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1
DeFea, K. A., J. Zalevsky, M. S. Thoma, O. Déry, R. D. Mullins, and N. W. Bunnett. "β-Arrestin–Dependent Endocytosis of Proteinase-Activated Receptor 2 Is Required for Intracellular Targeting of Activated Erk1/2." Journal of Cell Biology 148, no. 6 (March 20, 2000): 1267–82. http://dx.doi.org/10.1083/jcb.148.6.1267.
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Recently, a requirement for β-arrestin–mediated endocytosis in the activation of extracellular signal–regulated kinases 1 and 2 (ERK1/2) by several G protein–coupled receptors (GPCRs) has been proposed. However, the importance of this requirement for function of ERK1/2 is unknown. We report that agonists of Gαq-coupled proteinase–activated receptor 2 (PAR2) stimulate formation of a multiprotein signaling complex, as detected by gel filtration, immunoprecipitation and immunofluorescence. The complex, which contains internalized receptor, β-arrestin, raf-1, and activated ERK, is required for ERK1/2 activation. However, ERK1/2 activity is retained in the cytosol and neither translocates to the nucleus nor causes proliferation. In contrast, a mutant PAR2 (PAR2δST363/6A), which is unable to interact with β-arrestin and, thus, does not desensitize or internalize, activates ERK1/2 by a distinct pathway, and fails to promote both complex formation and cytosolic retention of the activated ERK1/2. Whereas wild-type PAR2 activates ERK1/2 by a PKC-dependent and probably a ras-independent pathway, PAR2(δST363/6A) appears to activate ERK1/2 by a ras-dependent pathway, resulting in increased cell proliferation. Thus, formation of a signaling complex comprising PAR2, β-arrestin, raf-1, and activated ERK1/2 might ensure appropriate subcellular localization of PAR2-mediated ERK activity, and thereby determine the mitogenic potential of receptor agonists.
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Jastrzebska, Beata, Yaroslav Tsybovsky, and Krzysztof Palczewski. "Complexes between photoactivated rhodopsin and transducin: progress and questions." Biochemical Journal 428, no. 1 (April 28, 2010): 1–10. http://dx.doi.org/10.1042/bj20100270.
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Activation of GPCRs (G-protein-coupled receptors) leads to conformational changes that ultimately initiate signal transduction. Activated GPCRs transiently combine with and activate heterotrimeric G-proteins resulting in GTP replacement of GDP on the G-protein α subunit. Both the detailed structural changes essential for productive GDP/GTP exchange on the G-protein α subunit and the structure of the GPCR–G-protein complex itself have yet to be elucidated. Nevertheless, transient GPCR–G-protein complexes can be trapped by nucleotide depletion, yielding an empty-nucleotide G-protein–GPCR complex that can be isolated. Whereas early biochemical studies indicated formation of a complex between G-protein and activated receptor only, more recent results suggest that G-protein can bind to pre-activated states of receptor or even couple transiently to non-activated receptor to facilitate rapid responses to stimuli. Efficient and reproducible formation of physiologically relevant, conformationally homogenous GPCR–G-protein complexes is a prerequisite for structural studies designed to address these possibilities.
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Klein, N. P., and R. J. Schneider. "Activation of Src family kinases by hepatitis B virus HBx protein and coupled signaling to Ras." Molecular and Cellular Biology 17, no. 11 (November 1997): 6427–36. http://dx.doi.org/10.1128/mcb.17.11.6427.
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The HBx protein of hepatitis B virus (HBV) is a small transcriptional transactivator that is essential for infection by the mammalian hepadnaviruses and is thought to be a cofactor in HBV-mediated liver cancer. HBx stimulates signal transduction pathways by acting in the cytoplasm, which accounts for many but not all of its transcriptional activities. Studies have shown that HBx protein activates Ras and downstream Ras signaling pathways including Raf, mitogen-activated protein (MAP) kinase kinase kinase (MEK), and MAP kinases. In this study, we investigated the mechanism of activation of Ras by HBx because it has been found to be central to the ability of HBx protein to stimulate transcription and to release growth arrest in quiescent cells. In contrast to the transient but strong stimulation of Ras typical of autocrine factors, activation of Ras by HBx protein was found to be constitutive but moderate. HBx induced the association of Ras upstream activating proteins Shc, Grb2, and Sos and stimulated GTP loading onto Ras, but without directly participating in complex formation. Instead, HBx is shown to stimulate Ras-activating proteins by functioning as an intracellular cytoplasmic activator of the Src family of tyrosine kinases, which can signal to Ras. HBx protein stimulated c-Src and Fyn kinases for a prolonged time. Activation of Src is shown to be indispensable for a number of HBx activities, including activation of Ras and the Ras-Raf-MAP kinase pathway and stimulation of transcription mediated by transcription factor AP-1. Importantly, HBx protein expressed in cultured cells during HBV replication is shown to activate the Ras signaling pathway. Mechanisms by which HBx protein might activate Src kinases are discussed.
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He, John Cijiang, Susana R. Neves, J. Dedrick Jordan, and Ravi Iyengar. "Role of the Go/i signaling network in the regulation of neurite outgrowthThis paper is one of a selection of papers published in this Special issue, entitled Second Messengers and Phosphoproteins—12th International Conference." Canadian Journal of Physiology and Pharmacology 84, no. 7 (July 2006): 687–94. http://dx.doi.org/10.1139/y06-025.
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Neurite outgrowth is a complex differentiation process stimulated by many neuronal growth factors and transmitters and by electrical activity. Among these stimuli are ligands for G-protein-coupled receptors (GPCR) that function as neurotransmitters. The pathways involved in GPCR-triggered neurite outgrowth are not fully understood. Many of these receptors couple to Gαo, one of the most abundant proteins in the neuronal growth cones. We have studied the Go signaling network involved in neurite outgrowth in Neuro2A cells. Gαo can induce neurite outgrowth. The CB1 cannabinoid receptor, a Go/i-coupled receptor expressed endogenously in Neuro2A cells, triggers neurite outgrowth by activating Rap1, which promotes the Gαo-stimulated proteasomal degradation of Rap1GAPII. CB1-receptor-mediated Rap1 activation leads to the activation of a signaling network that includes the small guanosine triphosphate (GTP)ases Ral and Rac, the protein kinases Src, and c-Jun N-terminal kinase (JNK), which converge onto the activation of signal transducer and activator of transcription 3 (Stat3), a key transcription factor that mediates the gene expression process of neurite outgrowth in Neuro2A cells. This review describes current findings from our laboratory and also discusses alternative pathways that Go/i might mediate to trigger neurite outgrowth. We also analyze the role neurotransmitters, which stimulate Go/i to activate a complex signaling network controlling neurite outgrowth, play in regeneration after neuronal injury.
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Falati, Shahrokh, Christine E. Edmead, and Alastair W. Poole. "Glycoprotein Ib-V-IX, a Receptor for von Willebrand Factor, Couples Physically and Functionally to the Fc Receptor γ-Chain, Fyn, and Lyn to Activate Human Platelets." Blood 94, no. 5 (September 1, 1999): 1648–56. http://dx.doi.org/10.1182/blood.v94.5.1648.
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Abstract The adhesion molecule von Willebrand factor (vWF) activates platelets upon binding 2 surface receptors, glycoprotein (GP) Ib-V-IX and integrin IIbβ3. We have used 2 approaches to selectively activate GP Ib using either the snake venom lectin alboaggregin-A or mutant recombinant forms of vWF (▵A1-vWF and RGGS-vWF) with selective binding properties to its 2 receptors. We show that activation of GP Ib induces platelet aggregation, secretion of 5-hydroxy tryptamine (5-HT), and an increase in cytosolic calcium. Syk becomes tyrosine phosphorylated and activated downstream of GP Ib, and associates with several tyrosine-phosphorylated proteins including the Fc receptor γ-chain through interaction with Syk SH2 domains. GP Ib physically associates with the γ-chain in GST-Syk-SH2 precipitates from platelets stimulated through GP Ib, and 2 Src family kinases, Lyn and Fyn, also associate with this signaling complex. In addition, GP Ib stimulation couples to tyrosine phosphorylation of phospholipase Cγ2. The Src family-specific inhibitor PP1 dose-dependently inhibits phosphorylation of Syk, its association with tyrosine-phosphorylated γ-chain, phosphorylation of PLCγ2, platelet aggregation, and 5-HT release. The results indicate that, upon activation, GP Ib is physically associated with FcR γ-chain and members of the Src family kinases, leading to phosphorylation of the γ-chain, recruitment, and activation of Syk. Phosphorylation of PLCγ2 also lies downstream of Src kinase activation and may critically couple early signaling events to functional platelet responses.
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Falati, Shahrokh, Christine E. Edmead, and Alastair W. Poole. "Glycoprotein Ib-V-IX, a Receptor for von Willebrand Factor, Couples Physically and Functionally to the Fc Receptor γ-Chain, Fyn, and Lyn to Activate Human Platelets." Blood 94, no. 5 (September 1, 1999): 1648–56. http://dx.doi.org/10.1182/blood.v94.5.1648.417k31_1648_1656.
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The adhesion molecule von Willebrand factor (vWF) activates platelets upon binding 2 surface receptors, glycoprotein (GP) Ib-V-IX and integrin IIbβ3. We have used 2 approaches to selectively activate GP Ib using either the snake venom lectin alboaggregin-A or mutant recombinant forms of vWF (▵A1-vWF and RGGS-vWF) with selective binding properties to its 2 receptors. We show that activation of GP Ib induces platelet aggregation, secretion of 5-hydroxy tryptamine (5-HT), and an increase in cytosolic calcium. Syk becomes tyrosine phosphorylated and activated downstream of GP Ib, and associates with several tyrosine-phosphorylated proteins including the Fc receptor γ-chain through interaction with Syk SH2 domains. GP Ib physically associates with the γ-chain in GST-Syk-SH2 precipitates from platelets stimulated through GP Ib, and 2 Src family kinases, Lyn and Fyn, also associate with this signaling complex. In addition, GP Ib stimulation couples to tyrosine phosphorylation of phospholipase Cγ2. The Src family-specific inhibitor PP1 dose-dependently inhibits phosphorylation of Syk, its association with tyrosine-phosphorylated γ-chain, phosphorylation of PLCγ2, platelet aggregation, and 5-HT release. The results indicate that, upon activation, GP Ib is physically associated with FcR γ-chain and members of the Src family kinases, leading to phosphorylation of the γ-chain, recruitment, and activation of Syk. Phosphorylation of PLCγ2 also lies downstream of Src kinase activation and may critically couple early signaling events to functional platelet responses.
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Son, Ga-Yeon, Krishna Prasad Subedi, Hwei Ling Ong, Lucile Noyer, Hassan Saadi, Changyu Zheng, Rajesh Bhardwaj, Stefan Feske, and Indu Suresh Ambudkar. "STIM2 targets Orai1/STIM1 to the AKAP79 signaling complex and confers coupling of Ca2+entry with NFAT1 activation." Proceedings of the National Academy of Sciences 117, no. 28 (June 29, 2020): 16638–48. http://dx.doi.org/10.1073/pnas.1915386117.
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The Orai1 channel is regulated by stromal interaction molecules STIM1 and STIM2 within endoplasmic reticulum (ER)-plasma membrane (PM) contact sites. Ca2+signals generated by Orai1 activate Ca2+-dependent gene expression. When compared with STIM1, STIM2 is a weak activator of Orai1, but it has been suggested to have a unique role in nuclear factor of activated T cells 1 (NFAT1) activation triggered by Orai1-mediated Ca2+entry. In this study, we examined the contribution of STIM2 in NFAT1 activation. We report that STIM2 recruitment of Orai1/STIM1 to ER-PM junctions in response to depletion of ER-Ca2+promotes assembly of the channel with AKAP79 to form a signaling complex that couples Orai1 channel function to the activation of NFAT1. Knockdown of STIM2 expression had relatively little effect on Orai1/STIM1 clustering or local and global [Ca2+]iincreases but significantly attenuated NFAT1 activation and assembly of Orai1 with AKAP79. STIM1ΔK, which lacks the PIP2-binding polybasic domain, was recruited to ER-PM junctions following ER-Ca2+depletion by binding to Orai1 and caused local and global [Ca2+]iincreases comparable to those induced by STIM1 activation of Orai1. However, in contrast to STIM1, STIM1ΔK induced less NFAT1 activation and attenuated the association of Orai1 with STIM2 and AKAP79. Orai1-AKAP79 interaction and NFAT1 activation were recovered by coexpressing STIM2 with STIM1ΔK. Replacing the PIP2-binding domain of STIM1 with that of STIM2 eliminated the requirement of STIM2 for NFAT1 activation. Together, these data demonstrate an important role for STIM2 in coupling Orai1-mediated Ca2+influx to NFAT1 activation.
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Jaiswal, R. K., S. A. Moodie, A. Wolfman, and G. E. Landreth. "The mitogen-activated protein kinase cascade is activated by B-Raf in response to nerve growth factor through interaction with p21ras." Molecular and Cellular Biology 14, no. 10 (October 1994): 6944–53. http://dx.doi.org/10.1128/mcb.14.10.6944.
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Nerve growth factor (NGF) activates the mitogen-activated protein (MAP) kinase cascade through a p21ras-dependent signal transduction pathway in PC12 cells. The linkage between p21ras and MEK1 was investigated to identify those elements which participate in the regulation of MEK1 activity. We have screened for MEK activators using a coupled assay in which the MAP kinase cascade has been reconstituted in vitro. We report that we have detected a single NGF-stimulated MEK-activating activity which has been identified as B-Raf. PC12 cells express both B-Raf and c-Raf1; however, the MEK-activating activity was found only in fractions containing B-Raf. c-Raf1-containing fractions did not exhibit a MEK-activating activity. Gel filtration analysis revealed that the B-Raf eluted with an apparent M(r) of 250,000 to 300,000, indicating that it is present within a stable complex with other unidentified proteins. Immunoprecipitation with B-Raf-specific antisera quantitatively precipitated all MEK activator activity from these fractions. We also demonstrate that B-Raf, as well as c-Raf1, directly interacted with activated p21ras immobilized on silica beads. NGF treatment of the cells had no effect on the ability of B-Raf or c-Raf1 to bind to activated p21ras. These data indicate that this interaction was not dependent upon the activation state of these enzymes; however, MEK kinase activity was found to be associated with p21ras following incubation with NGF-treated samples at levels higher than those obtained from unstimulated cells. These data provide direct evidence that NGF-stimulated B-Raf is responsible for the activation of the MAP kinase cascade in PC12 cells, whereas c-Raf1 activity was not found to function within this pathway.
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Jaiswal, R. K., S. A. Moodie, A. Wolfman, and G. E. Landreth. "The mitogen-activated protein kinase cascade is activated by B-Raf in response to nerve growth factor through interaction with p21ras." Molecular and Cellular Biology 14, no. 10 (October 1994): 6944–53. http://dx.doi.org/10.1128/mcb.14.10.6944-6953.1994.
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Nerve growth factor (NGF) activates the mitogen-activated protein (MAP) kinase cascade through a p21ras-dependent signal transduction pathway in PC12 cells. The linkage between p21ras and MEK1 was investigated to identify those elements which participate in the regulation of MEK1 activity. We have screened for MEK activators using a coupled assay in which the MAP kinase cascade has been reconstituted in vitro. We report that we have detected a single NGF-stimulated MEK-activating activity which has been identified as B-Raf. PC12 cells express both B-Raf and c-Raf1; however, the MEK-activating activity was found only in fractions containing B-Raf. c-Raf1-containing fractions did not exhibit a MEK-activating activity. Gel filtration analysis revealed that the B-Raf eluted with an apparent M(r) of 250,000 to 300,000, indicating that it is present within a stable complex with other unidentified proteins. Immunoprecipitation with B-Raf-specific antisera quantitatively precipitated all MEK activator activity from these fractions. We also demonstrate that B-Raf, as well as c-Raf1, directly interacted with activated p21ras immobilized on silica beads. NGF treatment of the cells had no effect on the ability of B-Raf or c-Raf1 to bind to activated p21ras. These data indicate that this interaction was not dependent upon the activation state of these enzymes; however, MEK kinase activity was found to be associated with p21ras following incubation with NGF-treated samples at levels higher than those obtained from unstimulated cells. These data provide direct evidence that NGF-stimulated B-Raf is responsible for the activation of the MAP kinase cascade in PC12 cells, whereas c-Raf1 activity was not found to function within this pathway.
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Luttrell, LM. "'Location, location, location': activation and targeting of MAP kinases by G protein-coupled receptors." Journal of Molecular Endocrinology 30, no. 2 (April 1, 2003): 117–26. http://dx.doi.org/10.1677/jme.0.0300117.
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A growing body of data supports the conclusion that G protein-coupled receptors can regulate cellular growth and differentiation by controlling the activity of MAP kinases. The activation of heterotrimeric G protein pools initiates a complex network of signals leading to MAP kinase activation that frequently involves cross-talk between G protein-coupled receptors and receptor tyrosine kinases or focal adhesions. The dominant mechanism of MAP kinase activation varies significantly between receptor and cell type. Moreover, the mechanism of MAP kinase activation has a substantial impact on MAP kinase function. Some signals lead to the targeting of activated MAP kinase to specific extranuclear locations, while others activate a MAP kinase pool that is free to translocate to the nucleus and contribute to a mitogenic response.
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Zhao, Zhou-shen, Edward Manser, Tsui-Han Loo, and Louis Lim. "Coupling of PAK-Interacting Exchange Factor PIX to GIT1 Promotes Focal Complex Disassembly." Molecular and Cellular Biology 20, no. 17 (September 1, 2000): 6354–63. http://dx.doi.org/10.1128/mcb.20.17.6354-6363.2000.
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ABSTRACT The p21-activated kinase PAK is targeted to focal complexes (FCs) through interactions with the SH3 domains of the PAK-interacting exchange factor PIX and Nck. PIX is a Rac GTP exchange factor that also binds the G-protein-coupled receptor kinase-interacting protein known as GIT1. Overexpression of GIT1 in fibroblasts or epithelial cells causes a loss of paxillin from FCs and stimulates cell motility. This is due to the direct interaction of a C-terminal 125-residue domain of GIT1 with paxillin, under the regulation of PIX. In its activated state, GIT1 can promote FC disassembly independent of actin-myosin contractile events. Additionally, GIT directly couples to a key component of FCs, focal adhesion kinase (FAK), via a conserved Spa2 homology domain. We propose that GIT1 and FAK cooperate to promote motility both by directly regulating focal complex dynamics and by the activation of Rac.
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Sieng, Monita, Arielle F. Selvia, Elisabeth E. Garland-Kuntz, Jesse B. Hopkins, Isaac J. Fisher, Andrea T. Marti, and Angeline M. Lyon. "Functional and structural characterization of allosteric activation of phospholipase Cε by Rap1A." Journal of Biological Chemistry 295, no. 49 (September 18, 2020): 16562–71. http://dx.doi.org/10.1074/jbc.ra120.015685.
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Phospholipase Cε (PLCε) is activated downstream of G protein–coupled receptors and receptor tyrosine kinases through direct interactions with small GTPases, including Rap1A and Ras. Although Ras has been reported to allosterically activate the lipase, it is not known whether Rap1A has the same ability or what its molecular mechanism might be. Rap1A activates PLCε in response to the stimulation of β-adrenergic receptors, translocating the complex to the perinuclear membrane. Because the C-terminal Ras association (RA2) domain of PLCε was proposed to the primary binding site for Rap1A, we first confirmed using purified proteins that the RA2 domain is indeed essential for activation by Rap1A. However, we also showed that the PLCε pleckstrin homology (PH) domain and first two EF hands (EF1/2) are required for Rap1A activation and identified hydrophobic residues on the surface of the RA2 domain that are also necessary. Small-angle X-ray scattering showed that Rap1A binding induces and stabilizes discrete conformational states in PLCε variants that can be activated by the GTPase. These data, together with the recent structure of a catalytically active fragment of PLCε, provide the first evidence that Rap1A, and by extension Ras, allosterically activate the lipase by promoting and stabilizing interactions between the RA2 domain and the PLCε core.
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Luo, LuGuang, Naohiro Yano, and John Z. Q. Luo. "The molecular mechanism of EGF receptor activation in pancreatic β-cells by thyrotropin-releasing hormone." American Journal of Physiology-Endocrinology and Metabolism 290, no. 5 (May 2006): E889—E899. http://dx.doi.org/10.1152/ajpendo.00466.2005.
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Thyrotropin-releasing hormone (TRH) and its receptor subtype TRH receptor-1 (TRHR1) are found in pancreatic β-cells, and it has been shown that TRH might have potential for autocrine/paracrine regulation through the TRHR1 receptor. In this paper, TRHR1 is studied to find whether it can initiate multiple signal transduction pathways to activate the epidermal growth factor (EGF) receptor in pancreatic β-cells. By initiating TRHR1 G protein-coupled receptor (GPCR) and dissociated αβγ-complex, TRH (200 nM) activates tyrosine residues at Tyr845 (a known target for Src) and Tyr1068 in the EGF receptor complex of an immortalized mouse β-cell line, βTC-6. Through manipulating the activation of Src, PKC, and heparin-binding EGF-like growth factor (HB-EGF), with corresponding individual inhibitors and activators, multiple signal transduction pathways linking TRH to EGF receptors in βTC-6 cell line have been revealed. The pathways include the activation of Src kinase and the release of HB-EGF as a consequence of matrix metalloproteinase (MMP)-3 activation. Alternatively, TRH inhibited PKC activity by reducing the EGF receptor serine/threonine phosphorylation, thereby enhancing tyrosine phosphorylation. TRH receptor activation of Src may have a central role in mediating the effects of TRH on the EGF receptor. The activation of the EGF receptor by TRH in multiple circumstances may have important implications for pancreatic β-cell biology.
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Qureshi, Bilal M., Elmar Behrmann, Johannes Schöneberg, Justus Loerke, Jörg Bürger, Thorsten Mielke, Jan Giesebrecht, et al. "It takes two transducins to activate the cGMP-phosphodiesterase 6 in retinal rods." Open Biology 8, no. 8 (August 2018): 180075. http://dx.doi.org/10.1098/rsob.180075.
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Among cyclic nucleotide phosphodiesterases (PDEs), PDE6 is unique in serving as an effector enzyme in G protein-coupled signal transduction. In retinal rods and cones, PDE6 is membrane-bound and activated to hydrolyse its substrate, cGMP, by binding of two active G protein α-subunits (Gα*). To investigate the activation mechanism of mammalian rod PDE6, we have collected functional and structural data, and analysed them by reaction–diffusion simulations. Gα* titration of membrane-bound PDE6 reveals a strong functional asymmetry of the enzyme with respect to the affinity of Gα* for its two binding sites on membrane-bound PDE6 and the enzymatic activity of the intermediary 1 : 1 Gα* · PDE6 complex. Employing cGMP and its 8-bromo analogue as substrates, we find that Gα* · PDE6 forms with high affinity but has virtually no cGMP hydrolytic activity. To fully activate PDE6, it takes a second copy of Gα* which binds with lower affinity, forming Gα* · PDE6 · Gα*. Reaction–diffusion simulations show that the functional asymmetry of membrane-bound PDE6 constitutes a coincidence switch and explains the lack of G protein-related noise in visual signal transduction. The high local concentration of Gα* generated by a light-activated rhodopsin molecule efficiently activates PDE6, whereas the low density of spontaneously activated Gα* fails to activate the effector enzyme.
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Wang, Jehng-Kang, Ming-Shyue Lee, I.-Chu Tseng, Feng-Pai Chou, Ya-Wen Chen, Amy Fulton, Herng-Sheng Lee, Cheng-Jueng Chen, Michael D. Johnson, and Chen-Yong Lin. "Polarized epithelial cells secrete matriptase as a consequence of zymogen activation and HAI-1-mediated inhibition." American Journal of Physiology-Cell Physiology 297, no. 2 (August 2009): C459—C470. http://dx.doi.org/10.1152/ajpcell.00201.2009.
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Matriptase, a transmembrane serine protease, is broadly expressed by, and crucial for the integrity of, the epithelium. Matriptase is synthesized as a zymogen and undergoes autoactivation to become an active protease that is immediately inhibited by, and forms complexes with, hepatocyte growth factor activator inhibitor (HAI-1). To investigate where matriptase is activated and how it is secreted in vivo, we determined the expression and activation status of matriptase in seminal fluid and urine and the distribution and subcellular localization of the protease in the prostate and kidney. The in vivo studies revealed that while the latent matriptase is localized at the basolateral surface of the ductal epithelial cells of both organs, only matriptase-HAI-1 complexes and not latent matriptase are detected in the body fluids, suggesting that activation, inhibition, and transcytosis of matriptase would have to occur for the secretion of matriptase. These complicated processes involved in the in vivo secretion were also observed in polarized Caco-2 intestinal epithelial cells. The cells target latent matriptase to the basolateral plasma membrane where activation, inhibition, and secretion of matriptase appear to take place. However, a proportion of matriptase-HAI-1 complexes, but not the latent matriptase, appears to undergo transcytosis to the apical plasma membrane for secretion. When epithelial cells lose their polarity, they secrete both latent and activated matriptase. Although most epithelial cells retain very low levels of matriptase-HAI-1 complex by rapidly secreting the complex, gastric chief cells may activate matriptase and store matriptase-HAI-1 complexes in the pepsinogen-secretory granules, suggesting an intracellular activation and regulated secretion in these cells. Taken together, while zymogen activation and closely coupled HAI-1-mediated inhibition are common features for matriptase regulation, the cellular location of matriptase activation and inhibition, and the secretory route for matriptase-HAI-1 complex may vary along with the functional divergence of different epithelial cells.
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Mazzieri, Roberta, Silvia D'Alessio, Richard Kamgang Kenmoe, Liliana Ossowski, and Francesco Blasi. "An Uncleavable uPAR Mutant Allows Dissection of Signaling Pathways in uPA-dependent Cell Migration." Molecular Biology of the Cell 17, no. 1 (January 2006): 367–78. http://dx.doi.org/10.1091/mbc.e05-07-0635.
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Urokinase-type plasminogen activator (uPA) binding to uPAR induces migration, adhesion, and proliferation through multiple interactions with G proteins-coupled receptor FPRL1, integrins, or the epidermal growth factor (EGF) receptor (EGFR). At least two forms of uPAR are present on the cell surface: full-length and cleaved uPAR, each specifically interacting with one or more transmembrane proteins. The connection between these interactions and the effects on the signaling pathways activation is not clear. We have exploited an uPAR mutant (hcr, human cleavage resistant) to dissect the pathways involved in uPA-induced cell migration. This mutant is not cleaved by proteases, is glycosylphosphatidylinositol anchored, and binds uPA with a normal Kd. Both wild-type (wt) and hcr-uPAR are able to mediate uPA-induced migration, are constitutively associated with the EGFR, and associate with α3β1 integrin upon uPA binding. However, they engage different pathways in response to uPA. wt-uPAR requires both integrins and FPRL1 to mediate uPA-induced migration, and association of wt-uPAR to α3β1 results in uPAR cleavage and extracellular signal-regulated kinase (ERK) activation. On the contrary, hcr-uPAR does not activate ERK and does not engage FPRL1 or any other G protein-coupled receptor, but it activates an alternative pathway initiated by the formation of a triple complex (uPAR-α3β1-EGFR) and resulting in the autotyrosine phosphorylation of EGFR.
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Huang, RiYun, Jian P. Lian, Dwight Robinson, and John A. Badwey. "Neutrophils Stimulated with a Variety of Chemoattractants Exhibit Rapid Activation of p21-Activated Kinases (Paks): Separate Signals Are Required for Activation and Inactivation of Paks." Molecular and Cellular Biology 18, no. 12 (December 1, 1998): 7130–38. http://dx.doi.org/10.1128/mcb.18.12.7130.
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ABSTRACT Activation of the p21-activated protein kinases (Paks) was compared in neutrophils stimulated with a wide variety of agonists that bind to receptors coupled to heterotrimeric G proteins. Neutrophils stimulated with sulfatide, a ligand for the L-selectin receptor, or the chemoattractant fMet-Leu-Phe (fMLP), platelet-activating factor, leukotriene B4, interleukin-8, or the chemokine RANTES exhibited a rapid and transient activation of the 63- and 69-kDa Paks. These kinases exhibited maximal activation with each of these agonists within 15 s followed by significant inactivation at 3 min. In contrast, neutrophils treated with the chemoattractant and anaphylatoxin C5a exhibited a prolonged activation (>15 min) of these Paks even though the receptor for this ligand may activate the same overall population of complex G proteins as the fMLP receptor. Addition of fMLP to neutrophils already stimulated with C5a resulted in the inactivation of the 63- and 69-kDa Paks. Optimal activation of Paks could be observed at concentrations of these agonists that elicited only shape changes and chemotaxis in neutrophils. While all of the agonists listed above triggered quantitatively similar activation of the 63- and 69-kDa Paks, fMLP was far superior to the other stimuli in triggering activation of the c-Jun N-terminal kinase (JNK) and the p38 mitogen-activated protein kinase (MAPK). These data indicate that separate signals are required for activation and inactivation of Paks and that, in contrast to other cell types, activated Pak does not trigger activation of JNK or p38-MAPK in neutrophils. These results are consistent with the recent hypothesis that G-protein-coupled receptors may initiate signals independent of those transmitted by the α and βγ subunits of complex G proteins.
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Stathopulos, Peter B., and Mitsuhiko Ikura. "Partial unfolding and oligomerization of stromal interaction molecules as an initiation mechanism of store operated calcium entryThis paper is one of a selection of papers published in this special issue entitled “Canadian Society of Biochemistry, Molecular & Cellular Biology 52nd Annual Meeting — Protein Folding: Principles and Diseases” and has undergone the Journal's usual peer review process." Biochemistry and Cell Biology 88, no. 2 (April 2010): 175–83. http://dx.doi.org/10.1139/o09-125.
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Spatiotemporally discrete cytoplasmic Ca2+ fluctuations are fundamental eukaryotic signals in myriad physiological and pathophysiological functions. Store-operated Ca2+ entry is the process whereby a decrease in endoplasmic reticulum (ER) luminal Ca2+ levels activates Ca2+ release activated calcium (CRAC) channels on the plasma membrane (PM), providing a sustained Ca2+ elevation to the cytoplasm and ultimately replenishing the ER lumen Ca2+ supply. Stromal interaction molecules (STIMs) are the Ca2+ sensors of the ER lumen, which macromolecularly couple depleted ER Ca2+ to the assembly and opening of PM CRAC channels. The considerable stability difference caused by Ca2+ loading and depletion within the luminal portion of STIMs modulates intramolecular cytoplasmic domain interactions essential to the assembly of PM CRAC channels. Thus, the action of the entire complex is tightly regulated through the Ca2+ sensitivity of luminal STIM domains. Recent structural and biochemical studies suggest that partial unfolding – coupled oligomerization of STIMs is a crucial step in CRAC channel activation. Based on these and other published data, this minireview discusses what is currently known about the molecular mechanism of ER Ca2+ sensing by STIMs.
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Agarwal, Nitin, Chae Hwa Kim, Kranthi Kunkalla, Gloria Yang, Youley Tjendra, Yadong Liu, Changju Qu, and Francisco Vega. "Smoothened (SMO) Is an Adaptor Protein That Recruits TRAF6 and Phospholipase C Gamma 2 (PLCg2) to Enhance the Activation of NF-Kb Signaling Pathway." Blood 126, no. 23 (December 3, 2015): 3907. http://dx.doi.org/10.1182/blood.v126.23.3907.3907.
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Abstract Background: Constitutive activation of NF-κB signaling is a hallmark of DLBCL. Activation of NF-κB is a multifactorial process resulting from oncogenic mutations (CARD11, MYD88…), chromosomal abnormalities, chronic activation of B-cell receptor signaling (BCR) as well as stimuli from the microenvironment. Chronic activation of BCR is not only the result of gene mutations (e.g.CD79B) but also is the result of stimuli generated from the lymphoma microenvironment. We previously found that smoothened (SMO), transducer of hedgehog (Hh) signaling, enhanced NF-κB activation in lymphoma cells, independently of the presence of oncogenic mutations (Changju et al., Blood 2013). Hh ligands are provided by the lymphoma microenvironment (e.g. stromal cells) to activate SMO in the lymphoma cells. Activated SMO, recruits and activates trimeric-G-coupled proteins to activate PKCβ/CARMA1/TRAF6/NEMO axis, followed by assembling of the CARMA1/BCL10/MALT1/TRAF6 complex to SMO resulting in NF-κB activation. Now, we reveal an additional mechanism by which SMO further contributes to the activation of NF-κB in DLBCL. Summary of results: We explored if recombinant Hh ligand can activate NF-κB signaling in the presence of the BTK inhibitor (PCI-32765). Hh stimulation resulted in phosphorylation of PLCg2 (downstream of BTK) and partially rescued the inhibitory effect of the BTK inhibitor on phosphorylation of PLCg2 suggesting a crosstalk between SMO and BCR receptor signaling. We identified that SMO forms a complex with TRAF-6 and PLCg2 using immunoprecipitation and Duolink in situ proximity ligation assays. We found that SMO stabilizes and protects TRAF-6 from proteosomal degradation. SMO-dependent TRAF6 stabilization is mediated by the ubiquitin-specific protease-8 (USP-8) by removing ubiquitin from K48-linked lysine of TRAF6. Conclusions: Collectively, our data reveals multilayer crosstalk between Hh and BCR/NF-κB pathways and provide significant insights into how SMO contributes to chemotolerance and progression of DLBCL. We expect that our results will delineate novel molecular mechanisms involved in the pathobiology of DLBCL that may serve as therapeutic targets. Disclosures Vega: Seatle Genetics: Honoraria; NIH: Research Funding.
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Ezumi, Yasuharu, Keisuke Shindoh, Masaaki Tsuji, and Hiroshi Takayama. "Physical and Functional Association of the Src Family Kinases Fyn and Lyn with the Collagen Receptor Glycoprotein VI-Fc Receptor γ Chain Complex on Human Platelets." Journal of Experimental Medicine 188, no. 2 (July 20, 1998): 267–76. http://dx.doi.org/10.1084/jem.188.2.267.
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We have previously shown that uncharacterized glycoprotein VI (GPVI), which is constitutively associated and coexpressed with Fc receptor γ chain (FcRγ) in human platelets, is essential for collagen-stimulated tyrosine phosphorylation of FcRγ, Syk, and phospholipase Cγ2 (PLCγ2), leading to platelet activation. Here we investigated involvement of the Src family in the proximal signals through the GPVI–FcRγ complex, using the snake venom convulxin from Crotalus durissus terrificus, which specifically recognizes GPVI and activates platelets through cross-linking GPVI. Convulxin-coupled beads precipitated the GPVI–FcRγ complex from platelet lysates. Collagen and convulxin induced tyrosine phosphorylation of FcRγ, Syk, and PLCγ2 and recruited tyrosine-phosphorylated Syk to the GPVI–FcRγ complex. Using coprecipitation methods with convulxin-coupled beads and antibodies against FcRγ and the Src family, we showed that Fyn and Lyn, but not Yes, Src, Fgr, Hck, and Lck, were physically associated with the GPVI–FcRγ complex irrespective of stimulation. Furthermore, Fyn was rapidly activated by collagen or cross-linking GPVI. The Src family–specific inhibitor PP1 dose-dependently inhibited collagen- or convulxin-induced tyrosine phosphorylation of proteins including FcRγ, Syk, and PLCγ2, accompanied by a loss of aggregation and ATP release reaction. These results indicate that the Src family plays a critical role in platelet activation via the collagen receptor GPVI–FcRγ complex.
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Xu, Hao, Baohua Gu, B. Tracy Nixon, and Timothy R. Hoover. "Purification and Characterization of the AAA+ Domain of Sinorhizobium meliloti DctD, a σ54-Dependent Transcriptional Activator." Journal of Bacteriology 186, no. 11 (June 1, 2004): 3499–507. http://dx.doi.org/10.1128/jb.186.11.3499-3507.2004.
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ABSTRACT Activators of σ54-RNA polymerase holoenzyme couple ATP hydrolysis to formation of an open complex between the promoter and RNA polymerase. These activators are modular, consisting of an N-terminal regulatory domain, a C-terminal DNA-binding domain, and a central activation domain belonging to the AAA+ superfamily of ATPases. The AAA+ domain of Sinorhizobium meliloti C4-dicarboxylic acid transport protein D (DctD) is sufficient to activate transcription. Deletion analysis of the 3′ end of dctD identified the minimal functional C-terminal boundary of the AAA+ domain of DctD as being located between Gly-381 and Ala-384. Histidine-tagged versions of the DctD AAA+ domain were purified and characterized. The DctD AAA+ domain was significantly more soluble than DctD( Δ 1-142), a truncated DctD protein consisting of the AAA+ and DNA-binding domains. In addition, the DctD AAA+ domain was more homogeneous than DctD( Δ 1-142) when analyzed by native gel electrophoresis, migrating predominantly as a single high-molecular-weight species, while DctD( Δ 1-142) displayed multiple species. The DctD AAA+ domain, but not DctD( Δ 1-142), formed a stable complex with σ54 in the presence of the ATP transition state analogue ADP-aluminum fluoride. The DctD AAA+ domain activated transcription in vitro, but many of the transcripts appeared to terminate prematurely, suggesting that the DctD AAA+ domain interfered with transcription elongation. Thus, the DNA-binding domain of DctD appears to have roles in controlling the oligomerization of the AAA+ domain and modulating interactions with σ54 in addition to its role in recognition of upstream activation sequences.
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David-Dufilho, Monique, Elisabeth Millanvoye-Van Brussel, Gokce Topal, Laurence Walch, Annie Brunet, and Francine Rendu. "Endothelial Thrombomodulin Induces Ca2+ Signals and Nitric Oxide Synthesis through Epidermal Growth Factor Receptor Kinase and Calmodulin Kinase II." Journal of Biological Chemistry 280, no. 43 (August 26, 2005): 35999–6006. http://dx.doi.org/10.1074/jbc.m506374200.
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Endothelial membrane-bound thrombomodulin is a high affinity receptor for thrombin to inhibit coagulation. We previously demonstrated that the thrombin-thrombomodulin complex restrains cell proliferation mediated through protease-activated receptor (PAR)-1. We have now tested the hypothesis that thrombomodulin transduces a signal to activate the endothelial nitric-oxide synthase (NOS3) and to modulate G protein-coupled receptor signaling. Cultured human umbilical vein endothelial cells were stimulated with thrombin or a mutant of thrombin that binds to thrombomodulin and has no catalytic activity on PAR-1. Thrombin and its mutant dose dependently activated NO release at cell surface. Pretreatment with anti-thrombomodulin antibody suppressed NO response to the mutant and to low thrombin concentration and reduced by half response to high concentration. Thrombin receptor-activating peptide that only activates PAR-1 and high thrombin concentration induced marked biphasic Ca2+ signals with rapid phosphorylation of PLCβ3 and NOS3 at both serine 1177 and threonine 495. The mutant thrombin evoked a Ca2+ spark and progressive phosphorylation of Src family kinases at tyrosine 416 and NOS3 only at threonine 495. It activated rapid phosphatidylinositol-3 kinase-dependent NO synthesis and phosphorylation of epidermal growth factor receptor and calmodulin kinase II. Complete epidermal growth factor receptor inhibition only partly reduced the activation of phospholipase Cγ1 and NOS3. Prestimulation of thrombomodulin did not affect NO release but reduced Ca2+ responses to thrombin and histamine, suggesting cross-talks between thrombomodulin and G protein-coupled receptors. This is the first demonstration of an outside-in signal mediated by the cell surface thrombomodulin receptor to activate NOS3 through tyrosine kinase-dependent pathway. This signaling may contribute to thrombomodulin function in thrombosis, inflammation, and atherosclerosis.
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Hesketh, Geoffrey G., Fotini Papazotos, Judy Pawling, Dushyandi Rajendran, James D. R. Knight, Sebastien Martinez, Mikko Taipale, Daniel Schramek, James W. Dennis, and Anne-Claude Gingras. "The GATOR–Rag GTPase pathway inhibits mTORC1 activation by lysosome-derived amino acids." Science 370, no. 6514 (October 15, 2020): 351–56. http://dx.doi.org/10.1126/science.aaz0863.
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The mechanistic target of rapamycin complex 1 (mTORC1) couples nutrient sufficiency to cell growth. mTORC1 is activated by exogenously acquired amino acids sensed through the GATOR–Rag guanosine triphosphatase (GTPase) pathway, or by amino acids derived through lysosomal degradation of protein by a poorly defined mechanism. Here, we revealed that amino acids derived from the degradation of protein (acquired through oncogenic Ras-driven macropinocytosis) activate mTORC1 by a Rag GTPase–independent mechanism. mTORC1 stimulation through this pathway required the HOPS complex and was negatively regulated by activation of the GATOR-Rag GTPase pathway. Therefore, distinct but functionally coordinated pathways control mTORC1 activity on late endocytic organelles in response to distinct sources of amino acids.
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Cai, Huaqing, Satarupa Das, Yoichiro Kamimura, Yu Long, Carole A. Parent, and Peter N. Devreotes. "Ras-mediated activation of the TORC2–PKB pathway is critical for chemotaxis." Journal of Cell Biology 190, no. 2 (July 26, 2010): 233–45. http://dx.doi.org/10.1083/jcb.201001129.
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In chemotactic cells, G protein–coupled receptors activate Ras proteins, but it is unclear how Ras-associated pathways link extracellular signaling to cell migration. We show that, in Dictyostelium discoideum, activated forms of RasC prolong the time course of TORC2 (target of rapamycin [Tor] complex 2)-mediated activation of a myristoylated protein kinase B (PKB; PKBR1) and the phosphorylation of PKB substrates, independently of phosphatidylinositol-(3,4,5)-trisphosphate. Paralleling these changes, the kinetics of chemoattractant-induced adenylyl cyclase activation and actin polymerization are extended, pseudopodial activity is increased and mislocalized, and chemotaxis is impaired. The effects of activated RasC are suppressed by deletion of the TORC2 subunit PiaA. In vitro RasCQ62L-dependent PKB phosphorylation can be rapidly initiated by the addition of a PiaA-associated immunocomplex to membranes of TORC2-deficient cells and blocked by TOR-specific inhibitor PP242. Furthermore, TORC2 binds specifically to the activated form of RasC. These results demonstrate that RasC is an upstream regulator of TORC2 and that the TORC2–PKB signaling mediates effects of activated Ras proteins on the cytoskeleton and cell migration.
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Pakharukova, Natalia, Ali Masoudi, Biswaranjan Pani, Dean P. Staus, and Robert J. Lefkowitz. "Allosteric activation of proto-oncogene kinase Src by GPCR–beta-arrestin complexes." Journal of Biological Chemistry 295, no. 49 (September 25, 2020): 16773–84. http://dx.doi.org/10.1074/jbc.ra120.015400.
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G protein–coupled receptors (GPCRs) initiate signaling cascades via G-proteins and beta-arrestins (βarr). βarr-dependent actions begin with recruitment of βarr to the phosphorylated receptor tail and are followed by engagement with the receptor core. βarrs are known to act as adaptor proteins binding receptors and various effectors, but it is unclear whether in addition to the scaffolding role βarrs can allosterically activate their downstream targets. Here we demonstrate the direct allosteric activation of proto-oncogene kinase Src by GPCR–βarr complexes in vitro and establish the conformational basis of the activation. Whereas free βarr1 had no effect on Src activity, βarr1 in complex with M2 muscarinic or β2-adrenergic receptors reconstituted in lipid nanodiscs activate Src by reducing the lag phase in Src autophosphorylation. Interestingly, receptor–βarr1 complexes formed with a βarr1 mutant, in which the finger-loop, required to interact with the receptor core, has been deleted, fully retain the ability to activate Src. Similarly, βarr1 in complex with only a phosphorylated C-terminal tail of the vasopressin 2 receptor activates Src as efficiently as GPCR–βarr complexes. In contrast, βarr1 and chimeric M2 receptor with nonphosphorylated C-terminal tail failed to activate Src. Taken together, these data demonstrate that the phosphorylated GPCR tail interaction with βarr1 is necessary and sufficient to empower it to allosterically activate Src. Our findings may have implications for understanding more broadly the mechanisms of allosteric activation of downstream targets by βarrs.
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Jang, Wonjo, C. Elizabeth Adams, Heng Liu, Cheng Zhang, Finn Olav Levy, Kjetil Wessel Andressen, and Nevin A. Lambert. "An inactive receptor-G protein complex maintains the dynamic range of agonist-induced signaling." Proceedings of the National Academy of Sciences 117, no. 48 (November 16, 2020): 30755–62. http://dx.doi.org/10.1073/pnas.2010801117.
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Agonist binding promotes activation of G protein-coupled receptors (GPCRs) and association of active receptors with G protein heterotrimers. The resulting active-state ternary complex is the basis for conventional stimulus-response coupling. Although GPCRs can also associate with G proteins before agonist binding, the impact of such preassociated complexes on agonist-induced signaling is poorly understood. Here we show that preassociation of 5-HT7serotonin receptors with Gsheterotrimers is necessary for agonist-induced signaling. 5-HT7receptors in their inactive state associate with Gs, as these complexes are stabilized by inverse agonists and receptor mutations that favor the inactive state. Inactive-state 5-HT7–Gscomplexes dissociate in response to agonists, allowing the formation of conventional agonist–5-HT7–Gsternary complexes and subsequent Gsactivation. Inactive-state 5-HT7–Gscomplexes are required for the full dynamic range of agonist-induced signaling, as 5-HT7receptors spontaneously activate Gsvariants that cannot form inactive-state complexes. Therefore, agonist-induced signaling in this system involves two distinct receptor-G protein complexes, a conventional ternary complex that activates G proteins and an inverse-coupled binary complex that maintains the inactive state when agonist is not present.
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Sellers, Lynda A., Forbes Alderton, Alan M. Carruthers, Marcus Schindler, and Patrick P. A. Humphrey. "Receptor Isoforms Mediate Opposing Proliferative Effects through Gβγ-Activated p38 or Akt Pathways." Molecular and Cellular Biology 20, no. 16 (August 15, 2000): 5974–85. http://dx.doi.org/10.1128/mcb.20.16.5974-5985.2000.
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ABSTRACT The opposing effects on proliferation mediated by G-protein-coupled receptor isoforms differing in their COOH termini could be correlated with the abilities of the receptors to differentially activate p38, implicated in apoptotic events, or phosphatidylinositol 3-kinase (PI 3-K), which provides a source of survival signals. These contrasting growth responses of the somatostatin sst2 receptor isoforms, which couple to identical Gα subunit pools (Gαi3 > Gαi2 >> Gα0), were both inhibited following βγ sequestration. The sst2(a) receptor-mediated ATF-2 activation and inhibition of proliferation induced by basic fibroblast growth factor (bFGF) were dependent on prolonged phosphorylation of p38. In contrast, cell proliferation and the associated transient phosphorylation of Akt and p70 rsk induced by sst2(b) receptors were blocked by the PI 3-K inhibitor LY 294002. Stimulation with bFGF alone had no effect on the activity of either p38 or Akt but markedly enhanced p38 phosphorylation mediated by sst2(a) receptors, suggesting that a complex interplay exists between the transduction cascades activated by these distinct receptor types. In addition, although all receptors mediated a sustained activation of extracellular signal-regulated kinases (ERK1 and ERK2), induction of the tumor suppressor p21 cip1 was detected only following amplification of ERK and p38 phosphorylation by concomitant bFGF and sst2(a) receptor activation. Expression of constitutively active Akt in the presence of a p38 inhibitor enabled a proliferative response to be detected in sst2(a) receptor-expressing cells. These findings demonstrate that the duration of activation and a critical balance between the mitogen-activated protein kinase and PI 3-K pathways are important for controlling cell proliferation and that the COOH termini of the sst2 receptor isoforms may determine the selection of appropriate βγ-pairings necessary for interaction with distinct kinase cascades.
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Tweardy, DJ, TM Wright, SF Ziegler, H. Baumann, A. Chakraborty, SM White, KF Dyer, and KA Rubin. "Granulocyte colony-stimulating factor rapidly activates a distinct STAT- like protein in normal myeloid cells." Blood 86, no. 12 (December 15, 1995): 4409–16. http://dx.doi.org/10.1182/blood.v86.12.4409.bloodjournal86124409.
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Binding of granulocyte colony-stimulating factor (G-CSF) to normal myeloid cells activates the protein tyrosine kinases Lyn and Syk and results in the immediate early upregulation of G-CSF receptor (R) mRNA. In our studies of the signaling pathways activated by G-CSF that are coupled to proliferation and differentiation of myeloid cells, we examined whether G-CSF activated a latent transcription factor belonging to the STAT protein family. Electrophoretic mobility shift assays (EMSAs) of nuclear extracts from G-CSF-stimulated human myeloid cells showed the rapid activation of a DNA-binding protein that bound to the high-affinity serum-inducible element (hSIE) and migrated with mobility similar to serum inducible factor (SIF)-A (Stat3 homodimer). The G-CSF-stimulated SIF-A complex (G-SIF-A) did not bind to duplex oligonucleotides used to purify and characterize other Stat proteins (Stat1–6). In addition, antibodies raised against Stat1–6 failed to supershift the G-SIF-A complex or interfere with its formation. Based on its binding to the hSIE and lack of antigenic cross-reactivity with other known STAT proteins that bind to this element, it is likely that G-SIF-A is composed of a distinct member of the STAT protein family. EMSAs of whole-cell extracts prepared from cell lines containing full- length and truncated mutants of the G-CSFR showed that activation of G- SIF-A did not correlate with proliferation; rather, optimal activation requires the distal half of the cytosolic domain of the G-CSFR that is essential for differentiation. Activation of G-SIF-A, therefore, may be an early G-CSFR-coupled event that is critical for myeloid maturation.
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Beckers, Cora, Victor van Hinsbergh, and Geerten van Nieuw Amerongen. "Driving Rho GTPase activity in endothelial cells regulates barrier integrity." Thrombosis and Haemostasis 103, no. 01 (2010): 40–55. http://dx.doi.org/10.1160/th09-06-0403.
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SummaryIn the past decade understanding of the role of the Rho GTPases RhoA, Rac1 and Cdc42 has been developed from regulatory proteins that regulate specific actin cytoskeletal structures – stress fibers, lamellipodia and filopodia – to complex integrators of cytoskeletal structures that can exert multiple functions depending on the cellular context. Fundamental to these functions are three-dimensional complexes between the individual Rho GTPases, their specific activators (GEFs) and inhibitors (GDIs and GAPs), which greatly outnumber the Rho GTPases themselves, and additional regulatory proteins. By this complexity of regulation different vasoactive mediators can induce various cytoskeletal structures that enable the endothelial cell (EC) to respond adequately. In this review we have focused on this complexity and the consequences of Rho GTPase regulation for endothelial barrier function. The permeability inducers thrombin and VEGF are presented as examples of G-protein coupled receptor- and tyrosine kinase receptormediated Rho GTPase activation, respectively. These mediators induce complex but markedly different networks of activators, inhibitors and effectors of Rho GTPases, which alter the endothelial barrier function. An interesting feature in this regulation is that Rho GTPases often have both barrier-protecting and barrier-disturbing functions. While Rac1 enforces the endothelial junctions, it becomes part of a barrier-disturbing mechanism as activator of reactive oxygen species generating NADPH oxidase. Similarly RhoA is protective under basal conditions, but becomes involved in barrier dysfunction after activation of ECs by thrombin. The challenge and promise lies in unfolding this complex regulation, as this will provide leads for new therapeutic opportunities.
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Thiel, Gerald, Andrea Lesch, and Anja Keim. "Transcriptional Response to Calcium-Sensing Receptor Stimulation." Endocrinology 153, no. 10 (October 1, 2012): 4716–28. http://dx.doi.org/10.1210/en.2012-1343.
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Abstract Elevated extracellular Ca2+ concentrations stimulate the G-protein coupled receptor calcium-sensing receptor. Here we show that this stimulation induces the expression of biologically active early growth response protein 1 (Egr-1), a zinc finger transcription factor. Expression of a dominant-negative mutant of the ternary complex factor Ets-like protein-1 (Elk-1), a key transcriptional regulator of serum response element-driven gene transcription, prevented Egr-1 expression, indicating that Elk-1 or related ternary complex factors connect the intracellular signaling cascade elicited by activation of calcium-sensing receptors with transcription of the Egr-1 gene. These data were corroborated by the fact that stimulation of calcium-sensing receptors increased the transcriptional activation potential of Elk-1. In addition, activator protein-1 (AP-1) transcriptional activity was significantly elevated after the stimulation of calcium-sensing receptors. The expression of a dominant-negative mutant of Elk-1 reduced c-Fos expression and prevented the up-regulation of AP-1 activity as a result of calcium-sensing receptor stimulation, indicating that ternary complex factors control both Egr-1- and AP-1-regulated transcription. In addition, AP-1 activity was reduced after the expression of a dominant-negative mutant of c-Jun in cells expressing an activated calcium-sensing receptor. Stimulus-transcription coupling leading to the up-regulation of Egr-1 and AP-1 controlled transcription in cells expressing calcium-sensing receptors required the protein kinases Raf and ERK, whereas the overexpression of MAPK phosphatase-1 interrupted the signaling cascade connecting calcium-sensing receptor stimulation with transcription of Egr-1 and AP-1 controlled genes. The fact that calcium-sensing receptor stimulation activates the transcription factors Egr-1, Elk-1, and AP-1 indicates that regulation of gene transcription is an integral part of calcium-sensing receptor induced signaling.
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Zhang, Wenwu, Youliang Huang, Yidi Wu, and Susan J. Gunst. "A novel role for RhoA GTPase in the regulation of airway smooth muscle contraction." Canadian Journal of Physiology and Pharmacology 93, no. 2 (February 2015): 129–36. http://dx.doi.org/10.1139/cjpp-2014-0388.
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Recent studies have demonstrated a novel molecular mechanism for the regulation of airway smooth muscle (ASM) contraction by RhoA GTPase. In ASM tissues, both myosin light chain (MLC) phosphorylation and actin polymerization are required for active tension generation. RhoA inactivation dramatically suppresses agonist-induced tension development and completely inhibits agonist-induced actin polymerization, but only slightly reduces MLC phosphorylation. The inhibition of MLC phosphatase does not reverse the effects of RhoA inactivation on contraction or actin polymerization. Thus, RhoA regulates ASM contraction through its effects on actin polymerization rather than MLC phosphorylation. Contractile stimulation of ASM induces the recruitment and assembly of paxillin, vinculin, and focal adhesion kinase (FAK) into membrane adhesion complexes (adhesomes) that regulate actin polymerization by catalyzing the activation of cdc42 GTPase by the G-protein-coupled receptor kinase-interacting target (GIT) – p21-activated kinase (PAK) – PAK-interacting exchange factor (PIX) complex. Cdc42 is a necessary and specific activator of the actin filament nucleation activator, N-WASp. The recruitment and activation of paxillin, vinculin, and FAK is prevented by RhoA inactivation, thus preventing cdc42 and N-WASp activation. We conclude that RhoA regulates ASM contraction by catalyzing the assembly and activation of membrane adhesome signaling modules that regulate actin polymerization, and that the RhoA-mediated assembly of adhesome complexes is a fundamental step in the signal transduction process in response to a contractile agonist.
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Ma, Xiuyan, Yunfei Hu, Hossein Batebi, Jie Heng, Jun Xu, Xiangyu Liu, Xiaogang Niu, et al. "Analysis of β2AR-Gs and β2AR-Gi complex formation by NMR spectroscopy." Proceedings of the National Academy of Sciences 117, no. 37 (August 31, 2020): 23096–105. http://dx.doi.org/10.1073/pnas.2009786117.
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The β2-adrenergic receptor (β2AR) is a prototypical G protein-coupled receptor (GPCR) that preferentially couples to the stimulatory G protein Gs and stimulates cAMP formation. Functional studies have shown that the β2AR also couples to inhibitory G protein Gi, activation of which inhibits cAMP formation [R. P. Xiao, Sci. STKE 2001, re15 (2001)]. A crystal structure of the β2AR-Gs complex revealed the interaction interface of β2AR-Gs and structural changes upon complex formation [S. G. Rasmussen et al., Nature 477, 549–555 (2011)], yet, the dynamic process of the β2AR signaling through Gs and its preferential coupling to Gs over Gi is still not fully understood. Here, we utilize solution nuclear magnetic resonance (NMR) spectroscopy and supporting molecular dynamics (MD) simulations to monitor the conformational changes in the G protein coupling interface of the β2AR in response to the full agonist BI-167107 and Gs and Gi1. These results show that BI-167107 stabilizes conformational changes in four transmembrane segments (TM4, TM5, TM6, and TM7) prior to coupling to a G protein, and that the agonist-bound receptor conformation is different from the G protein coupled state. While most of the conformational changes observed in the β2AR are qualitatively the same for Gs and Gi1, we detected distinct differences between the β2AR-Gs and the β2AR-Gi1 complex in intracellular loop 2 (ICL2). Interactions with ICL2 are essential for activation of Gs. These differences between the β2AR-Gs and β2AR-Gi1 complexes in ICL2 may be key determinants for G protein coupling selectivity.
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Vizurraga, Alexander, Rashmi Adhikari, Jennifer Yeung, Maiya Yu, and Gregory G. Tall. "Mechanisms of adhesion G protein–coupled receptor activation." Journal of Biological Chemistry 295, no. 41 (August 6, 2020): 14065–83. http://dx.doi.org/10.1074/jbc.rev120.007423.
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Adhesion G protein–coupled receptors (AGPCRs) are a thirty-three-member subfamily of Class B GPCRs that control a wide array of physiological processes and are implicated in disease. AGPCRs uniquely contain large, self-proteolyzing extracellular regions that range from hundreds to thousands of residues in length. AGPCR autoproteolysis occurs within the extracellular GPCR autoproteolysis-inducing (GAIN) domain that is proximal to the N terminus of the G protein–coupling seven-transmembrane–spanning bundle. GAIN domain–mediated self-cleavage is constitutive and produces two-fragment holoreceptors that remain bound at the cell surface. It has been of recent interest to understand how AGPCRs are activated in relation to their two-fragment topologies. Dissociation of the AGPCR fragments stimulates G protein signaling through the action of the tethered-peptide agonist stalk that is occluded within the GAIN domain in the holoreceptor form. AGPCRs can also signal independently of fragment dissociation, and a few receptors possess GAIN domains incapable of self-proteolysis. This has resulted in complex theories as to how these receptors are activated in vivo, complicating pharmacological advances. Currently, there is no existing structure of an activated AGPCR to support any of the theories. Further confounding AGPCR research is that many of the receptors remain orphans and lack identified activating ligands. In this review, we provide a detailed layout of the current theorized modes of AGPCR activation with discussion of potential parallels to mechanisms used by other GPCR classes. We provide a classification means for the ligands that have been identified and discuss how these ligands may activate AGPCRs in physiological contexts.
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ROLFE, Mark, Laura E. McLEOD, Phillip F. PRATT, and Christopher G. PROUD. "Activation of protein synthesis in cardiomyocytes by the hypertrophic agent phenylephrine requires the activation of ERK and involves phosphorylation of tuberous sclerosis complex 2 (TSC2)." Biochemical Journal 388, no. 3 (June 7, 2005): 973–84. http://dx.doi.org/10.1042/bj20041888.
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The hypertrophic Gq-protein-coupled receptor agonist PE (phenylephrine) activates protein synthesis. We showed previously that activation of protein synthesis by PE requires MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase] and mTOR (mammalian target of rapamycin). However, it remained unclear whether ERK activation was required and which downstream components were involved in activating mTOR and protein synthesis. Using an adenovirus encoding the MKP3 (MAPK phosphatase 3) to inhibit ERK activity, we demonstrate that ERK is essential for the activation of protein synthesis by PE. Activation and phosphorylation of S6K1 (ribosomal protein S6 kinase 1) and phosphorylation of eIF4E (eukaryotic initiation factor 4E)-binding protein (both are mTOR targets) were also inhibited by MKP3, suggesting that ERK is also required for the activation of mTOR signalling. PE stimulation of cardiomyocytes induced the phosphorylation of TSC2 (tuberous sclerosis complex 2), a negative regulator of mTOR activity. TSC2 was phosphorylated only weakly at Thr1462, but phosphorylated at additional sites within the sequence RXRXX(S/T). This differs from the phosphorylation induced by insulin, indicating that MEK/ERK signalling targets distinct sites in TSC2. This phosphorylation may be mediated by p90RSK (90 kDa ribosomal protein S6K), which is activated by ERK, and appears to involve phosphorylation at Ser1798. Activation of protein synthesis by PE is partially insensitive to the mTOR inhibitor rapamycin. Inhibition of the MAPK-interacting kinases by CGP57380 decreases the phosphorylation of eIF4E and PE-induced protein synthesis. Moreover, CGP57380+rapamycin inhibited protein synthesis to the same extent as blocking ERK activation, suggesting that MAPK-interacting kinases and regulation of mTOR each contribute to the activation of protein synthesis by PE in cardiomyocytes.
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Lin, Hung-Yun, Faith B. Davis, Jennifer K. Gordinier, Leon J. Martino, and Paul J. Davis. "Thyroid hormone induces activation of mitogen-activated protein kinase in cultured cells." American Journal of Physiology-Cell Physiology 276, no. 5 (May 1, 1999): C1014—C1024. http://dx.doi.org/10.1152/ajpcell.1999.276.5.c1014.
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Thyroid hormone [l-thyroxine (T4)] rapidly induced phosphorylation and nuclear translocation (activation) of mitogen-activated protein kinase (MAPK) in HeLa and CV-1 cells in the absence of cytokine or growth factor. A pertussis toxin-sensitive and guanosine 5′- O-(3-thiotriphosphate)-sensitive cell surface mechanism responsive to T4and agarose-T4, suggesting a G protein-coupled receptor, was implicated. Cells depleted of MAPK or treated with MAPK pathway inhibitors showed reduced activation of MAPK and of the signal transducer and activator of transcription STAT1α by T4; they also showed reduced T4potentiation of the antiviral action of interferon-γ (IFN-γ). T4treatment caused tyrosine-phosphorylated MAPK-STAT1α nuclear complex formation and enhanced Ser-727 phosphorylation of STAT1α, in the presence or absence of IFN-γ. STAT1α-deficient cells transfected with STAT1α containing an alanine-for-serine substitution at residue 727 (STAT1αA727) showed minimal T4-stimulated STAT1α activation. IFN-γ induced the antiviral state in cells containing wild-type STAT1α (STAT1αwt) or STAT1αA727; T4potentiated IFN-γ action in STAT1αwtcells but not in STAT1αA727cells. T4-directed STAT1α Ser-727 phosphorylation is MAPK mediated and results in potentiated STAT1α activation and enhanced IFN-γ activity.
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Yang, Teddy T. C., Qiufang Xiong, Isabella A. Graef, Gerald R. Crabtree, and Chi-Wing Chow. "Recruitment of the Extracellular Signal-Regulated Kinase/Ribosomal S6 Kinase Signaling Pathway to the NFATc4 Transcription Activation Complex." Molecular and Cellular Biology 25, no. 3 (February 1, 2005): 907–20. http://dx.doi.org/10.1128/mcb.25.3.907-920.2005.
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ABSTRACT Integration of protein kinases into transcription activation complexes influences the magnitude of gene expression. The nuclear factor of activated T cells (NFAT) group of proteins are critical transcription factors that direct gene expression in immune and nonimmune cells. A balance of phosphotransferase activity is necessary for optimal NFAT activation. Activation of NFAT requires dephosphorylation by the calcium-mediated calcineurin phosphatase to promote NFAT nuclear accumulation, and the Ras-activated extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase, which targets NFAT partners, to potentiate transcription. Whether protein kinases operate on NFAT and contribute positively to transcription activation is not clear. Here, we coupled DNA affinity isolation with in-gel kinase assays to avidly pull down the activated NFAT and identify its associated protein kinases. We demonstrate that p90 ribosomal S6 kinase (RSK) is recruited to the NFAT-DNA transcription complex upon activation. The formation of RSK-NFATc4-DNA transcription complex is also apparent upon adipogenesis. Bound RSK phosphorylates Ser676 and potentiates NFATc4 DNA binding by escalating NFAT-DNA association. Ser676 is also targeted by the ERK MAP kinase, which interacts with NFAT at a distinct region than RSK. Thus, integration of the ERK/RSK signaling pathway provides a mechanism to modulate NFATc4 transcription activity.
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Becker, Elena, Uyen Huynh-Do, Sacha Holland, Tony Pawson, Tom O. Daniel, and Edward Y. Skolnik. "Nck-Interacting Ste20 Kinase Couples Eph Receptors to c-Jun N-Terminal Kinase and Integrin Activation." Molecular and Cellular Biology 20, no. 5 (March 1, 2000): 1537–45. http://dx.doi.org/10.1128/mcb.20.5.1537-1545.2000.
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ABSTRACT The mammalian Ste20 kinase Nck-interacting kinase (NIK) specifically activates the c-Jun amino-terminal kinase (JNK) mitogen-activated protein kinase module. NIK also binds the SH3 domains of the SH2/SH3 adapter protein Nck. To determine whether Nck functions as an adapter to couple NIK to a receptor tyrosine kinase signaling pathway, we determined whether NIK is activated by Eph receptors (EphR). EphRs constitute the largest family of receptor tyrosine kinases (RTK), and members of this family play important roles in patterning of the nervous and vascular systems. In this report, we show that NIK kinase activity is specifically increased in cells stimulated by two EphRs, EphB1 and EphB2. EphB1 kinase activity and phosphorylation of a juxtamembrane tyrosine (Y594), conserved in all Eph receptors, are both critical for NIK activation by EphB1. Although pY594 in the EphB1R has previously been shown to bind the SH2 domain of Nck, we found that stimulation of EphB1 and EphB2 led predominantly to a complex between NIK/Nck, p62 dok , RasGAP, and an unidentified 145-kDa tyrosine-phosphorylated protein. Tyrosine-phosphorylated p62 dok most probably binds directly to the SH2 domain of Nck and RasGAP and indirectly to NIK bound to the SH3 domain of Nck. We found that NIK activation is also critical for coupling EphB1R to biological responses that include the activation of integrins and JNK by EphB1. Taken together, these findings support a model in which the recruitment of the Ste20 kinase NIK to phosphotyrosine-containing proteins by Nck is an important proximal step in the signaling cascade downstream of EphRs.
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Ferreira, Monica E., Stefan Hermann, Philippe Prochasson, Jerry L. Workman, Kurt D. Berndt, and Anthony P. H. Wright. "Mechanism of Transcription Factor Recruitment by Acidic Activators." Journal of Biological Chemistry 280, no. 23 (April 11, 2005): 21779–84. http://dx.doi.org/10.1074/jbc.m502627200.
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Many transcriptional activators are intrinsically unstructured yet display unique, defined conformations when bound to target proteins. Target-induced folding provides a mechanism by which activators could form specific interactions with an array of structurally unrelated target proteins. Evidence for such a binding mechanism has been reported previously in the context of the interaction between the cancer-related c-Myc protein and the TATA-binding protein, which can be modeled as a two-step process in which a rapidly forming, low affinity complex slowly converts to a more stable form, consistent with a coupled binding and folding reaction. To test the generality of the target-induced folding model, we investigated the binding of two widely studied acidic activators, Gal4 and VP16, to a set of target proteins, including TATA-binding protein and the Swi1 and Snf5 subunits of the Swi/Snf chromatin remodeling complex. Using surface plasmon resonance, we show that these activator-target combinations also display bi-phasic kinetics suggesting two distinct steps. A fast initial binding phase that is inhibited by high ionic strength is followed by a slow phase that is favored by increased temperature. In all cases, overall affinity increases with temperature and, in most cases, with increased ionic strength. These results are consistent with a general mechanism for recruitment of transcriptional components to promoters by naturally occurring acidic activators, by which the initial contact is mediated predominantly through electrostatic interactions, whereas subsequent target-induced folding of the activator results in a stable complex.
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Matsumura, Nobutoshi, Ian M. Robertson, Shereen M. Hamza, Carrie-Lynn M. Soltys, Miranda M. Sung, Grant Masson, Donna L. Beker, and Jason R. B. Dyck. "A novel complex I inhibitor protects against hypertension-induced left ventricular hypertrophy." American Journal of Physiology-Heart and Circulatory Physiology 312, no. 3 (March 1, 2017): H561—H570. http://dx.doi.org/10.1152/ajpheart.00604.2016.
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Since left ventricular hypertrophy (LVH) increases the susceptibility for the development of other cardiac conditions, pharmacotherapy that mitigates pathological cardiac remodeling may prove to be beneficial in patients with LVH. Previous work has shown that the activation of the energy-sensing kinase AMP-activated protein kinase (AMPK) can inhibit some of the molecular mechanisms that are involved in LVH. Of interest, metformin activates AMPK through its inhibition of mitochondrial complex I in the electron transport chain and can prevent LVH induced by pressure overload. However, metformin has additional cellular effects unrelated to AMPK activation, raising questions about whether mitochondrial complex I inhibition is sufficient to reduce LVH. Herein, we characterize the cardiac effects of a novel compound (R118), which is a more potent complex I inhibitor than metformin and is thus used at a much lower concentration. We show that R118 activates AMPK in the cardiomyocyte, inhibits multiple signaling pathways involved in LVH, and prevents Gq protein-coupled receptor agonist-induced prohypertrophic signaling. We also show that in vivo administration of R118 prevents LVH in a mouse model of hypertension, suggesting that R118 can directly modulate the response of the cardiomyocyte to stress. Of importance, we also show that while R118 treatment prevents adaptive remodelling in response to elevated afterload, it does so without compromising systolic function, improves myocardial energetics, and prevents a decline in diastolic function in hypertensive mice. Taken together, our data suggest that inhibition of mitochondrial complex I may be worthy of future investigation for the treatment of LVH. NEW & NOTEWORTHY Inhibition of mitochondrial complex I by R118 reduces left ventricular hypertrophy (LVH) and improves myocardial energetics as well as diastolic function without compromising systolic function. Together, these effects demonstrate the therapeutic potential of complex I inhibitors in the treatment of LVH, even in the presence of persistent hypertension.
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Wright, Shane C., Maria Consuelo Alonso Cañizal, Tobias Benkel, Katharina Simon, Christian Le Gouill, Pierre Matricon, Yoon Namkung, et al. "FZD5 is a Gαq-coupled receptor that exhibits the functional hallmarks of prototypical GPCRs." Science Signaling 11, no. 559 (December 4, 2018): eaar5536. http://dx.doi.org/10.1126/scisignal.aar5536.
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Frizzleds (FZDs) are a group of seven transmembrane–spanning (7TM) receptors that belong to class F of the G protein–coupled receptor (GPCR) superfamily. FZDs bind WNT proteins to stimulate diverse signaling cascades involved in embryonic development, stem cell regulation, and adult tissue homeostasis. Frizzled 5 (FZD5) is one of the most studied class F GPCRs that promote the functional inactivation of the β-catenin destruction complex in response to WNTs. However, whether FZDs function as prototypical GPCRs has been heavily debated and, in particular, FZD5 has not been shown to activate heterotrimeric G proteins. Here, we show that FZD5 exhibited a conformational change after the addition of WNT-5A, which is reminiscent of class A and class B GPCR activation. In addition, we performed several live-cell imaging and spectrometric-based approaches, such as dual-color fluorescence recovery after photobleaching (dcFRAP) and resonance energy transfer (RET)–based assays that demonstrated that FZD5 activated Gαq and its downstream effectors upon stimulation with WNT-5A. Together, these findings suggest that FZD5 is a 7TM receptor with a bona fide GPCR activation profile and suggest novel targets for drug discovery in WNT-FZD signaling.
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Efimova, Tatiana, Ann-Marie Broome, and Richard L. Eckert. "Protein Kinase Cδ Regulates Keratinocyte Death and Survival by Regulating Activity and Subcellular Localization of a p38δ-Extracellular Signal-Regulated Kinase 1/2 Complex." Molecular and Cellular Biology 24, no. 18 (September 15, 2004): 8167–83. http://dx.doi.org/10.1128/mcb.24.18.8167-8183.2004.
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ABSTRACT Protein kinase Cδ (PKCδ) is an important regulator of apoptosis in epidermal keratinocytes. However, little information is available regarding the downstream kinases that mediate PKCδ-dependent keratinocyte death. This study implicates p38δ mitogen-activated protein kinase (MAPK) as a downstream carrier of the PKCδ-dependent death signal. We show that coexpression of PKCδ with p38δ produces profound apoptosis-like morphological changes. These morphological changes are associated with increased sub-G1 cell population, cytochrome c release, loss of mitochondrial membrane potential, caspase activation, and PARP cleavage. This death response is specific for the combination of PKCδ and p38δ and is not produced by replacing PKCδ with PKCα or p38δ with p38α. A constitutively active form of MEK6, an upstream activator of p38δ, can also produce cell death when coupled with p38δ. In addition, concurrent p38δ activation and extracellular signal-regulated kinase 1/2 (ERK1/2) inactivation are required for apoptosis. Regarding this inverse regulation, we describe a p38δ-ERK1/2 complex that may coordinate these changes in activity. We further show that this p38δ-ERK1/2 complex relocates into the nucleus in response to PKCδ expression. This regulation appears to be physiological, since H2O2, a known inducer of keratinocyte apoptosis, promotes identical PKCδ and p38δ-ERK1/2 activity changes, leading to similar morphological changes.
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Eng, Francis J., and Scott L. Friedman. "Fibrogenesis I. New insights into hepatic stellate cell activation: the simple becomes complex." American Journal of Physiology-Gastrointestinal and Liver Physiology 279, no. 1 (July 1, 2000): G7—G11. http://dx.doi.org/10.1152/ajpgi.2000.279.1.g7.
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Hepatic stellate cell activation is a complex process. Paradoxes and controversies include the origin(s) of hepatic stellate cells, the regulation of membrane receptor signaling and transcription, and the fate of the cells once liver injury resolves. Major themes have emerged, including the dominance of autocrine signaling and the identification of counterregulatory stimuli that oppose key features of activated cells. Advances in analytical methods including proteomics and gene array, coupled with powerful bioinformatics, promise to revolutionize how we view cellular responses. Our understanding of stellate cell activation is likely to benefit from these advances, unearthing modes of regulating cellular behavior that are not even conceivable on the basis of current paradigms.
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Skov, Søren, Mette Nielsen, Søren Bregenholt, Niels Ødum, and Mogens H. Claesson. "Activation of Stat-3 Is Involved in the Induction of Apoptosis After Ligation of Major Histocompatibility Complex Class I Molecules on Human Jurkat T Cells." Blood 91, no. 10 (May 15, 1998): 3566–73. http://dx.doi.org/10.1182/blood.v91.10.3566.
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Abstract Activation of Janus tyrosine kinases (Jak) and Signal transducers and activators of transcription (Stat) after ligation of major histocompatibility complex class I (MHC-I) was explored in Jurkat T cells. Cross-linking of MHC-I mediated tyrosine phosphorylation of Tyk2, but not Jak1, Jak2, and Jak3. In addition, the transcription factor Stat-3 was tyrosine phosphorylated in the cytoplasma and subsequently translocated to the cell nucleus. Data obtained by electrophoretic mobility shift assay suggested that the activated Stat-3 protein associates with the human serum-inducible element (hSIE) DNA-probe derived from the interferon-γ activated site (GAS) in the c-fos promoter, a common DNA sequence for Stat protein binding. An association between hSIE and Stat-3 after MHC-I ligation was directly demonstrated by precipitating Stat-3 from nuclear extracts with biotinylated hSIE probe and avidin-coupled agarose. To investigate the function of the activated Stat-3, Jurkat T cells were transiently transfected with a Stat-3 isoform lacking the transactivating domain. This dominant-negative acting Stat-3 isoform significantly inhibited apoptosis induced by ligation of MHC-I. In conclusion, our data suggest the involvement of the Jak/Stat signal pathway in MHC-I–induced signal transduction in T cells.
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Skov, Søren, Mette Nielsen, Søren Bregenholt, Niels Ødum, and Mogens H. Claesson. "Activation of Stat-3 Is Involved in the Induction of Apoptosis After Ligation of Major Histocompatibility Complex Class I Molecules on Human Jurkat T Cells." Blood 91, no. 10 (May 15, 1998): 3566–73. http://dx.doi.org/10.1182/blood.v91.10.3566.3566_3566_3573.
Full textAbstract:
Activation of Janus tyrosine kinases (Jak) and Signal transducers and activators of transcription (Stat) after ligation of major histocompatibility complex class I (MHC-I) was explored in Jurkat T cells. Cross-linking of MHC-I mediated tyrosine phosphorylation of Tyk2, but not Jak1, Jak2, and Jak3. In addition, the transcription factor Stat-3 was tyrosine phosphorylated in the cytoplasma and subsequently translocated to the cell nucleus. Data obtained by electrophoretic mobility shift assay suggested that the activated Stat-3 protein associates with the human serum-inducible element (hSIE) DNA-probe derived from the interferon-γ activated site (GAS) in the c-fos promoter, a common DNA sequence for Stat protein binding. An association between hSIE and Stat-3 after MHC-I ligation was directly demonstrated by precipitating Stat-3 from nuclear extracts with biotinylated hSIE probe and avidin-coupled agarose. To investigate the function of the activated Stat-3, Jurkat T cells were transiently transfected with a Stat-3 isoform lacking the transactivating domain. This dominant-negative acting Stat-3 isoform significantly inhibited apoptosis induced by ligation of MHC-I. In conclusion, our data suggest the involvement of the Jak/Stat signal pathway in MHC-I–induced signal transduction in T cells.
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De Koninck, Yves, and Istvan Mody. "Endogenous GABA Activates Small-Conductance K+ Channels Underlying Slow IPSCs in Rat Hippocampal Neurons." Journal of Neurophysiology 77, no. 4 (April 1, 1997): 2202–8. http://dx.doi.org/10.1152/jn.1997.77.4.2202.
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De Koninck, Yves and Istvan Mody. Endogenous GABA activates small-conductance K+ channels underlying slow IPSCs in rat hippocampal neurons. J. Neurophysiol. 77: 2202–2208, 1997. The objective of this study was to determine the properties of K+ channels activated by endogenously released trasmitter under synaptic conditions. First, the levels of γ-aminobutyric acid (GABA) were depleted in hippocampal nerve endings to establish the relative contribution of endogenously released GABA to the activation of GABAB receptors mediating slow inhibitory postsynaptic currents (IPSCs). Inhibition of glutamic acid decarboxylase and GABA reuptake effectively depleted >85% of the releasable GABA pool, producing parallel reductions of GABAA and GABAB receptor-mediated IPSCs, indicating that both classes of receptors are activated synaptically by endogenously released GABA. Whole cell patch-clamp recordings of stimulus-evoked slow IPSCs at potentials hyperpolarized from the potassium reversal potential were consistent with the activation of a nonrectifying ( n = 3) or slightly outwardly rectifying ( n = 4) K+ conductance by the endogenously released GABA. Spectral analysis of the decay phase of GABAB IPSCs revealed several time constants indicating complex underlying channel kinetics. Nonstationary variance analysis yielded a small unitary conductance in the range of 5–13 pS, consistent with a large number of channels activated during evoked currents. These results indicate that in granule cells of the dentate gyrus, GABA released synaptically from interneuron terminals activates an unusually small K+ conductance, with no or slight outward rectification. This conductance is therefore unlike those typically reported for neuronal G protein-coupled K+ channels or those activated by exogenously applied baclofen with larger, inwardly rectifying conductances.
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Kasthuri, Raj S., Mark B. Taubman, and Nigel Mackman. "Role of Tissue Factor in Cancer." Journal of Clinical Oncology 27, no. 29 (October 10, 2009): 4834–38. http://dx.doi.org/10.1200/jco.2009.22.6324.
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Tissue factor (TF) is a transmembrane glycoprotein that localizes the coagulation serine protease factor VII/VIIa (FVII/VIIa) to the cell surface. The primary function of TF is to activate the clotting cascade. The TF:FVIIa complex also activates cells by cleavage of a G-protein coupled receptor called protease-activated receptor 2 (PAR2). TF is expressed by tumor cells and contributes to a variety of pathologic processes, such as thrombosis, metastasis, tumor growth, and tumor angiogenesis. For instance, tumor cells release TF-positive procoagulant microparticles into the circulation and these may trigger venous thromboembolism in patients with cancer. TF on circulating tumor cells also leads to the coating of the cells with fibrin that traps them within the microvasculature and facilitates hematogenous metastasis. In addition, TF:FVIIa-dependent activation of PAR2 on tumor cells increases tumor growth via an undefined mechanism. One possibility is that PAR2-dependent signaling increases the expression of proangiogenic proteins. Other studies have reported that endothelial cells in the tumor vasculature express TF and this may enhance angiogenesis. These results suggest that inhibition of TF should reduce several pathologic pathways that increase tumor growth and metastasis. This would represent a novel approach to anticancer therapy. Initial studies using inhibitors of the TF:FVIIa complex in mouse tumor models have produced encouraging results. Nevertheless, additional studies are needed to determine if this strategy can be successfully translated to the treatment of cancer patients.
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MA, Hong-Tao, Kartik VENKATACHALAM, Krystyna E. RYS-SIKORA, Li-Ping HE, Fei ZHENG, and Donald L. GILL. "Modification of phospholipase C-γ-induced Ca2+ signal generation by 2-aminoethoxydiphenyl borate." Biochemical Journal 376, no. 3 (December 15, 2003): 667–76. http://dx.doi.org/10.1042/bj20031345.
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The mechanisms by which Ca2+-store-release channels and Ca2+-entry channels are coupled to receptor activation are poorly understood. Modification of Ca2+ signals by 2-aminoethoxydiphenyl borate (2-APB), suggests the agent may target entry channels or the machinery controlling their activation. In DT40 B-cells and Jurkat T-cells, complete Ca2+ store release was induced by 2-APB (EC50 10–20 µM). At 75 µM, 2-APB emptied stores completely in both lymphocyte lines, but had no such effect on other cells. In DT40 cells, 2-APB mimicked B-cell receptor (BCR) cross-linking, but no effect was observed in mutant DT40 lines devoid of inositol 1,4,5-trisphosphate (InsP3) receptors (InsP3Rs) or phospholipase C-γ2 (PLC-γ2). Like the BCR, 2-APB activated transfected TRPC3 (canonical transient receptor potential) channels, which acted as sensors for PLC-γ2-generated diacylglycerol in DT40 cells. The action of 2-APB on InsP3Rs and TRPC3 channels was prevented by PLC-inhibition, and required PLC-γ2 catalytic activity. However, unlike BCR activation, no increased InsP3 level could be measured in response to 2-APB. Also, calyculin A-induced cytoskeletal reorganization prevented 2-APB-induced InsP3R and TRPC3-channel activation, but not that induced by the BCR. 2-APB still activated TRPC3 channels in DT40 cells with fully depleted Ca2+ stores, indicating its action was not via Ca2+ release. Significantly, 2-APB-induced InsP3R and TRPC3 activation was prevented in DT40 knockout cells devoid of the BCR- and PLC-γ2-coupled adaptor/kinases, Syk, Lyn, Btk or BLNK. The results suggest that 2-APB activates Ca2+ signals in lymphocytes by initiating and enhancing coupling between components of the BCR–PLC-γ2 complex and both Ca2+-entry and Ca2+-release channels.
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Kino, Tomoshige, Anatoly Tiulpakov, Takamasa Ichijo, Ly Chheng, Tohru Kozasa, and George P. Chrousos. "G protein β interacts with the glucocorticoid receptor and suppresses its transcriptional activity in the nucleus." Journal of Cell Biology 169, no. 6 (June 13, 2005): 885–96. http://dx.doi.org/10.1083/jcb.200409150.
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Extracellular stimuli that activate cell surface receptors modulate glucocorticoid actions via as yet unclear mechanisms. Here, we report that the guanine nucleotide-binding protein (G protein)–coupled receptor-activated WD-repeat Gβ interacts with the glucocorticoid receptor (GR), comigrates with it into the nucleus and suppresses GR-induced transactivation of the glucocorticoid-responsive genes. Association of Gγ with Gβ is necessary for this action of Gβ. Both endogenous and enhanced green fluorescent protein (EGFP)–fused Gβ2 and Gγ2 proteins were detected in the nucleus at baseline, whereas a fraction of EGFP-Gβ2 and DsRed2-GR comigrated to the nucleus or the plasma membrane, depending on the exposure of cells to dexamethasone or somatostatin, respectively. Gβ2 was associated with GR/glucocorticoid response elements (GREs) in vivo and suppressed activation function-2–directed transcriptional activity of the GR. We conclude that the Gβγ complex interacts with the GR and suppresses its transcriptional activity by associating with the transcriptional complex formed on GR-responsive promoters.
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Sharma, Vivek, Galina Belevich, Ana P. Gamiz-Hernandez, Tomasz Róg, Ilpo Vattulainen, Marina L. Verkhovskaya, Mårten Wikström, Gerhard Hummer, and Ville R. I. Kaila. "Redox-induced activation of the proton pump in the respiratory complex I." Proceedings of the National Academy of Sciences 112, no. 37 (September 1, 2015): 11571–76. http://dx.doi.org/10.1073/pnas.1503761112.
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Complex I functions as a redox-linked proton pump in the respiratory chains of mitochondria and bacteria, driven by the reduction of quinone (Q) by NADH. Remarkably, the distance between the Q reduction site and the most distant proton channels extends nearly 200 Å. To elucidate the molecular origin of this long-range coupling, we apply a combination of large-scale molecular simulations and a site-directed mutagenesis experiment of a key residue. In hybrid quantum mechanics/molecular mechanics simulations, we observe that reduction of Q is coupled to its local protonation by the His-38/Asp-139 ion pair and Tyr-87 of subunit Nqo4. Atomistic classical molecular dynamics simulations further suggest that formation of quinol (QH2) triggers rapid dissociation of the anionic Asp-139 toward the membrane domain that couples to conformational changes in a network of conserved charged residues. Site-directed mutagenesis data confirm the importance of Asp-139; upon mutation to asparagine the Q reductase activity is inhibited by 75%. The current results, together with earlier biochemical data, suggest that the proton pumping in complex I is activated by a unique combination of electrostatic and conformational transitions.
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Krupitza, G., and G. Thireos. "Translational activation of GCN4 mRNA in a cell-free system is triggered by uncharged tRNAs." Molecular and Cellular Biology 10, no. 8 (August 1990): 4375–78. http://dx.doi.org/10.1128/mcb.10.8.4375.
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Translation of GCN4 mRNA is activated when yeast cells are grown under conditions of amino acid limitation. In this study, we established the conditions through which translation of the GCN4 mRNA could be activated in a homologous in vitro system. This activation paralleled the in vivo situation: it required the small open reading frames located in the 5' untranslated region of the GCN4 mRNA, and it was coupled with reduced rates of 43S preinitiation complex formation. Translational derepression in vitro was triggered by uncharged tRNA molecules, demonstrating that deacylated tRNAs are more proximal signals for translational activation of the GCN4 mRNA.