共查询到20条相似文献,搜索用时 0 毫秒
1.
Although the N-methyl-D-aspartate (NMDA) receptor plays a critical role in the central nervous system, many questions remain regarding the relationship between its structure and functional properties. In particular, the involvement of ligand-binding domain closure in determining agonist efficacy, which has been reported in other glutamate receptor subtypes, remains unresolved. To address this question, we designed dual cysteine point mutations spanning the NR1 and NR2 ligand-binding clefts, aiming to stabilize these domains in closed cleft conformations. Two mutants, E522C/I691C in NR1 (EI) and K487C/N687C in NR2 (KN) were found to exhibit significant glycine- and glutamate-independent activation, respectively, and co-expression of the two subunits produced a constitutively active channel. However, both individual mutants could be activated above constitutive levels in a concentration-dependent manner, indicating that cleft closure does not completely prevent agonist association. Interestingly, whereas the NR2 KN disulfide was found to potentiate channel gating and M3 accessibility, NR1 EI exhibited the opposite phenotype, suggesting that the EI disulfide may trap the NR1 ligand-binding domain in a lower efficacy conformation. Furthermore, both mutants affected agonist sensitivity at the opposing subunit, suggesting that closed cleft stabilization may contribute to coupling between the subunits. These results support a correlation between cleft stability and receptor activation, providing compelling evidence for the Venus flytrap mechanism of glutamate receptor domain closure. 相似文献
2.
M N Berry R B Gregory A R Grivell D C Henly J W Phillips P G Wallace G R Welch 《FEBS letters》1988,231(1):19-24
We have studied the stimulatory effects of palmitate on the rate of glucose synthesis from lactate in isolated hepatocytes. Control of the metabolic flow was achieved by modulating the activity of enolase using graded concentrations of fluoride. Unexpectedly, palmitate stimulated gluconeogenesis even when enolase was rate-limiting. This stimulation was also observed when the activities of phosphoenolpyruvate carboxykinase and aspartate aminotransferase were modulated using graded concentrations of quinolinate and aminooxyacetate, respectively. Linear force-flow relationships were found between the rate of gluconeogenesis and indicators of cellular energy status (i.e. mitochondrial membrane and redox potentials and cellular phosphorylation potential). These findings suggest that the fatty acid stimulation of glucose synthesis is in part mediated through thermodynamic mechanisms. 相似文献
3.
Direct hyposmotic stimulation of gastric acid secretion 总被引:1,自引:0,他引:1
T J Sernka 《Membrane biochemistry》1990,9(1):1-7
Gastric glands isolated from rabbit stomach were incubated in isosmotic medium or media made hyposmotic by 50-100 mOsm/kg. As indicated by radiolabeled aminopyrine accumulation, acid secretion was nearly 3 times greater in 200 mOsm/kg hyposmotic than in isosmotic medium after a 30-min incubation. The hyposmotic stimulation appeared within 2 min, peaked at 10-15 min and declined almost to the isosmotic control by 45 min. As estimated by the wet weight corrected for inulin extracellular space, the intracellular water of the glands also peaked at 15 min and returned to the isosmotic norm by 45 min. Hyposmotic stimulation of acid secretion directly involved the parietal cell, since parietal cells obtained from gastric glands were also stimulated. That the hyposmotic response was direct was indicated by omeprazole inhibition of aminopyrine accumulation in hyposmotic medium. 相似文献
4.
T J Sernka 《Membrane biochemistry》1989,8(2):107-114
Gastric glands incubated in hyposmotic medium (200 mOsm) accumulated aminopyrine, a measure of acid secretion, to the same extent as that of paired glands in isomotic medium containing histamine (10(-4) M). These maximal responses to hyposmolality and histamine were not additive. The hyposmotic response peaked earlier than the histamine response. Hyposmotic stimulation was nearly abolished by preincubation of the glands with metiamide and cimetidine, H-2 histamine antagonists. In the presence of histaminase, no hyposmotic stimulation occurred. The response to forskolin, a stimulant of adenylate cyclase, was equivalent in hyposmotic and isosmotic media. These results indicate that hyposmolality releases histamine from a paracrine cell in the gastric gland and that histamine binds to H-2 receptors on the parietal cell to initiate a cyclic AMP-mediated stimulation of acid secretion. 相似文献
5.
Busque SM Kerstetter JE Geibel JP Insogna K 《American journal of physiology. Gastrointestinal and liver physiology》2005,289(4):G664-G669
Parietal cells are the primary acid secretory cells of the stomach. We have previously shown that activation of the calcium-sensing receptor (CaSR) by divalent (Ca(2+)) or trivalent (Gd(3+)) ions stimulates acid production in the absence of secretagogues by increasing H(+),K(+)-ATPase activity. When overexpressed in HEK-293 cells, the CaSR can be allosterically activated by L-amino acids in the presence of physiological concentrations of extracellular Ca(2+) (Ca(o)(2+); 1.5-2.5 mM). To determine whether the endogenously expressed parietal cell CaSR is allosterically activated by L-amino acids, we examined the effect of the amino acids L-phenylalanine (L-Phe), L-tryptophan, and L-leucine on acid secretion. In ex vivo whole stomach preparations, exposure to L-Phe resulted in gastric luminal pH significantly lower than controls. Studies using D-Phe (inactive isomer) failed to elicit a response on gastric pH. H(+)-K(+)-ATPase activity was monitored by measuring the intracellular pH (pH(i)) of individual parietal cells in isolated rat gastric glands and calculating the rate of H(+) extrusion. We demonstrated that increasing Ca(o)(2+) in the absence of secretagogues caused a dose-dependent increase in H(+) extrusion. These effects were amplified by the addition of amino acids at various Ca(o)(2+) concentrations. Blocking the histamine-2 receptor with cimetidine or inhibiting system L-amino acid transport with 2-amino-2-norbornane-carboxylic acid did not affect the rate of H(+) extrusion in the presence of L-Phe. These data support the conclusion that amino acids, in conjunction with a physiological Ca(o)(2+) concentration, can induce acid secretion independent of hormonal stimulation via allosteric activation of the stomach CaSR. 相似文献
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The amino-terminal domain of class B G protein-coupled receptors contains six conserved cysteine residues involved in structurally and functionally critical disulfide bonds. The mapping of these bonds has been unclear, with one pattern based on biochemical and NMR structural characterizations of refolded, nonglycosylated amino-terminal fragments, and another pattern derived from functional characterizations of intact receptors having paired cysteine mutations. In the present study, we determined the disulfide bonding pattern of the prototypic class B secretin receptor by applying the same paired cysteine mutagenesis approach and confirming the predicted bonding pattern with proteolytic cleavage of intact functional receptor. As expected, systematic mutation to serine of the six conserved cysteine residues within this region of the secretin receptor singly and in pairs resulted in loss of function of most constructs. Notable exceptions were single mutations of the 4th and 6th cysteine residues and paired mutations involving the 1st and 3rd, 2nd and 5th, and 4th and 6th conserved cysteines, with secretin eliciting statistically significant cAMP responses above basal levels of activation for each of these constructs. Immunofluorescence microscopy confirmed similar levels of plasma membrane expression for each of the mutated receptors. Furthermore, cyanogen bromide cleaved a series of wild type and mutant secretin receptors, yielding patterns that agreed with our paired cysteine mutagenesis results. In conclusion, these data suggest the same pattern of disulfide bonding as that predicted previously by NMR and thus support a consistent pattern of amino-terminal disulfide bonds in class B G protein-coupled receptors. 相似文献
8.
Communication between receptor tyrosine kinase and G protein-coupled receptor (GPCR)-mediated signaling is recognized as a common integrator linking diverse aspects of intracellular signaling systems. Here, we report that G protein-coupled beta-adrenergic receptor activation leading to stimulation of salivary phospholipid release occurs with the involvement of epidermal growth factor receptor (EGFR). Using sublingual gland acinar cells, we show that prosecretory effect of isoproterenol on phospholipid release was subjected to suppression by EGFR kinase inhibitor, PD153035, and wortmannin, an inhibitor of PI3K, but not by PD98059, an inhibitor of extracellular signal regulated kinase (ERK). Furthermore, wortmannin, but not the ERK inhibitor, caused the reduction in the acinar cell secretory responses to beta-adrenergic agonist-generated cAMP as well as adenyl cyclase activator, forskolin. The acinar cell phospholipid secretory responses to isoproterenol, moreover, were inhibited by PP2, a selective inhibitor of tyrosine kinase Src responsible for ligand-independent EGFR phosphorylation. Taken together, our data are the first to demonstrate the requirement for Src kinase-dependent EGFR transactivation in regulation of salivary phospholipid secretion in response to beta-adrenergic GPCR activation. 相似文献
9.
Juan M. Duran Christophe Anjard Chris Stefan William F. Loomis Vivek Malhotra 《The Journal of cell biology》2010,188(4):527-536
Starving Dictyostelium
discoideum cells secrete AcbA, an acyl coenzyme A–binding protein (ACBP) that lacks a conventional signal sequence for entering the endoplasmic reticulum (ER). Secretion of AcbA in D. discoideum requires the Golgi-associated protein GRASP. In this study, we report that starvation-induced secretion of Acb1, the Saccharomyces cerevisiae ACBP orthologue, also requires GRASP (Grh1). This highlights the conserved function of GRASP in unconventional secretion. Although genes required for ER to Golgi or Golgi to cell surface transport are not required for Acb1 secretion in yeast, this process involves autophagy genes and the plasma membrane t-SNARE, Sso1. Inhibiting transport to vacuoles does not affect Acb1 secretion. In sum, our experiments reveal a unique secretory pathway where autophagosomes containing Acb1 evade fusion with the vacuole to prevent cargo degradation. We propose that these autophagosome intermediates fuse with recycling endosomes instead to form multivesicular body carriers that then fuse with the plasma membrane to release cargo. 相似文献
10.
Anticancer effect of retinoic acid via AP-1 activity repression is mediated by retinoic acid receptor alpha and beta in gastric cancer cells 总被引:2,自引:0,他引:2
To uncover the mechanisms relating to the anticancer effect of retinoic acids in gastric cancer cells, the mediation of activator protein-1 (AP-1) activity repression by retinoic acid receptors (RARs) was investigated. All-trans retinoic acid (ATRA) inhibited AP-1 activity in BGC-823 cells (RARalpha(+), RARbeta(+)), but not in MKN-45 cells (RARalpha(lo), RARbeta(-)). Transient transfection of RARbeta expression vector into MKN-45 cells significantly resulted in direct repression of AP-1 activity in a receptor concentration-dependent manner, and this could be strengthened by ATRA. Stable transfection of RARbeta into MKN-45 cells directly inhibited cell growth and colony formation, and ATRA also enhanced these effects. Transient transfection of RARalpha into MKN-45 cells however, displayed receptor concentration-dependent AP-1 activity inhibition only in the presence of ATRA. Stable transfection of RARalpha into MKN-45 cells resulted in ATRA-dependent inhibition of cell growth and colony formation. For AP-1 binding activity induced by TPA, the repressive effect of ATRA was only observed in BGC-823 and RARalpha and RARbeta stably transfected MKN-45 cells, but not in intact MKN-45 cells. This indicates the necessity for sufficient cellular RARalpha and/or RARbeta in order for AP-1 activity repression to occur. Deletion of DNA binding domain (DBD) of RARbeta, but not ligand binding domain (LBD), eliminated the anti-AP-1 function of RARbeta. It is therefore concluded that both RARalpha and RARbeta are mediators in the anticancer function of ATRA via AP-1 activity inhibition, and that RARbeta, not RARalpha, can inhibit AP-1 activity to a certain extent directly by itself. Thus DBD, not LBD, is critical for anti-AP-1 activity. 相似文献
11.
C1-inhibitor is a member of the serpin family of proteinase inhibitors and is an important inhibitor of complement and contact system proteinases. The native protein has the characteristic serpin feature of being in a kinetically trapped metastable state rather than in the most stable state it could adopt. A consequence of this is that it readily forms loop-sheet dimers and polymers, by a mechanism believed to be the same as observed with other serpins. An unusual feature of C1-inhibitor is that it has a unique amino-terminal domain, of unknown function, held to the serpin domain by two disulfide bonds not found in other serpins. We report here that reduction of these bonds by DTT, causes a conformational change such that the reactive center loop inserts into beta-sheet A. This form of C1-inhibitor is less stable to heat and urea than the native protein, and is more susceptible to extensive degradation by trypsin. These data show that the disulfide bonds in C1-inhibitor are required for the protein to be stabilized in the metastable state with the reactive center loop expelled from beta-sheet A. 相似文献
12.
Schmidt BZ Fowler NL Hidvegi T Perlmutter DH Colten HR 《The Journal of biological chemistry》1999,274(17):11782-11788
Factor H, a secretory glycoprotein composed of 20 short consensus repeat modules, is an inhibitor of the complement system. Previous studies of inherited factor H deficiency revealed single amino acid substitutions at conserved cysteine residues, on one allele arginine for cysteine 518 (C518R) and on the other tyrosine for cysteine 941 (C941Y) (Ault, B. H., Schmidt, B. Z., Fowler, N. L., Kashtan, C. E., Ahmed, A. E., Vogt, B. A., and Colten, H. R. (1997) J. Biol. Chem. 272, 25168-25175). To ascertain if the phenotype, impaired secretion of factor H, is due to the C518R substitution or the C941Y substitution and to ascertain the mechanism by which secretion is impaired, we studied COS-1 and HepG2 cells transfected with wild type and several mutant factor H molecules. The results showed markedly impaired secretion of both C518R and C941Y factor H as well as that of factor H molecules bearing alanine or arginine substitutions at the Cys518-Cys546 disulfide bond (C518A, C546A, C546R, C518A-C546A). In each case, mutant factor H was retained in the endoplasmic reticulum and degraded relatively slowly as compared with most other mutant secretory and membrane proteins that are retained in the endoplasmic reticulum. These data indicate that impaired secretion of the naturally occurring C518R and C941Y mutant factor H proteins is due to disruption of framework-specific disulfide bonds in factor H short consensus repeat modules. 相似文献
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14.
Philipp A Lang Roman Schniepp Philipp Kirchhoff Thenral Socrates Shafik M Sidani John P Geibel 《Cellular physiology and biochemistry》2007,20(5):527-534
Excessive gastric acid secretion plays an important role in the pathogenesis of peptic ulcers. Dexamethasone, a widely used drug, is known to stimulate gastric acid secretion and increase the incidence of peptic ulcers. However little is known about the mechanism of the dexamethasone's effect on parietal cells. The present study was performed to investigate the contribution of the phosphatidylinositol-3-kinase (PI3 kinase) to dexamethasone induced stimulation of gastric acid secretion. In vivo pretreatment with dexamethasone injections (150 microg/100g for 3 days) or in vitro exposure to (10 microM for > 20 minutes) significantly increased acid secretion in isolated gastric glands approximately 2-3 fold. The dexamethasone induced stimulation of gastric acid secretion was concentration dependent and significantly blunted by the H+/K2+ ATPase inhibitor omeprazole (200 microM), the PI3 kinase inhibitor Wortmannin (500 nM), the protein kinase inhibitor staurosporine (2.5 microM) and the Cl(-) channel blocker NPPB (100 microM); but not by the H(2) antagonist cimetidine (100 microM). In conclusion, it was observed that dexamethasone's effect on proton extrusion requires the activity of a PI3 kinase pathway, an apical Cl(-) channel and the H2+/K2+ ATPase. 相似文献
15.
The stomach divalent ion-sensing receptor scar is a modulator of gastric acid secretion 总被引:5,自引:0,他引:5
Geibel JP Wagner CA Caroppo R Qureshi I Gloeckner J Manuelidis L Kirchhoff P Radebold K 《The Journal of biological chemistry》2001,276(43):39549-39552
Divalent cation receptors have recently been identified in a wide variety of tissues and organs, yet their exact function remains controversial. We have previously identified a member of this receptor family in the stomach and have demonstrated that it is localized to the parietal cell, the acid secretory cell of the gastric gland. The activation of acid secretion has been classically defined as being regulated by two pathways: a neuronal pathway (mediated by acetylcholine) and an endocrine pathway (mediated by gastrin and histamine). Here, we identified a novel pathway modulating gastric acid secretion through the stomach calcium-sensing receptor (SCAR) located on the basolateral membrane of gastric parietal cells. Activation of SCAR in the intact rat gastric gland by divalent cations (Ca(2+) or Mg(2+)) or by the potent stimulator gadolinium (Gd(3+)) led to an increase in the rate of acid secretion through the apical H+,K+ -ATPase. Gd(3+) was able to activate acid secretion through the omeprazole-sensitive H+,K+ -ATPase even in the absence of the classical stimulator histamine. In contrast, inhibition of SCAR by reduction of extracellular cations abolished the stimulatory effect of histamine on gastric acid secretion, providing evidence for the regulation of the proton secretory transport protein by the receptor. These studies present the first example of a member of the divalent cation receptors modulating a plasma membrane transport protein and may lead to new insights into the regulation of gastric acid secretion. 相似文献
16.
In many systems, the integration of converging regulatory signals that relay on G protein-coupled receptor (GPCR) activation into functional cellular pathways requires the involvement of receptor tyrosine kinase. In this report, we provide evidence that activation of GPCR by beta-adrenergic agonist leading to stimulation in gastric mucin secretion requires epidermal growth factor receptor (EGFR) participation. Using [(3)H]glucosamine-labeled gastric mucosal cells, we show that stimulatory effect of beta-adrenergic agonist, isoproterenol, on mucin secretion was inhibited by EGFR kinase inhibitor, PD153035, as well as wortmannin, a specific inhibitor of PI3K. Both inhibitors, moreover, blunted the mucin secretory responses to beta-adrenergic agonist-generated second messenger, cAMP as well as adenylate cyclase activator, forskolin. The gastric mucin secretory responses to isoproterenol, furthermore, were inhibited by PP2, a selective inhibitor of tyrosine kinase Src responsible for ligand-independent EGFR autophosphorylation, but not by ERK inhibitor, PD98059. The inhibition of ERK, moreover, did not cause attenuation in mucin secretion in response to cAMP and forskolin. The findings underline the role of EGFR as a convergence point in gastric mucin secretion triggered by beta-adrenergic GPCR activation, and demonstrate the requirement for Src kinase in EGFR transactivation. 相似文献
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Biochemical evidence indicates that TGF-beta-activated kinase 1 (TAK1), a key modulator of the inflammatory response, exists in a complex with various adaptor proteins including the TAK1 binding protein 1 (TAB1). However, the physiological importance of TAB1 in TAK1 activation, and in the subsequent induction of proinflammatory mediators, remains unclear. In this study, a critical role for TAK1 in IL-1alpha or TNFalpha stimulated MAPK and NFkappaB activation was confirmed by inhibition of the nuclear accumulation of NFkappaB p65 and phosphorylated forms of c-Jun and p38 following siRNA mediated TAK1 silencing. These effects were associated with significant reductions in IL-1alpha stimulated levels of secreted IL-6, IL-8, MCP-1 and GM-CSF. In contrast, IL-1alpha or TNFalpha dependent cellular redistribution of NFkappaB p65 and phosphorylated c-Jun and p38 was not affected by 80% siRNA mediated knockdown of TAB1 protein levels. Interestingly, IL-6, IL-8 and GM-CSF release from TAB1 siRNA transfected cells was significantly reduced following IL-1alpha treatment, but was unchanged after TNFalpha stimulation, suggesting differential roles for TAB1 in IL-1alpha and TNFalpha signalling pathways. These findings may imply an as yet unidentified role for TAB1 in the inflammatory response, which is independent of the activation of classical TAK1 associated signalling cascades. 相似文献
19.
Mechanisms underlying intracisternal TRH-induced stimulation of gastric acid secretion in rats 总被引:1,自引:0,他引:1
The neurohumoral pathways mediating intracisternal TRH-induced stimulation of gastric acid secretion were investigated. In urethane-anesthetized rats, with gastric and intrajugular cannulas, TRH or the analog [N-Val2]-TRH (1 microgram) injected intracisternally increased gastric acid output for 90 min. Serum gastrin levels were not elevated significantly. Under these conditions the TRH analog, unlike TRH, was devoid of thyrotropin-releasing activity as measured by serum TSH levels. In pylorus-ligated rats, gastrin values were not modified 2 h after peptide injection whereas gastric acid output was enhanced. TRH (0.1-1 micrograms) stimulated vagal efferent discharge, recorded from a multifiber preparation of the cervical vagus in urethane-anesthetized rats and the response was dose-dependent. The time course of vagal activation was well correlated with the time profile of gastric stimulation measured every 2 min. These results demonstrated that gastric acid secretory stimulation elicited by intracisternal TRH is not related to changes in circulating levels of gastrin or TSH but is mediated by the activation of efferent vagal pathways that stimulated parietal cell secretion. 相似文献
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