Biomedical subjects

Tohru Kozasa

Publications and source records attributed to Tohru Kozasa.

At least 19 recordsLinked to original sources

PubMed2006

Dominant negative effects of a Gbeta mutant on G-protein coupled inward rectifier K+ channel.

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.

Animals↗

PubMed2006

A new approach to producing functional G alpha subunits yields the activated and deactivated structures of G alpha(12/13) proteins.

The oncogenic G(12/13) subfamily of heterotrimeric G proteins transduces extracellular signals that regulate the actin cytoskeleton, cell cycle progression, and gene transcription. Previously, structural analyses of fully functional G alpha(12/13) subunits have been hindered by insufficient amounts of homogeneous, functional protein. Herein, we report that substitution of the N-terminal helix of G alpha(i1) for the corresponding region of G alpha12 or G alpha13 generated soluble chimeric subunits (G alpha(i/12) and G alpha(i/13)) that could be purified in sufficient amounts for crystallographic studies. Each chimera bound guanine nucleotides, G betagamma subunits, and effector proteins and exhibited GAP responses to p115RhoGEF and leukemia-associated RhoGEF. Like their wild-type counterparts, G alpha(i/13), but not G alpha(i/12), stimulated the activity of p115RhoGEF. Crystal structures of the G alpha(i/12) x GDP x AlF4(-) and G alpha(i/13) x GDP complexes were determined using diffraction data extending to 2.9 and 2.0 A, respectively. These structures reveal not only the native structural features of G alpha12 and G alpha13 subunits, which are expected to be important for their interactions with GPCRs and effectors such as G alpha-regulated RhoGEFs, but also novel conformational changes that are likely coupled to GTP hydrolysis in the G alpha(12/13) class of heterotrimeric G proteins.

Animals↗

PubMed2006

Activation state-dependent interaction between Galphai and p67phox.

The phagocyte NADPH oxidase consists of multiple protein subunits that interact with each other to form a functional superoxide-generating complex. Although the essential components for superoxide production have been well characterized, other proteins potentially involved in the regulation of NADPH oxidase activation remain to be identified. We report here that the Galphai subunit of heterotrimeric G proteins is a novel binding partner for p67phox in transfected HEK293T cells and peripheral blood polymorphonuclear leukocytes. p67phox preferably interacted with inactive Galphai. Expression of p67phox caused a dose-dependent decrease in intracellular cyclic AMP concentration, suggesting altered function of Galphai. We identified a fragment of p67phox, consisting of the PB1 domain and the C-terminal SH3 domain, to be critical for the interaction with Galphai. Because these domains are involved in the interaction with p47phox and p40phox, the relationship between the respective binding events was investigated. Wild-type Galphai, but not its QL mutant, could promote the interaction between p67phox and p47phox. However, the interaction between p67phox and p40phox was not affected by either Galphai form. These results provide the first evidence for an interaction between p67phox and an alpha subunit of heterotrimeric G proteins, suggesting a potential role for Galphai in the regulation or activation of NADPH oxidase.

Animals↗

PubMed2005

Snapshot of activated G proteins at the membrane: the Galphaq-GRK2-Gbetagamma complex.

G protein-coupled receptor kinase 2 (GRK2) plays a key role in the desensitization of G protein-coupled receptor signaling by phosphorylating activated heptahelical receptors and by sequestering heterotrimeric G proteins. We report the atomic structure of GRK2 in complex with Galphaq and Gbetagamma, in which the activated Galpha subunit of Gq is fully dissociated from Gbetagamma and dramatically reoriented from its position in the inactive Galphabetagamma heterotrimer. Galphaq forms an effector-like interaction with the GRK2 regulator of G protein signaling (RGS) homology domain that is distinct from and does not overlap with that used to bind RGS proteins such as RGS4.

Amino Acid Sequence↗

PubMed2005

G protein beta interacts with the glucocorticoid receptor and suppresses its transcriptional activity in the nucleus.

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 Gbeta interacts with the glucocorticoid receptor (GR), comigrates with it into the nucleus and suppresses GR-induced transactivation of the glucocorticoid-responsive genes. Association of Ggamma with Gbeta is necessary for this action of Gbeta. Both endogenous and enhanced green fluorescent protein (EGFP)-fused Gbeta2 and Ggamma2 proteins were detected in the nucleus at baseline, whereas a fraction of EGFP-Gbeta2 and DsRed2-GR comigrated to the nucleus or the plasma membrane, depending on the exposure of cells to dexamethasone or somatostatin, respectively. Gbeta2 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 Gbetagamma complex interacts with the GR and suppresses its transcriptional activity by associating with the transcriptional complex formed on GR-responsive promoters.

Active Transport, Cell Nucleus↗

PubMed2005

G alpha 12/13- and reactive oxygen species-dependent activation of c-Jun NH2-terminal kinase and p38 mitogen-activated protein kinase by angiotensin receptor stimulation in rat neonatal cardiomyocytes.

In the present study, we examined signal transduction mechanism of reactive oxygen species (ROS) production and the role of ROS in angiotensin II-induced activation of mitogen-activated protein kinases (MAPKs) in rat neonatal cardiomyocytes. Among three MAPKs, c-Jun NH(2)-terminal kinase (JNK) and p38 MAPK required ROS production for activation, as an NADPH oxidase inhibitor, diphenyleneiodonium, inhibited the activation. The angiotensin II-induced activation of JNK and p38 MAPK was also inhibited by the expression of the Galpha(12/13)-specific regulator of G protein signaling (RGS) domain, a specific inhibitor of Galpha(12/13), but not by an RGS domain specific for Galpha(q). Constitutively active Galpha(12)- or Galpha(13)-induced activation of JNK and p38 MAPK, but not extracellular signal-regulated kinase (ERK), was inhibited by diphenyleneiodonium. Angiotensin II receptor stimulation rapidly activated Galpha(13), which was completely inhibited by the Galpha(12/13)-specific RGS domain. Furthermore, the Galpha(12/13)-specific but not the Galpha(q)-specific RGS domain inhibited angiotensin II-induced ROS production. Dominant negative Rac inhibited angiotensin II-stimulated ROS production, JNK activation, and p38 MAPK activation but did not affect ERK activation. Rac activation was mediated by Rho and Rho kinase, because Rac activation was inhibited by C3 toxin and a Rho kinase inhibitor, Y27632. Furthermore, angiotensin II-induced Rho activation was inhibited by Galpha(12/13)-specific RGS domain but not dominant negative Rac. An inhibitor of epidermal growth factor receptor kinase AG1478 did not affect angiotensin II-induced JNK activation cascade. These results suggest that Galpha(12/13)-mediated ROS production through Rho and Rac is essential for JNK and p38 MAPK activation.

Angiotensin Receptor Antagonists↗

PubMed2005

Signal transduction pathway for the substance P-induced inhibition of rat Kir3 (GIRK) channel.

Certain transmitters inhibit Kir3 (GIRK) channels, resulting in neuronal excitation. We analysed signalling mechanisms for substance P (SP)-induced Kir3 inhibition in relation to the role of phosphatidylinositol 4,5-bisphosphate (PIP(2)). SP rapidly - with a half-time of approximately 10 s with intracellular GTPgammaS and approximately 14 s with intracellular GTP - inhibits a robustly activated Kir3.1/Kir3.2 current. A mutant Kir3 channel, Kir3.1(M223L)/Kir3.2(I234L), which has a stronger binding to PIP(2) than does the wild type Kir3.1/Kir3.2, is inhibited by SP as rapidly as the wild type Kir3.1/Kir3.2. This result contradicts the idea that Kir3 inhibition originates from the depletion of PIP(2). A Kir2.1 (IRK1) mutant, Kir2.1(R218Q), despite having a weaker binding to PIP(2) than wild type Kir3.1/Kir3.2, shows a SP-induced inhibition slower than the wild type Kir3.1/Kir3.2 channel, again conflicting with the PIP(2) theory of channel inhibition. Co-immunoprecipitation reveals that Galpha(q) binds with Kir3.2, but not with Kir2.2 or Kir2.1. These functional results and co-immunoprecipitation data suggest that G(q) activation rapidly inhibits Kir3 (but not Kir2), possibly by direct binding of Galpha(q) to the channel.

Animals↗

PubMed2004

Functional characterization of Galphao signaling through G protein-regulated inducer of neurite outgrowth 1.

G protein-regulated inducer of neurite outgrowth 1 (GRIN1) was initially identified as a binding protein for guanosine 5'-3-O-(thio)triphosphate-bound Galphaz. GRIN1 is specifically expressed in brain and interacts selectively with activated alpha subunits of the Gi subfamily. GRIN1 colocalizes with Galphao at the growth cone of neuronal cells and promotes neurite extension in Neuro2a cells when coexpressed with constitutively active mutant GalphaoQ205L. These results suggest that GRIN1 functions as a downstream target for Galphao. However, GRIN1 does not contain domains that are homologous to known signaling motifs. To understand the mechanisms of Galphao-GRIN1 pathway, we analyzed functional domains of GRIN1 that are involved in binding with Galphao or with its targeting to the plasma membrane. Using pull-down assays with glutathione S-transferase-fused GRIN1 deletion mutants, Galphao binding regions were localized to amino acid residues 716 to 746 and 797 to 827 of GRIN1. The Galphao binding region of GRIN1 did not demonstrate GTPase accelerating activity for Galphao. GRIN1 localized in the cell periphery in Neuro2a cells, and two cysteine residues at C-terminal region of GRIN1 (Cys818 and Cys819) were shown to be critical for its membrane targeting. Coexpression of GRIN1 with GalphaoQ205L or GRIN1Delta(717-827), which lacks Galphao binding region, promoted microspike formation in Swiss 3T3 cells or neurite extension in Neuro2a cells. The dominant-negative mutant of Cdc42 blocked these morphological changes. Coexpression of GRIN1 and GalphaoQ205L stimulated the formation of GTP-bound Cdc42 in Swiss 3T3 cells. These results suggest that the binding of activated Galphao to GRIN1 induces activation of Cdc42, which leads to morphological changes in neuronal cells.

Amino Acid Sequence↗

PubMed2004

Critical role of lysine 204 in switch I region of Galpha13 for regulation of p115RhoGEF and leukemia-associated RhoGEF.

Heterotrimeric G proteins of the G12 family regulate the Rho GTPase through RhoGEFs that contain an amino-terminal regulator of G protein signaling (RGS) domain (RGS-RhoGEFs). Direct regulation of the activity of RGS-RhoGEFs p115 or leukemia-associated RhoGEF (LARG) by Galpha13 has previously been demonstrated. However, the precise biochemical mechanism by which Galpha13 stimulates the RhoGEF activity of these proteins has not yet been well understood. Based on the crystal structure of Galphai1 in complex with RGS4, we mutated the Galpha13 residue lysine 204 to alanine (Galpha13K204A) and characterized the effect of this mutation in its regulation of RGS-RhoGEFs p115 or LARG. Compared with wild-type Galpha13, Galpha13K204A induced much less serum-response factor activation when expressed in HeLa cells. Recombinant Galpha13K204A exhibits normal function in terms of nucleotide binding, basal GTP hydrolysis, and formation of heterotrimer with betagamma. We found that lysine 204 of Galpha13 is important for interaction with the RGS domain of p115 or LARG and for the GTPase-activating protein activity of these proteins. In addition, the K204A mutation of Galpha13 impaired its regulation of the RhoGEF activity of p115 or LARG. We conclude that lysine 204 of Galpha13 is important for interaction with RGS-RhoGEFs and is critically involved in the regulation of their activity.

Alanine↗

PubMed2004

Thrombin and lysophosphatidic acid receptors utilize distinct rhoGEFs in prostate cancer cells.

Thrombin and lysophosphatidic acid (LPA) receptors play important roles in vascular biology, development, and cancer. These receptors activate rho via G(12/13) family heterotrimeric G proteins, which are known to directly activate three distinct rho guanine nucleotide exchange factors (rhoGEFs) that contain a regulator of G protein signaling (RGS) domain (RGS-rhoGEFs). However, it is not known which, if any, of these RGS-rhoGEFs (LARG (leukemia-associated rhoGEF), p115rhoGEF, or PDZrhoGEF) plays a role in G protein-coupled receptor-stimulated rho signaling. Using oligonucleotide small interfering RNAs that suppress specific RGS-rhoGEF expression, we show that thrombin receptor stimulation of rho is primarily mediated by LARG in HEK293T and PC-3 prostate cancer cell lines. In contrast, the LPA-stimulated rho response in PC-3 cells is dependent on PDZrhoGEF expression. Suppression of p115rhoGEF had no effect. Thus different rhoGEFs (LARG and PDZrhoGEF) mediate downstream rho signaling by the thrombin and LPA receptors.

Cell Line, Tumor↗

PubMed2004

The structure of GRK2-G beta gamma complex: intimate association of G-protein signaling modules.

G-protein-mediated signaling is the most widely used signaling mechanism in cells and its regulation is crucial for various physiological functions. G-protein-coupled receptor (GPCR) kinases (GRKs) are involved in the desensitization of GPCR signals. Recently, the X-ray crystal structure of GRK2 complexed with G beta gamma was demonstrated and revealed the intimate association of three important signaling modules with G beta gamma to regulate GRK2 activity.

Cyclic AMP-Dependent Protein Kinases↗

PubMed2004

Ribozyme- and siRNA-mediated suppression of RGS-containing RhoGEF proteins.

Given recent efforts to determine the sequence information on thousands of genes in the human genome, the current challenge is to identify the functions of these genes, including those encoding the regulator of G-protein signaling protein gene superfamily, and to establish their roles in particular signaling pathways in a native system. Increasingly, reverse genetic approaches are being used to address these questions. This article compares two powerful approaches [ribozyme and "short interfering" RNA (siRNA) techniques] under identical conditions for the first report on the suppression of endogenous RGS domain-containing RhoGEFs. The siRNA technique was found to be much more potent than ribozyme targeting at the same mRNA site of RGS-RhoGEFs. Also, the three siRNAs targeting LARG, PDZ-RhoGEF, and p115-RhoGEF are able to discriminate the closely related sequences within this RGS-RhoGEF gene family.

Amino Acid Sequence↗

PubMed2004

Regulation of RGS-RhoGEFs by Galpha12 and Galpha13 proteins.

Three mammalian Rho guanine nucleotide exchange factors (RhoGEFs), leukemia-associated RhoGEF (LARG), p115RhoGEF, and PDZ-RhoGEF, contain regulator of G-protein signaling (RGS) domains within their amino-terminal regions. These RhoGEFs link signals from heterotrimeric G12/13 protein-coupled receptors to Rho GTPase activation, leading to various cellular responses, such as actin reorganization and gene expression. The activity of these RhoGEFs is regulated by Galpha12/13 through their RGS domains. Because RhoGEFs stimulate guanine nucleotide exchange by Rho GTPases, RhoGEF activation can be measured by monitoring GTP binding to or GDP dissociation from Rho GTPases. This article describes methods used to perform reconstitution assays to measure the activity of RhoGEFs regulated by Galpha12/13.

Animals↗

PubMed2004

Identification and biochemical analysis of GRIN1 and GRIN2.

We have identified the novel Galphaz-binding protein, which is referred to as the G-protein-regulated inducer of neurite outgrowth (GRIN1) using the far-western method. GRIN1 is expressed specifically in brain and binds preferentially to the activated form of alpha subunits of Gz, Gi, and Go. Coexpression of GRIN1 and the activated form of Galphao induce neurite outgrowth in Neuro2a cells. We have further identified two human GRIN1 homologs, GRIN2 and GRIN3, in the database. This article shows that GRIN2 can also bind to the GTP-bound form of Galphao. These findings suggest that the GRIN1 family may function as a downstream effector for Galphao to regulate neurite growth.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Selective inhibition of G protein-mediated pathways using RGS domains.

Regulators of G protein signaling (RGS) proteins are recently identified regulators of G protein-mediated pathways. These proteins recognize specific members of G protein alpha subunits and, in most cases, act as GTPase activating proteins (GAPs). RGS proteins interact with activated forms of G alpha subunits through RGS domain of about 120 amino acids. Thus, the overexpression of the RGS domain in cells can specifically block the signalling pathways that are mediated by the interacting G proteins. The method is particularly useful to differentiate between Gq-mediated and G12/13-mediated pathways.

Cloning, Molecular↗

PubMed2004

Purification of G protein subunits from Sf9 insect cells using hexahistidine-tagged alpha and beta gamma subunits.

G protein-mediated pathways are the most fundamental mechanisms of cell signaling. In order to analyze these pathways, the availability of purified recombinant G proteins are critically important. Using Sf9-Baculovirus expression system, a general and simplified method to purify various G protein subunits is described in this chapter. This method is useful for purification of most of G protein subunits.

Animals↗

PubMed2003

Identification and molecular characterization of the G alpha12-Rho guanine nucleotide exchange factor pathway in Caenorhabditis elegans.

G alpha 12/13-mediated pathways have been shown to be involved in various fundamental cellular functions in mammalian cells such as axonal guidance, apoptosis, and chemotaxis. Here, we identified a homologue of Rho-guanine nucleotide exchange factor (GEF) in Caenorhabditis elegans (CeRhoGEF), which functions downstream of gpa-12, the C. elegans homologue of G alpha 12/13. CeRhoGEF contains a PSD-95/Dlg/ZO-1 domain and a regulator of G protein signaling (RGS) domain upstream of the Dbl homology-pleckstrin homology region similar to mammalian RhoGEFs with RGS domains, PSD-95/Dlg/ZO-1-RhoGEF and leukemia-associated RhoGEF. It has been shown in mammalian cells that these RhoGEFs interact with activated forms of G alpha 12 or G alpha 13 through their RGS domains. We demonstrated by coimmunoprecipitation that the RGS domain of CeRhoGEF interacts with GPA-12 in an AIF4- activation-dependent manner and confirmed that the Dbl homology-pleckstrin homology domain of CeRhoGEF was capable of Rho-dependent signaling. These results proved conservation of the G alpha 12-RhoGEF pathway in C. elegans. Expression of DsRed or GFP under the control of the promoter of CeRhoGEF or gpa-12 revealed an overlap of their expression patterns in ventral cord motor neurons and several neurons in the head. RNA-mediated gene interference for CeRhoGEF and gpa-12 resulted in similar phenotypes such as embryonic lethality and sensory and locomotive defects in adults. Thus, the G alpha 12/13-RhoGEF pathway is likely to be involved in embryonic development and neuronal function in C. elegans.

Allergy and Immunology↗

PubMed2003

RGS16 inhibits signalling through the G alpha 13-Rho axis.

G alpha 13 stimulates the guanine nucleotide exchange factors (GEFs) for Rho, such as p115Rho-GEF. Activated Rho induces numerous cellular responses, including actin polymerization, serum response element (SRE)-dependent gene transcription and transformation. p115Rho-GEF contains a Regulator of G protein Signalling domain (RGS box) that confers GTPase activating protein (GAP) activity towards G alpha 12 and G alpha 13 (ref. 3). In contrast, classical RGS proteins (such as RGS16 and RGS4) exhibit RGS domain-dependent GAP activity on G alpha i and G alpha q, but not G alpha 12 or G alpha 13 (ref 4). Here, we show that RGS16 inhibits G alpha 13-mediated, RhoA-dependent reversal of stellation and SRE activation. The RGS16 amino terminus binds G alpha 13 directly, resulting in translocation of G alpha 13 to detergent-resistant membranes (DRMs) and reduced p115Rho-GEF binding. RGS4 does not bind G alpha 13 or attenuate G alpha 13-dependent responses, and neither RGS16 nor RGS4 affects G alpha 12-mediated signalling. These results elucidate a new mechanism whereby a classical RGS protein regulates G alpha 13-mediated signal transduction independently of the RGS box.

Cell Line, Tumor↗