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ABSTRACT: The heterotrimeric G protein Gα(q) is a key regulator of blood pressure, and excess Gα(q) signaling leads to hypertension. A specific inhibitor of Gα(q) is the GTPase activating protein (GAP) known as regulator of G protein signaling 2 (RGS2). The molecular basis for how Gα(q/11) subunits serve as substrates for RGS proteins and how RGS2 mandates its selectivity for Gα(q) is poorly understood. In crystal structures of the RGS2-Gα(q) complex, RGS2 docks to Gα(q) in a different orientation from that observed in RGS-Gα(i/o) complexes. Despite its unique pose, RGS2 maintains canonical interactions with the switch regions of Gα(q) in part because its α6 helix adopts a distinct conformation. We show that RGS2 forms extensive interactions with the α-helical domain of Gα(q) that contribute to binding affinity and GAP potency. RGS subfamilies that do not serve as GAPs for Gα(q) are unlikely to form analogous stabilizing interactions.
Structure 02/2013; · 6.35 Impact Factor
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ABSTRACT: G protein-mediated signal transduction can transduce signals from a large variety of extracellular stimuli into cells and is the most widely used mechanism for cell communication at the membrane. The RhoGTPase family has been well established as key regulators of cell growth, differentiation and cell shape changes. Among G protein-mediated signal transduction, G12/13-mediated signalling is one mechanism to regulate RhoGTPase activity in response to extracellular stimuli. The alpha subunits of G12 or G13 have been shown to interact with members of the RH domain containing guanine nucleotide exchange factors for Rho (RH-RhoGEF) family of proteins to directly connect G protein-mediated signalling and RhoGTPase signalling. The G12/13-RH-RhoGEF signalling mechanism is well conserved over species and is involved in critical steps for cell physiology and disease conditions, including embryonic development, oncogenesis and cancer metastasis. In this review, we will summarize current progress on this important signalling mechanism.
Journal of biochemistry 08/2011; 150(4):357-69. · 1.95 Impact Factor
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ABSTRACT: RH-RhoGEFs are a family of guanine nucleotide exchange factors that contain a regulator of G protein signaling homology (RH) domain. The heterotrimeric G protein Gα(13) stimulates the guanine nucleotide exchange factor (GEF) activity of RH-RhoGEFs, leading to activation of RhoA. The mechanism by which Gα(13) stimulates the GEF activity of RH-RhoGEFs, such as p115RhoGEF, has not yet been fully elucidated. Here, specific residues in Gα(13) that mediate activation of p115RhoGEF are identified. Mutation of these residues significantly impairs binding of Gα(13) to p115RhoGEF as well as stimulation of GEF activity. These data suggest that the exchange activity of p115RhoGEF is stimulated allosterically by Gα(13) and not through its interaction with a secondary binding site. A crystal structure of Gα(13) bound to the RH domain of p115RhoGEF is also presented, which differs from a previously crystallized complex with a Gα(13)-Gα(i1) chimera. Taken together, these data provide new insight into the mechanism by which p115RhoGEF is activated by Gα(13).
Journal of Biological Chemistry 06/2011; 286(23):20625-36. · 4.77 Impact Factor
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ABSTRACT: RH-RhoGEFs are a family of guanine nucleotide exchange factors that contain a regulator of G protein signaling homology (RH)
domain. The heterotrimeric G protein Gα13 stimulates the guanine nucleotide exchange factor (GEF) activity of RH-RhoGEFs, leading to activation of RhoA. The mechanism
by which Gα13 stimulates the GEF activity of RH-RhoGEFs, such as p115RhoGEF, has not yet been fully elucidated. Here, specific residues
in Gα13 that mediate activation of p115RhoGEF are identified. Mutation of these residues significantly impairs binding of Gα13 to p115RhoGEF as well as stimulation of GEF activity. These data suggest that the exchange activity of p115RhoGEF is stimulated
allosterically by Gα13 and not through its interaction with a secondary binding site. A crystal structure of Gα13 bound to the RH domain of p115RhoGEF is also presented, which differs from a previously crystallized complex with a Gα13-Gαi1 chimera. Taken together, these data provide new insight into the mechanism by which p115RhoGEF is activated by Gα13.
Journal of Biological Chemistry 06/2011; 286(23):20625-20636. · 4.77 Impact Factor
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Gary L. Waldo,
Tiffany K. Ricks,
Stephanie N. Hicks,
Matthew L. Cheever,
Takeharu Kawano,
Kazuhito Tsuboi,
Xiaoyue Wang,
Craig Montell, Tohru Kozasa,
John Sondek,
T. Kendall Harden
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ABSTRACT: Transmembrane signals initiated by a broad range of extracellular stimuli converge on nodes that regulate phospholipase C
(PLC)–dependent inositol lipid hydrolysis for signal propagation. We describe how heterotrimeric guanine nucleotide–binding
proteins (G proteins) activate PLC-βs and in turn are deactivated by these downstream effectors. The 2.7-angstrom structure
of PLC-β3 bound to activated Gαq reveals a conserved module found within PLC-βs and other effectors optimized for rapid engagement of activated G proteins.
The active site of PLC-β3 in the complex is occluded by an intramolecular plug that is likely removed upon G protein–dependent
anchoring and orientation of the lipase at membrane surfaces. A second domain of PLC-β3 subsequently accelerates guanosine
triphosphate hydrolysis by Gαq, causing the complex to dissociate and terminate signal propagation. Mutations within this domain dramatically delay signal
termination in vitro and in vivo. Consequently, this work suggests a dynamic catch-and-release mechanism used to sharpen spatiotemporal
signals mediated by diverse sensory inputs.
Science 11/2010; 330(6006):974-980. · 31.20 Impact Factor
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Gary L Waldo,
Tiffany K Ricks,
Stephanie N Hicks,
Matthew L Cheever,
Takeharu Kawano,
Kazuhito Tsuboi,
Xiaoyue Wang,
Craig Montell, Tohru Kozasa,
John Sondek,
T Kendall Harden
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ABSTRACT: Transmembrane signals initiated by a broad range of extracellular stimuli converge on nodes that regulate phospholipase C (PLC)-dependent inositol lipid hydrolysis for signal propagation. We describe how heterotrimeric guanine nucleotide-binding proteins (G proteins) activate PLC-βs and in turn are deactivated by these downstream effectors. The 2.7-angstrom structure of PLC-β3 bound to activated Gα(q) reveals a conserved module found within PLC-βs and other effectors optimized for rapid engagement of activated G proteins. The active site of PLC-β3 in the complex is occluded by an intramolecular plug that is likely removed upon G protein-dependent anchoring and orientation of the lipase at membrane surfaces. A second domain of PLC-β3 subsequently accelerates guanosine triphosphate hydrolysis by Gα(q), causing the complex to dissociate and terminate signal propagation. Mutations within this domain dramatically delay signal termination in vitro and in vivo. Consequently, this work suggests a dynamic catch-and-release mechanism used to sharpen spatiotemporal signals mediated by diverse sensory inputs.
Science 10/2010; 330(6006):974-80. · 31.20 Impact Factor
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ABSTRACT: Integrins mediate cell adhesion to the extracellular matrix and transmit signals within the cell that stimulate cell spreading, retraction, migration, and proliferation. The mechanism of integrin outside-in signaling has been unclear. We found that the heterotrimeric guanine nucleotide-binding protein (G protein) Galpha13 directly bound to the integrin beta3 cytoplasmic domain and that Galpha13-integrin interaction was promoted by ligand binding to the integrin alphaIIbbeta3 and by guanosine triphosphate (GTP) loading of Galpha13. Interference of Galpha13 expression or a myristoylated fragment of Galpha13 that inhibited interaction of alphaIIbbeta3 with Galpha13 diminished activation of protein kinase c-Src and stimulated the small guanosine triphosphatase RhoA, consequently inhibiting cell spreading and accelerating cell retraction. We conclude that integrins are noncanonical Galpha13-coupled receptors that provide a mechanism for dynamic regulation of RhoA.
Science 01/2010; 327(5963):340-3. · 31.20 Impact Factor
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Kristin K Jernigan,
Christopher S Cselenyi,
Curtis A Thorne,
Alison J Hanson,
Emilios Tahinci,
Nicole Hajicek,
William M Oldham,
Laura A Lee,
Heidi E Hamm,
John R Hepler, Tohru Kozasa,
Maurine E Linder,
Ethan Lee
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ABSTRACT: Evidence from Drosophila and cultured cell studies supports a role for heterotrimeric guanosine triphosphate-binding proteins (G proteins) in Wnt signaling. Wnt inhibits the degradation of the transcriptional regulator beta-catenin. We screened the alpha and betagamma subunits of major families of G proteins in a Xenopus egg extract system that reconstitutes beta-catenin degradation. We found that Galpha(o), Galpha(q), Galpha(i2), and Gbetagamma inhibited beta-catenin degradation. Gbeta(1)gamma(2) promoted the phosphorylation and activation of the Wnt co-receptor low-density lipoprotein receptor-related protein 6 (LRP6) by recruiting glycogen synthase kinase 3 (GSK3) to the membrane and enhancing its kinase activity. In both a reporter gene assay and an in vivo assay, c-betaARK (C-terminal domain of beta-adrenergic receptor kinase), an inhibitor of Gbetagamma, blocked LRP6 activity. Several components of the Wnt-beta-catenin pathway formed a complex: Gbeta(1)gamma(2), LRP6, GSK3, axin, and dishevelled. We propose that free Gbetagamma and Galpha subunits, released from activated G proteins, act cooperatively to inhibit beta-catenin degradation and activate beta-catenin-mediated transcription.
Science Signaling 01/2010; 3(121):ra37. · 7.50 Impact Factor
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ABSTRACT: The transforming heterotrimeric G proteins Gα12 and Gα13 have been implicated in the regulation of various cellular processes,
such as gene transcription and rearrangement of the actin-based cytoskeleton. Many of these functions are dependent on the
activation of the monomeric GTPase RhoA. The RGS-RhoGEFs, a distinct family of guanine nucleotide exchange factors, which
contain RGS-like domains in their N-termini, serve as direct links between Gα12/13 and RhoA. The focus of this chapter is
on the molecular mechanisms that underlie and regulate signaling from Gα12/13 to RhoA via the RGS-RhoGEFs. The physiological
significance of this signaling pathway is also discussed.
12/2009: pages 59-76;
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ABSTRACT: Accumulating data indicate that G12 subfamily (Galpha12/13)-mediated signaling pathways play pivotal roles in a variety of physiological processes, while aberrant regulation of this pathway has been identified in various human diseases. It has been demonstrated that Galpha12/13-mediated signals form networks with other signaling proteins at various levels, from cell surface receptors to transcription factors, to regulate cellular responses. Galpha12/13 have slow rates of nucleotide exchange and GTP hydrolysis, and specifically target RhoGEFs containing an amino-terminal RGS homology domain (RH-RhoGEFs), which uniquely function both as a GAP and an effector for Galpha12/13. In this review, we will focus on the mechanisms regulating the Galpha12/13 signaling system, particularly the Galpha12/13-RH-RhoGEF-Rho pathway, which can regulate a wide variety of cellular functions from migration to transformation.
Neurosignals 02/2009; 17(1):55-70. · 2.11 Impact Factor
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ABSTRACT: The transient protein-protein interactions induced by guanine nucleotide-dependent conformational changes of G proteins play central roles in G protein-coupled receptor-mediated signaling systems. Leukemia-associated RhoGEF (LARG), a guanine nucleotide exchange factor for Rho, contains an RGS homology (RH) domain and Dbl homology/pleckstrin homology (DH/PH) domains and acts both as a GTPase-activating protein (GAP) and an effector for Galpha(13). However, the molecular mechanism of LARG activation upon Galpha(13) binding is not yet well understood. In this study, we analyzed the Galpha(13)-LARG interaction using cellular and biochemical methods, including a surface plasmon resonance (SPR) analysis. The results obtained using various LARG fragments demonstrated that active Galpha(13) interacts with LARG through the RH domain, DH/PH domains, and C-terminal region. However, an alanine substitution at the RH domain contact position in Galpha(13) resulted in a large decrease in affinity. Thermodynamic analysis revealed that binding of Galpha(13) proceeds with a large negative heat capacity change (DeltaCp degrees ), accompanied by a positive entropy change (DeltaS degrees ). These results likely indicate that the binding of Galpha(13) with the RH domain triggers conformational rearrangements between Galpha(13) and LARG burying an exposed hydrophobic surface to create a large complementary interface, which facilitates complex formation through both GAP and effector interfaces, and activates the RhoGEF. We propose that LARG activation is regulated by an induced-fit mechanism through the GAP interface of Galpha(13).
Journal of Biological Chemistry 01/2009; 284(8):5000-9. · 4.77 Impact Factor
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ABSTRACT: G-protein (Gbetagamma)-mediated voltage-dependent inhibition of N- and P/Q-type Ca(2+) channels contributes to presynaptic inhibition and short-term synaptic plasticity. The voltage dependence derives from the dissociation of Gbetagamma from the inhibited channels, but the underlying molecular and biophysical mechanisms remain largely unclear. In this study we investigated the role in this process of Ca(2+) channel beta subunit (Ca(v)beta) and a rigid alpha-helical structure between the alpha-interacting domain (AID), the primary Ca(v)beta docking site on the channel alpha(1) subunit, and the pore-lining IS6 segment. Gbetagamma inhibition of P/Q-type channels was reconstituted in giant inside-out membrane patches from Xenopus oocytes. Large populations of channels devoid of Ca(v)beta were produced by washing out a mutant Ca(v)beta with a reduced affinity for the AID. These beta-less channels were still inhibited by Gbetagamma, but without any voltage dependence, indicating that Ca(v)beta is indispensable for voltage-dependent Gbetagamma inhibition. A truncated Ca(v)beta containing only the AID-binding guanylate kinase (GK) domain could fully confer voltage dependence to Gbetagamma inhibition. Gbetagamma did not alter inactivation properties, and channels recovered from Gbetagamma inhibition exhibited the same activation property as un-inhibited channels, indicating that Gbetagamma does not dislodge Ca(v)beta from the inhibited channel. Furthermore, voltage-dependent Gbetagamma inhibition was abolished when the rigid alpha-helix between the AID and IS6 was disrupted by insertion of multiple glycines, which also eliminated Ca(v)beta regulation of channel gating, revealing a pivotal role of this rigid alpha-helix in both processes. These results suggest that depolarization-triggered movement of IS6, coupled to the subsequent conformational change of the Gbetagamma-binding pocket through a rigid alpha-helix induced partly by the Ca(v)beta GK domain, causes the dissociation of Gbetagamma and is fundamental to voltage-dependent Gbetagamma inhibition.
Journal of Neuroscience 01/2009; 28(52):14176-88. · 7.11 Impact Factor
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ABSTRACT: Transmembrane signaling through G alpha(q)-coupled receptors is linked to physiological processes such as cardiovascular development and smooth muscle function. Recent crystallographic studies have shown how G alpha(q) interacts with two activation-dependent targets, p63RhoGEF and G protein-coupled receptor kinase 2 (GRK2). These proteins bind to the effector-binding site of G alpha(q) in a manner that does not appear to physically overlap with the site on G alpha(q) bound by regulator of G-protein signaling (RGS) proteins, which function as GTPase-activating proteins (GAPs). Herein we confirm the formation of RGS-G alpha(q)-GRK2/p63RhoGEF ternary complexes using flow cytometry protein interaction and GAP assays. RGS2 and, to a lesser extent, RGS4 are negative allosteric modulators of Galpha(q) binding to either p63RhoGEF or GRK2. Conversely, GRK2 enhances the GAP activity of RGS4 but has little effect on that of RGS2. Similar but smaller magnitude responses are induced by p63RhoGEF. The fact that GRK2 and p63RhoGEF respond similarly to these RGS proteins supports the hypothesis that GRK2 is a bona fide G alpha(q) effector. The results also suggest that signal transduction pathways initiated by GRK2, such as the phosphorylation of G protein-coupled receptors, and by p63RhoGEF, such as the activation of gene transcription, can be regulated by RGS proteins via both allosteric and GAP mechanisms.
Journal of Biological Chemistry 11/2008; 283(50):34923-34. · 4.77 Impact Factor
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ABSTRACT: GRIN1 (Gprin 1) is a signaling molecule coexpression of which with constitutively active form of Galphao can stimulate neurite extensions in Neuro2a cells, yet its in vivo roles remain elusive. Here, we examine expression profiles of GRIN1 during mouse development by in situ hybridization (ISH) and immunohistochemistry. ISH analysis revealed that GRIN1 expression was limited to the nervous system at all developmental stages tested: in the central nervous system, GRIN1 expression occurred within the entire embryonic mantle zones, while it became restricted to sets of nuclei at postnatal to adult stages. Immunohistochemistry using a GRIN1-specific antibody demonstrated that GRIN1 colocalized with Galphao at neuronal dendrites and axons, but it was not detected in glial cells. These results suggest that Galphao-GRIN1 pathway could mediate significant roles in neuronal migration and differentiation at embryonic stages and exert functions in wiring and/or maintenance of specific neural circuitries at postnatal to adult stages.
Developmental Dynamics 10/2008; 237(9):2415-29. · 2.54 Impact Factor
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ABSTRACT: GRIN1 (Gprin 1) is a signaling molecule coexpression of which with constitutively active form of Gαo can stimulate neurite extensions in Neuro2a cells, yet its in vivo roles remain elusive. Here, we examine expression profiles of GRIN1 during mouse development by in situ hybridization (ISH) and immunohistochemistry. ISH analysis revealed that GRIN1 expression was limited to the nervous system at all developmental stages tested: in the central nervous system, GRIN1 expression occurred within the entire embryonic mantle zones, while it became restricted to sets of nuclei at postnatal to adult stages. Immunohistochemistry using a GRIN1-specific antibody demonstrated that GRIN1 colocalized with Gαo at neuronal dendrites and axons, but it was not detected in glial cells. These results suggest that Gαo-GRIN1 pathway could mediate significant roles in neuronal migration and differentiation at embryonic stages and exert functions in wiring and/or maintenance of specific neural circuitries at postnatal to adult stages. Developmental Dynamics 237:2415–2429, 2008. © 2008 Wiley-Liss, Inc.
Developmental Dynamics 08/2008; 237(9):2415 - 2429. · 2.54 Impact Factor
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ABSTRACT: The coordinated cross-talk from heterotrimeric G proteins to Rho GTPases is essential during a variety of physiological processes. Emerging data suggest that members of the Galpha(12/13) and Galpha(q/11) families of heterotrimeric G proteins signal downstream to RhoA via distinct pathways. Although studies have elucidated mechanisms governing Galpha(12/13)-mediated RhoA activation, proteins that functionally couple Galpha(q/11) to RhoA activation have remained elusive. Recently, the Dbl-family guanine nucleotide exchange factor (GEF) p63RhoGEF/GEFT has been described as a novel mediator of Galpha(q/11) signaling to RhoA based on its ability to synergize with Galpha(q/11) resulting in enhanced RhoA signaling in cells. We have used biochemical/biophysical approaches with purified protein components to better understand the mechanism by which activated Galpha(q) directly engages and stimulates p63RhoGEF. Basally, p63RhoGEF is autoinhibited by the Dbl homology (DH)-associated pleckstrin homology (PH) domain; activated Galpha(q) relieves this autoinhibition by interacting with a highly conserved C-terminal extension of the PH domain. This unique extension is conserved in the related Dbl-family members Trio and Kalirin and we show that the C-terminal Rho-specific DH-PH cassette of Trio is similarly activated by Galpha(q).
Journal of Biological Chemistry 11/2007; 282(40):29201-10. · 4.77 Impact Factor
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ABSTRACT: Galpha12 and Galpha13 transduce signals from G protein-coupled receptors to RhoA through RhoGEFs containing an RGS homology (RH) domain, such as p115 RhoGEF or leukemia-associated RhoGEF (LARG). The RH domain of p115 RhoGEF or LARG binds with high affinity to active forms of Galpha12 and Galpha13 and confers specific GTPase-activating protein (GAP) activity, with faster GAP responses detected in Galpha13 than in Galpha12. At the same time, Galpha13, but not Galpha12, directly stimulates the RhoGEF activity of p115 RhoGEF or nonphosphorylated LARG in reconstitution assays. In order to better understand the molecular mechanism by which Galpha13 regulates RhoGEF activity through interaction with RH-RhoGEFs, we sought to identify the region(s) of Galpha13 involved in either the GAP response or RhoGEF activation. For this purpose, we generated chimeras between Galpha12 and Galpha13 subunits and characterized their biochemical activities. In both cell-based and reconstitution assays of RhoA activation, we found that replacing the carboxyl-terminal region of Galpha12 (residues 267-379) with that of Galpha13 (residues 264-377) conferred gain-of-function to the resulting chimeric subunit, Galpha12C13. The inverse chimera, Galpha13C12, exhibited basal RhoA activation which was similar to Galpha12. In contrast to GEF assays, GAP assays showed that Galpha12C13 or Galpha13C12 chimeras responded to the GAP activity of p115 RhoGEF or LARG in a manner similar to Galpha12 or Galpha13, respectively. We conclude from these results that the carboxyl-terminal region of Galpha13 (residues 264-377) is essential for its RhoGEF stimulating activity, whereas the amino-terminal alpha helical and switch regions of Galpha12 and Galpha13 are responsible for their differential GAP responses to the RH domain.
Cellular Signalling 09/2007; 19(8):1681-9. · 4.06 Impact Factor
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ABSTRACT: Activation of substance P receptors, which are coupled to Galpha(q), inhibits the Kir3.1/3.2 channels, resulting in neuronal excitation. We have shown previously that this channel inactivation is not caused by reduction of the phosphatidylinositol 4,5-bisphosphate level in membrane. Moreover, Galpha(q) immunoprecipitates with Kir3.2 (J Physiol 564:489-500, 2005), suggesting that Galpha(q) interacts with Kir3.2. Positive immunoprecipitation, however, does not necessarily indicate direct interaction between the two proteins. Here, the glutathione transferase pull-down assay was used to investigate interaction between Galpha(q) and the K(+) channels. We found that Galpha(q) interacted with N termini of Kir3.1, Kir3.2, and Kir3.4. However, Galpha(q) did not interact with the C terminus of any Kir3 or with the C or N terminus of Kir2.1. TRPC6 is regulated by the signal initiated by Galpha(q). Immunoprecipitation, however, showed that Galpha(q) did not interact with TRPC6. Thus, the interaction between Galpha(q) and the Kir3 N terminus is quite specific. This interaction occurred in the presence of GDP or GDP-AlF(-)(4). The Galpha(q) binding could take place somewhere between residues 51 to 90 of Kir3.2; perhaps the segment between 81 to 90 residues is crucial. Gbetagamma, which is known to bind to N terminus of Kir3, did not compete with Galpha(q) for the binding, suggesting that these two binding regions are different. These findings agree with the hypothesis (J Physiol 564:489-500, 2005) that the signal to inactivate the Kir3 channel could be mainly transmitted directly from Galpha(q) to Kir3.
Molecular Pharmacology 05/2007; 71(4):1179-84. · 4.88 Impact Factor
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ABSTRACT: This study investigated the involvement of Galpha(13) switch region I (SRI) in protease-activated receptor 1 (PAR1)-mediated platelet function and signaling. To this end, myristoylated peptides representing the Galpha(13) SRI (Myr-G(13)SRI(pep)) and its random counterpart were evaluated for their effects on PAR1 activation. Initial studies demonstrated that Myr-G(13)SRI(pep) and Myr-G(13)SRI(Random-pep) were equally taken up by human platelets and did not interfere with PAR1-ligand interaction. Subsequent experiments revealed that Myr-G(13)SRI(pep) specifically bound to platelet RhoA guanine nucleotide exchange factor (p115RhoGEF) and blocked PAR1-mediated RhoA activation in platelets and human embryonic kidney cells. These results suggest a direct interaction of Galpha(13) SRI with p115RhoGEF and a mechanism for Myr-G(13)SRI(pep) inhibition of RhoA activation. Platelet function studies demonstrated that Myr-G(13)SRI(pep) specifically inhibited PAR1-stimulated shape change, aggregation, and secretion in a dose-dependent manner but did not inhibit platelet activation induced by either ADP or A23187. It was also found that Myr-G(13)SRI(pep) inhibited low dose, but not high dose, thrombin-induced aggregation. Additional experiments showed that PAR1-mediated calcium mobilization was partially blocked by Myr-G(13)SRI(pep) but not by the Rho kinase inhibitor Y-27632. Finally, Myr-G(13)SRI(pep) effectively inhibited PAR1-induced stress fiber formation and cell contraction in endothelial cells. Collectively, these results suggest the following: 1) interaction of Galpha(13) SRI with p115RhoGEF is required for G(13)-mediated RhoA activation in platelets; 2) signaling through the G(13) pathway is critical for PAR1-mediated human platelet functional changes and low dose thrombin-induced aggregation; and 3) G(13) signaling elicits calcium mobilization in human platelets through a Rho kinase-independent mechanism.
Journal of Biological Chemistry 05/2007; 282(14):10210-22. · 4.77 Impact Factor
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ABSTRACT: HEK293 cells were transfected with cDNAs for Gbeta1(W332A) [a mutant Gbeta1], Ggamma2, and inward rectifier K+ channels (Kir3.1/Kir3.2). Application of Gbeta1gamma2 protein to these cells activated the K+ channels only slightly. When mu-opioid receptors and Kir3.1/Kir3.2 were transfected, application of a mu-opioid agonist induced a Kir3 current. However, co-expression of Gbeta1(W332A) suppressed this current. Most likely, Gbeta1(W332A) inhibited the action of the endogenous Gbeta. Such a dominant negative effect of Gbeta1(W332A) was also observed in neuronal Kir3 channels in locus coeruleus. The mutant, Gbeta1(W332A) protein, although inactive, retains its ability to bind Kir3 and prevents the wild type Gbeta from activating the channel.
FEBS Letters 08/2006; 580(16):3879-82. · 3.54 Impact Factor
