desensitization. Taken together, our data suggest that these
HA-α2AAR knock-in mice will serve as an important model in
developing ligands to favor endocytosis or nonendocytosis of receptors,
depending on the target cell and pathophysiology being addressed.G protein-coupled receptors
(GPCRs)4 represent the
largest family of cell surface receptors mediating responses to hormones,
cytokines, neurotransmitters, and therapeutic agents
(1). In addition to regulating
downstream signaling, ligand binding to a receptor can initiate
phosphorylation of the active conformation of the receptor by G protein
receptor kinases (GRKs) and subsequent binding of arrestins, thus restricting
the magnitude and duration of the ligand-evoked signaling responses
(2,
3). Binding of arrestins to
GPCRs also leads to GPCR internalization
(4,
5), a process that has been
proposed as a means to desensitize receptor signaling at the cell surface,
resensitize receptors, and/or initiate intracellular signaling
(6,
7).Different ligands are able to induce distinct signaling and internalization
profiles of the same receptor
(8-14).
However, the lack of available tools to study trafficking of endogenous GPCRs
in native target cells has limited our understanding of ligand-selective
endocytosis profiles and the relative contribution of receptor endocytosis to
desensitization in native biological settings.To specifically test hypotheses regarding ligand-selective effects on GPCR
internalization, and functional consequences of this trafficking on signaling,
we utilized a homologous recombination gene targeting strategy to introduce a
hemagglutinin (HA) epitope-tagged wild type α2A-adrenergic
receptor (AR) into the mouse ADRA2A gene locus
(“knock-in”). The α2AAR is a prototypical GPCR
that couples to the Gi/o subfamily of G proteins
(15). Studies on genetically
engineered mice made null or mutant for the α2AAR have
revealed that this subtype mediates the therapeutic effects of
α2-adrenergic agents on blood pressure, pain perception,
volatile anesthetic sparing, analgesia, and working memory enhancement
(16-18).
Two classic α2-ligands, clonidine and guanfacine, have been
widely used to treat hypertension
(19), attention deficit and
hyperactivity disorder (20),
and to elicit analgesia (19,
21) mediated via the
α2AAR. Clinically guanfacine has a much longer duration of
action than clonidine
(22-24);
this longer duration of action cannot be accounted for by the pharmacokinetic
profile of these agents in human beings, as both drugs have a half-life of
12-14 h (25,
26). Because ligand-induced
desensitization and trafficking of GPCRs have been implicated as critical
mechanisms for modulating response duration in vivo
(3), one hypothesis underlying
the difference in duration between clonidine and guanfacine is that clonidine
provokes accelerated desensitization of the α2AAR via one or
several mechanisms, whereas guanfacine does not. Signaling desensitization in
response to these two agonists has not been compared under the same
experimental settings. To specifically test this hypothesis, we have exploited
our HA-α2AAR knock-in mice so that we could examine these
properties of guanfacine and clonidine in native target cells.We compared internalization of the α2AAR and inhibition of
Ca2+ currents induced by clonidine and guanfacine in primary
superior cervical ganglia (SCG) neurons, where the α2AAR is
the major adrenergic receptor subtype controlling norepinephrine release and
sympathetic tone (17,
27). Our data revealed a
differential regulation of α2AAR trafficking and signaling
duration by clonidine versus guanfacine, i.e. clonidine
induced a more dramatic desensitization of the α2AAR than
guanfacine, and this desensitization was largely because of
α2AAR internalization. These studies reveal the powerful tool
that the HA-α2AAR knock-in mice provide for identifying
endocytosis-dependent and -independent physiological phenomena for this
receptor subtype as a first step in defining novel loci for therapeutic
intervention in the α2AAR signaling/trafficking cascade. 相似文献
17.
Victoria L Alonso Carla Ritagliati Pamela Cribb Esteban C Serra 《Memórias do Instituto Oswaldo Cruz》2014,109(8):1081-1085
We present here three expression plasmids for Trypanosoma cruzi
adapted to the Gateway® recombination cloning system. Two of
these plasmids were designed to express trypanosomal proteins fused to a double tag
for tandem affinity purification (TAPtag). The TAPtag and Gateway®
cassette were introduced into an episomal (pTEX) and an integrative (pTREX) plasmid.
Both plasmids were assayed by introducing green fluorescent protein (GFP) by
recombination and the integrity of the double-tagged protein was determined by
western blotting and immunofluorescence microscopy. The third Gateway adapted vector
assayed was the inducible pTcINDEX. When tested with GFP,
pTcINDEX-GW showed a good response to tetracycline, being less
leaky than its precursor (pTcINDEX). 相似文献
18.
Haihong Zong Claire C. Bastie Jun Xu Reinhard Fassler Kevin P. Campbell Irwin J. Kurland Jeffrey E. Pessin 《The Journal of biological chemistry》2009,284(7):4679-4688
Integrin receptor plays key roles in mediating both inside-out and
outside-in signaling between cells and the extracellular matrix. We have
observed that the tissue-specific loss of the integrin β1 subunit in
striated muscle results in a near complete loss of integrin β1 subunit
protein expression concomitant with a loss of talin and to a lesser extent, a
reduction in F-actin content. Muscle-specific integrin β1-deficient mice
had no significant difference in food intake, weight gain, fasting glucose,
and insulin levels with their littermate controls. However, dynamic analysis
of glucose homeostasis using euglycemichyperinsulinemic clamps demonstrated a
44 and 48% reduction of insulin-stimulated glucose infusion rate and glucose
clearance, respectively. The whole body insulin resistance resulted from a
specific inhibition of skeletal muscle glucose uptake and glycogen synthesis
without any significant effect on the insulin suppression of hepatic glucose
output or insulin-stimulated glucose uptake in adipose tissue. The reduction
in skeletal muscle insulin responsiveness occurred without any change in GLUT4
protein expression levels but was associated with an impairment of the
insulin-stimulated protein kinase B/Akt serine 473 phosphorylation but not
threonine 308. The inhibition of insulin-stimulated serine 473 phosphorylation
occurred concomitantly with a decrease in integrin-linked kinase expression
but with no change in the mTOR·Rictor·LST8 complex (mTORC2).
These data demonstrate an in vivo crucial role of integrin β1
signaling events in mediating cross-talk to that of insulin action.Integrin receptors are a large family of integral membrane proteins
composed of a single α and β subunit assembled into a heterodimeric
complex. There are 19 α and 8 β mammalian subunit isoforms that
combine to form 25 distinct α,β heterodimeric receptors
(1-5).
These receptors play multiple critical roles in conveying extracellular
signals to intracellular responses (outside-in signaling) as well as altering
extracellular matrix interactions based upon intracellular changes (inside-out
signaling). Despite the large overall number of integrin receptor complexes,
skeletal muscle integrin receptors are limited to seven α subunit
subtypes (α1, α3, α4, α5, α6, α7, and
αν subunits), all associated with the β1 integrin subunit
(6,
7).Several studies have suggested an important cross-talk between
extracellular matrix and insulin signaling. For example, engagement of β1
subunit containing integrin receptors was observed to increase
insulin-stimulated insulin receptor substrate
(IRS)2
phosphorylation, IRS-associated phosphatidylinositol 3-kinase, and activation
of protein kinase B/Akt
(8-11).
Integrin receptor regulation of focal adhesion kinase was reported to modulate
insulin stimulation of glycogen synthesis, glucose transport, and cytoskeleton
organization in cultured hepatocytes and myoblasts
(12,
13). Similarly, the
integrin-linked kinase (ILK) was suggested to function as one of several
potential upstream kinases that phosphorylate and activate Akt
(14-18).
In this regard small interfering RNA gene silencing of ILK in fibroblasts and
conditional ILK gene knockouts in macrophages resulted in a near complete
inhibition of insulin-stimulated Akt serine 473 (Ser-473) phosphorylation
concomitant with an inhibition of Akt activity and phosphorylation of Akt
downstream targets (19).
However, a complex composed of mTOR·Rictor·LST8 (termed mTORC2)
has been identified in several other studies as the Akt Ser-473 kinase
(20,
21). In addition to Ser-473,
Akt protein kinase activation also requires phosphorylation on threonine 308
Thr-30 by phosphoinositide-dependent protein kinase, PDK1
(22-24).In vivo, skeletal muscle is the primary tissue responsible for
postprandial (insulin-stimulated) glucose disposal that results from the
activation of signaling pathways leading to the translocation of the
insulin-responsive glucose transporter, GLUT4, from intracellular sites to the
cell surface membranes (25,
26). Dysregulation of any step
of this process in skeletal muscle results in a state of insulin resistance,
thereby predisposing an individual for the development of diabetes
(27-33).
Although studies described above have utilized a variety of tissue culture
cell systems to address the potential involvement of integrin receptor
signaling in insulin action, to date there has not been any investigation of
integrin function on insulin action or glucose homeostasis in vivo.
To address this issue, we have taken advantage of Cre-LoxP technology to
inactivate the β1 integrin receptor subunit gene in striated muscle. We
have observed that muscle creatine kinase-specific integrin β1 knock-out
(MCKItgβ1 KO) mice display a reduction of insulin-stimulated glucose
infusion rate and glucose clearance. The impairment of insulin-stimulated
skeletal muscle glucose uptake and glycogen synthesis resulted from a decrease
in Akt Ser-473 phosphorylation concomitant with a marked reduction in ILK
expression. Together, these data demonstrate an important cross-talk between
integrin receptor function and insulin action and suggests that ILK may
function as an Akt Ser-473 kinase in skeletal muscle. 相似文献
19.
20.