共查询到20条相似文献,搜索用时 9 毫秒
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Nicholas W. Bellono Julia A. Najera Elena Oancea 《The Journal of general physiology》2014,143(2):203-214
While short exposure to solar ultraviolet radiation (UVR) can elicit increased skin pigmentation, a protective response mediated by epidermal melanocytes, chronic exposure can lead to skin cancer and photoaging. However, the molecular mechanisms that allow human skin to detect and respond to UVR remain incompletely understood. UVR stimulates a retinal-dependent signaling cascade in human melanocytes that requires GTP hydrolysis and phospholipase C β (PLCβ) activity. This pathway involves the activation of transient receptor potential A1 (TRPA1) ion channels, an increase in intracellular Ca2+, and an increase in cellular melanin content. Here, we investigated the identity of the G protein and downstream elements of the signaling cascade and found that UVR phototransduction is Gαq/11 dependent. Activation of Gαq/11/PLCβ signaling leads to hydrolysis of phosphatidylinositol (4,5)-bisphosphate (PIP2) to generate diacylglycerol (DAG) and inositol 1, 4, 5-trisphosphate (IP3). We found that PIP2 regulated TRPA1-mediated photocurrents, and IP3 stimulated intracellular Ca2+ release. The UVR-elicited Ca2+ response appears to involve both IP3-mediated release from intracellular stores and Ca2+ influx through TRPA1 channels, showing the fast rising phase of the former and the slow decay of the latter. We propose that melanocytes use a UVR phototransduction mechanism that involves the activation of a Gαq/11-dependent phosphoinositide cascade, and resembles light phototransduction cascades of the eye. 相似文献
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Hirosato Mashima Noboru Watanabe Masanari Sekine Satohiro Matsumoto Takeharu Asano Kazuhito Yuhashi Noriyoshi Sagihara Shunsuke Urayoshi Takeshi Uehara Junichi Fujiwara Takehiro Ishii Rumiko Tsuboi Hiroyuki Miyatani Hirohide Ohnishi 《Biochemistry and Biophysics Reports》2018
Intestinal homeostasis and the coordinated actions of digestion, absorption and excretion are tightly regulated by a number of gastrointestinal hormones. Most of them exert their actions through G-protein-coupled receptors. Recently, we showed that the absence of Gαq/Gα11 signaling impaired the maturation of Paneth cells, induced their differentiation toward goblet cells, and affected the regeneration of the colonic mucosa in an experimental model of colitis. Although an immunohistochemical study showed that Gαq/Gα11 were highly expressed in enterocytes, it seemed that enterocytes were not affected in Int-Gq/G11 double knock-out intestine. Thus, we used an intestinal epithelial cell line to examine the role of signaling through Gαq/Gα11 in enterocytes and manipulated the expression level of Gαq and/or Gα11. The proliferation was inhibited in IEC-6 cells that overexpressed Gαq/Gα11 and enhanced in IEC-6 cells in which Gαq/Gα11 was downregulated. The expression of T-cell factor 1 was increased according to the overexpression of Gαq/Gα11. The expression of Notch1 intracellular cytoplasmic domain was decreased by the overexpression of Gαq/Gα11 and increased by the downregulation of Gαq/Gα11. The relative mRNA expression of Muc2, a goblet cell marker, was elevated in a Gαq/Gα11 knock-down experiment. Our findings suggest that Gαq/Gα11-mediated signaling inhibits proliferation and may support a physiological function, such as absorption or secretion, in terminally differentiated enterocytes. 相似文献
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Mark R. Nance Barry Kreutz Valerie M. Tesmer Rachel Sterne-Marr Tohru Kozasa John J.G. Tesmer 《Structure (London, England : 1993)》2013,21(3):438-448
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Raja Bhattacharyya Jayashree Banerjee Kamel Khalili Philip B. Wedegaertner 《Cellular signalling》2009,21(6):996-1006
Regulator of G protein signaling domain-containing Rho guanine-nucleotide exchange factors (RGS-RhoGEFs) directly links activated forms of the G12 family of heterotrimeric G protein α subunits to the small GTPase Rho. Stimulation of G12/13-coupled GPCRs or expression of constitutively activated forms of α12 and α13 has been shown to induce the translocation of the RGS-RhoGEF, p115-RhoGEF, from the cytoplasm to the plasma membrane (PM). However, little is known regarding the functional importance and mechanisms of this regulated PM recruitment, and thus PM recruitment of p115-RhoGEF is the focus of this report. A constitutively PM-localized mutant of p115-RhoGEF shows a much greater activity compared to wild type p115-RhoGEF in promoting Rho-dependent neurite retraction of NGF-differentiated PC12 cells, providing the first evidence that PM localization can activate p115-RhoGEF signaling. Next, we uncovered the unexpected finding that Rho is required for α13-induced PM translocation of p115-RhoGEF. However, inhibition of Rho did not prevent α12-induced PM translocation of p115-RhoGEF. Additional differences between α13 and α12 in promoting PM recruitment of p115-RhoGEF were revealed by analyzing RGS domain mutants of p115-RhoGEF. Activated α12 effectively recruits the isolated RGS domain of p115-RhoGEF to the PM, whereas α13 only weakly does. On the other hand, α13 strongly recruits to the PM a p115-RhoGEF mutant containing amino acid substitutions in an acidic region at the N-terminus of the RGS domain; however, α12 is unable to recruit this p115-RhoGEF mutant to the PM. These studies provide new insight into the function and mechanisms of α12/13-mediated PM recruitment of p115-RhoGEF. 相似文献
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Dieter Paul Hoyer Sabine Grönke Konrad F. Frank Klaus Addicks Nina Wettschureck Stefan Offermanns Erland Erdmann Hannes Reuter 《Molecular and cellular biochemistry》2010,341(1-2):235-244
Neurohumoral stimulation of Gq-coupled receptors has been proposed as a central mechanism in the pathogenesis of diabetic heart disease. The resulting contractile dysfunction is closely related to abnormal intracellular Ca2+ handling with functional defects of the sarcoplasmic reticulum (SR). The present study was therefore designed to determine the role of Gq-protein signaling via Gα11 and Gαq in diabetes for the induction of functional and structural changes in the Ca2+ release complex of the SR. An experimental type 1-diabetes was induced in wild type, Gα11 knockout, and Gα11/q-knockout mice by injection of streptozotocin. Cardiac morphology and function was assessed in vivo by echocardiography. SR Ca2+ leak was tested in vitro based on a 45Ca2+ assay and protein densities as well as gene expression of ryanodine receptor (RyR2), FKBP12.6, sorcin, and annexin A7 were analyzed by immunoblot and RT-PCR. In wild type animals 8 weeks of diabetes resulted in cardiac hypertrophy and SR Ca2+ leak was increased. In addition, diabetic wild type animals showed reduced protein levels of FKBP12.6 and annexin A7. In Gα11- and Gα11/q-knockout animals, however, SR Ca2+ release and cardiac phenotype remained unchanged upon induction of diabetes. Densities of the proteins that we presently analyzed were also unaltered in Gα11-knockout mice. Gα11/q-knockout animals even showed increased expression of sorcin and annexin A7. Thus, based on the present study we suggest a signaling pathway via the Gq-proteins, Gα11 and Gαq, that could link increased neurohumoral stimulation in diabetes with defective RyR2 channel function by regulating protein expression of FKBP12.6, annexin A7, and sorcin. 相似文献
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Inga Preuß Barbara Kurig Bernd Nürnberg Joachim H.C. Orth Klaus Aktories 《Cellular signalling》2009,21(4):551-558
The mitogenic Pasteurella multocida toxin (PMT) is a major virulence factor of P. multocida, which causes Pasteurellosis in man and animals. The toxin activates the small GTPase RhoA, the MAP kinase ERK and STAT proteins via the stimulation of members of two G protein families, Gq and G12/13. PMT action also results in an increase in inositol phosphates, which is due to the stimulation of PLCβ via Gαq. Recent studies indicate that PMT additionally activates Gαi to inhibit adenylyl cyclase. Here we show that PMT acts not only via Gα but also through Gβγ signaling. Activation of Gβγ by PMT causes stimulation of phosphoinositide 3-kinase (PI3K) γ and formation of phosphatidylinositol-3,4,5-trisphosphate (PIP3) as indicated by the recruitment of a PIP3-binding pleckstrin homology (PH) domain-containing protein to the plasma membrane. Moreover, it is demonstrated that Gβγ is necessary for PMT-induced signaling via Gα. Mutants of Gαq incapable of binding or releasing Gβγ are not activated by PMT. Similarly, sequestration of Gβγ inhibits PMT-induced Gα-signaling. 相似文献
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The tumor suppressor Fhit protein is defective or absent in many tumor cells due to methylation, mutation or deletion of the FHIT gene. Despite numerous attempts to unravel the functions of Fhit, the mechanisms by which the function and expression of Fhit are regulated remain poorly understood. We have recently shown that activated Gαq subunits interact directly with Fhit and enhance its inhibitory effect on cell growth. Here we investigated the regulation of Fhit expression by Gq. Our results showed that Fhit was up-regulated specifically by activating Gα subunits of the Gq subfamily but not by those of the other G protein subfamilies. This up-regulation effect was mediated by a PKC/MEK pathway independent of Src-mediated Fhit Tyr114 phosphorylation. We further demonstrated that elevated Fhit expression was due to the specific regulation of Fhit protein synthesis in the ribosome by activated Gαq, where the regulations of cap-dependent protein synthesis were apparently not required. Moreover, we showed that activated Gαq could increase cell–cell adhesion through Fhit. These findings provide a possible handle to modulate the level of the Fhit tumor suppressor by manipulating the activity of Gq-coupled receptors. 相似文献
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Chen Z Guo L Hadas J Gutowski S Sprang SR Sternweis PC 《The Journal of biological chemistry》2012,287(30):25490-25500
RGS-containing RhoGEFs (RGS-RhoGEFs) represent a direct link between the G(12) class of heterotrimeric G proteins and the monomeric GTPases. In addition to the canonical Dbl homology (DH) and pleckstrin homology domains that carry out the guanine nucleotide exchange factor (GEF) activity toward RhoA, these RhoGEFs also possess RGS homology (RH) domains that interact with activated α subunits of G(12) and G(13). Although the GEF activity of p115-RhoGEF (p115), an RGS-RhoGEF, can be stimulated by Gα(13), the exact mechanism of the stimulation has remained unclear. Using combined studies with small angle x-ray scattering, biochemistry, and mutagenesis, we identify an additional binding site for activated Gα(13) in the DH domain of p115. Small angle x-ray scattering reveals that the helical domain of Gα(13) docks onto the DH domain, opposite to the surface of DH that binds RhoA. Mutation of a single tryptophan residue in the α3b helix of DH reduces binding to activated Gα(13) and ablates the stimulation of p115 by Gα(13). Complementary mutations at the predicted DH-binding site in the αB-αC loop of the helical domain of Gα(13) also affect stimulation of p115 by Gα(13). Although the GAP activity of p115 is not required for stimulation by Gα(13), two hydrophobic motifs in RH outside of the consensus RGS box are critical for this process. Therefore, the binding of Gα(13) to the RH domain facilitates direct association of Gα(13) to the DH domain to regulate its exchange activity. This study provides new insight into the mechanism of regulation of the RGS-RhoGEF and broadens our understanding of G protein signaling. 相似文献
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EM Myers 《PloS one》2012,7(7):e40368
Caenorhabditis elegans enter an alternate developmental stage called dauer in unfavorable conditions such as starvation, overcrowding, or high temperature. Several evolutionarily conserved signaling pathways control dauer formation. DAF-7/TGFβ and serotonin, important ligands in these signaling pathways, affect not only dauer formation, but also the expression of one another. The heterotrimeric G proteins GOA-1 (Gα(o)) and EGL-30 (Gα(q)) mediate serotonin signaling as well as serotonin biosynthesis in C. elegans. It is not known whether GOA-1 or EGL-30 also affect dauer formation and/or daf-7 expression, which are both modulated in part by serotonin. The purpose of this study is to better understand the relationship between proteins important for neuronal signaling and developmental plasticity in both C. elegans and humans. Using promoter-GFP transgenic worms, it was determined that both goa-1 and egl-30 regulate daf-7 expression during larval development. In addition, the normal daf-7 response to high temperature or starvation was altered in goa-1 and egl-30 mutants. Despite the effect of goa-1 and egl-30 mutations on daf-7 expression in various environmental conditions, there was no effect of the mutations on dauer formation. This paper provides evidence that while goa-1 and egl-30 are important for normal daf-7 expression, mutations in these genes are not sufficient to disrupt dauer formation. 相似文献
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Yan Zhang Ji-Yeon Kim Orapin Horst Yukiko Nakano Li Zhu Ralf J. Radlanski Sunita Ho Pamela K. Den Besten 《PloS one》2014,9(8)
Dental fluorosis is characterized by subsurface hypomineralization and increased porosity of enamel, associated with a delay in the removal of enamel matrix proteins. To investigate the effects of fluoride on ameloblasts, A/J mice were given 50 ppm sodium fluoride in drinking water for four weeks, resulting serum fluoride levels of 4.5 µM, a four-fold increase over control mice with no fluoride added to drinking water. MicroCT analyses showed delayed and incomplete mineralization of fluorosed incisor enamel as compared to control enamel. A microarray analysis of secretory and maturation stage ameloblasts microdissected from control and fluorosed mouse incisors showed that genes clustered with Mmp20 appeared to be less downregulated in maturation stage ameloblasts of fluorosed incisors as compared to control maturation ameloblasts. One of these Mmp20 co-regulated genes was the global chromatin organizer, special AT-rich sequence-binding protein-1 (SATB1). Immunohistochemical analysis showed increased SATB1 protein present in fluorosed ameloblasts compared to controls. In vitro, exposure of human ameloblast-lineage cells to micromolar levels of both NaF and AlF3 led to a significantly increase in SATB1 protein content, but not levels of Satb1 mRNA, suggesting a fluoride-induced mechanism protecting SABT1 from degradation. Consistent with this possibility, we used immunohistochemistry and Western blot to show that fluoride exposed ameloblasts had increased phosphorylated PKCα both in vivo and in vitro. This kinase is known to phosphorylate SATB1, and phosphorylation is known to protect SATB1 from degradation by caspase-6. In addition, production of cellular diacylglycerol (DAG) was significantly increased in fluorosed ameloblasts, suggesting that the increased phosphorylation of SATB1 may be related to an effect of fluoride to enhance Gαq activity of secretory ameloblasts. 相似文献
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mobilization of body fat is essential for energy homeostasis in animals. In insects, the adipokinetic hormone (Akh) systemically controls body fat mobilization. Biochemical evidence supports that Akh signals via a G protein-coupled receptor (GPCR) called Akh receptor (AkhR) using cyclic-AMP (cAMP) and Ca2+ second messengers to induce storage lipid release from fat body cells. Recently, we provided genetic evidence that the intracellular calcium (iCa2+) level in fat storage cells controls adiposity in the fruit fly Drosophila melanogaster. However, little is known about the genes, which mediate Akh signalling downstream of the AkhR to regulate changes in iCa2+. Here, we used thermogenetics to provide in vivo evidence that the GPCR signal transducers G protein α q subunit (Gαq), G protein γ1 (Gγ1) and Phospholipase C at 21C (Plc21C) control cellular and organismal fat storage in Drosophila. Transgenic modulation of Gαq, Gγ1 and Plc21C affected the iCa2+ of fat body cells and the expression profile of the lipid metabolism effector genes midway and brummer, which results in severely obese or lean flies. Moreover, functional impairment of Gαq, Gγ1 and Plc21C antagonised Akh-induced fat depletion. This study characterizes Gαq, Gγ1 and Plc21C as anti-obesity genes and supports the model that Akh employs the Gαq/Gγ1/Plc21C module of iCa2+ control to regulate lipid mobilization in adult Drosophila. 相似文献
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Cui R He J Mei R de Fromentel CC Martel-Planche G Taniere P Hainaut P 《Biochemical and biophysical research communications》2005,336(1):339-345
Diabetic microangiopathy is often observed in diabetic patients, but there is little evidence regarding the relationship between post-prandial glycemia or insulinemia and the incidence of diabetic microangiopathy. In this study, to elucidate the relationship between post-prandial glycemia (or insulinemia) and diabetic microangiopathy, we performed a cross-sectional study of 232 subjects with type 2 diabetes mellitus who were not being treated with insulin injections. A multiple regression analysis showed that post-prandial hyperglycemia independently correlated with the incidence of diabetic retinopathy and neuropathy. Post-prandial hyperglycemia also correlated, although not independently, with the incidence of diabetic nephropathy. In addition, interestingly, post-prandial hypoinsulinemia independently correlated with the incidence of diabetic retinopathy, although not correlated with diabetic neuropathy or nephropathy. In conclusion, post-prandial hyperglycemia, rather than fasting glycemia or hemoglobin A1c levels, is an important predictor of the incidence of diabetic microangiopathy in Japanese type 2 diabetic patients. 相似文献
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Aoyama C Sugimoto H Ando H Yamashita S Horibata Y Sugimoto S Satou M 《The Biochemical journal》2011,440(2):241-250
In a previous study we purified a novel lysoPLD (lysophospholipase D) which converts LPC (lysophosphatidylcholine) into a bioactive phospholipid, LPA (lysophosphatidic acid), from the rat brain. In the present study, we identified the purified 42 and 35 kDa proteins as the heterotrimeric G protein subunits Gα(q) and Gβ(1) respectively. When FLAG-tagged Gα(q) or Gβ(1) was expressed in cells and purified, significant lysoPLD activity was observed in the microsomal fractions. Levels of the hydrolysed product choline increased over time, and the Mg(2+) dependency and substrate specificity of Gα(q) were similar to those of lysoPLD purified from the rat brain. Mutation of Gα(q) at amino acids Lys(52), Thr(186) or Asp(205), residues that are predicted to interact with nucleotide phosphates or catalytic Mg(2+), dramatically reduced lysoPLD activity. GTP does not compete with LPC for the lysoPLD activity, indicating that these substrate-binding sites are not identical. Whereas the enzyme activity of highly purified FLAG-tagged Gα(q) overexpressed in COS-7 cells was ~4 nmol/min per mg, the activity from Neuro2A cells was 137.4 nmol/min per mg. The calculated K(m) and V(max) values for lysoPAF (1-O-hexadecyl-sn-glycero-3-phosphocholine) obtained from Neuro2A cells were 21 μM and 0.16 μmol/min per mg respectively, similar to the enzyme purified from the rat brain. These results reveal a new function for Gα(q) and Gβ(1) as an enzyme with lysoPLD activity. Tag-purified Gα(11) also exhibited a high lysoPLD activity, but Gα(i) and Gα(s) did not. The lysoPLD activity of the Gα subunit is strictly dependent on its subfamily and might be important for cellular responses. However, treatment of Hepa-1 cells with Gα(q) and Gα(11) siRNAs (small interfering RNAs) did not change lysoPLD activity in the microsomal fraction. Clarification of the physiological relevance of lysoPLD activity of these proteins will need further studies. 相似文献
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Waldron RT Innamorati G Torres-Marquez ME Sinnett-Smith J Rozengurt E 《Cellular signalling》2012,24(4):914-921
Protein kinase D (PKD) is activated within cells by stimulation of multiple G protein coupled receptors (GPCR). Earlier studies demonstrated a role for PKC to mediate rapid activation loop phosphorylation-dependent PKD activation. Subsequently, a novel PKC-independent pathway in response to Gαq-coupled GPCR stimulation was identified. Here, we examined further the specificity and PKC-dependence of PKD activation using COS-7 cells cotransfected with different Gq-family Gα and stimulated with aluminum fluoride (AlF4−). PKD activation was measured by kinase assays, and Western blot analysis of activation loop sites Ser744, a prominent and rapid PKC transphosphorylation site, and Ser748, a site autophosphorylated in the absence of PKC signaling. Treatment with AlF4− potently induced PKD activation and Ser744 and Ser748 phosphorylation, in the presence of cotransfected Gαq, Gα11, Gα14 or Gα15. These treatments achieved PKD activation loop phosphorylation similar to the maximal levels obtained by stimulation with the phorbol ester, PDBu. Preincubation with the PKC inhibitor GF1 potently blocked Gα11-, Gα14-, and Gα15-mediated enhancement of Ser748 phosphorylation induced by AlF4−, and largely abolished Ser744 phosphorylation. In contrast, Ser748 phosphorylation was almost completely intact, and Ser744 phosphorylation was significantly activated in cells cotransfected with Gαq. Importantly, the differential Ser748 phosphorylation was also promoted by treatment of Swiss 3T3 cells with Pasteurella multocida toxin, a selective activator of Gαq but not Gα11. Taken together, our results suggest that Gαq, but not the closely related Gα11, promotes PKD activation in response to GPCR ligands in a unique manner leading to PKD autophosphorylation at Ser748. 相似文献