Biomedical subjects

K Touhara

Publications and source records attributed to K Touhara.

29 records · Page 2Linked to original sources

PubMed1995

G beta gamma interactions with PH domains and Ras-MAPK signaling pathways.

G-protein-coupled receptor signaling and receptor tyrosine kinase (RTK) signaling are two mechanisms of transmembrane communication used by numerous extracellular agents and stimuli. The beta gamma-subunit complex of G proteins mediates many of the functions associated with G-protein-coupled receptor signaling and may even provide a means to link G proteins to RTK-initiated cascades. This connection may be mediated by the pleckstrin homology domain, a modular domain found in many signaling proteins that interact with G beta gamma.

Animals↗

PubMed1995

[PH domain: a new functional domain].

A pleckstrin homology (PH) domain is an approximately 100 amino acid region of sequence homology present in numerous proteins involved in signal transduction and growth control. The three dimensional structures of several PH domains demonstrate that they consist of a beta-barrel of seven antiparallel beta-sheets and a carboxyl-terminal amphiphilic alpha-helix. Several ligands capable of binding to PH domains have been identified including phosphatidylinositol 4,5-bisphosphate and the beta gamma subunits of heterotrimeric G proteins, which bind to the amino and carboxyl-termini of the PH domain, respectively. Furthermore, several isoforms of protein kinase C appear to bind to some PH domains. A general function of PH domains may be to anchor PH domain-containing proteins to the appropriate membrane-compartment. The membrane localization of PH domain-containing proteins may require cooperative multiple ligand binding to the PH domain. Finally, the heterogeneity of sequences among various PH domains may prove to be the basis for differences in the regulation and specificity of PH domain-ligand interaction in a fashion similar to SH2 and SH3 domains. The function of the PH domain and the mechanisms of PH domain action seem to be quite complex.

Animals↗

PubMed1994

Determination of the G beta gamma-binding domain of phosducin. A regulatable modulator of G beta gamma signaling.

Although a role for the beta gamma-subunits of heterotrimeric G proteins (G beta gamma) in signal transduction by several cellular systems has been established, the structural features of cellular proteins interacting with G beta gamma have yet to be fully elucidated. The G beta gamma-binding region of beta-adrenergic receptor kinase (beta ARK), a cytosolic enzyme recruited to the membrane receptor substrate by G beta gamma, has been localized to the carboxyl terminus of the enzyme. Here, we demonstrate that the amino terminus of phosducin, a 33-kDa G beta gamma-binding retinal phosphoprotein, contains sequences homologous with the G beta gamma-binding domain of beta ARK. Accordingly, a glutathione S-transferase-fusion protein containing only the amino-terminal 105 amino acids of phosducin displayed G beta gamma binding ability. This domain of phosducin contains a protein kinase A (PKA) phosphorylation site, and upon phosphorylation, the binding of full-length phosducin to G beta gamma is reduced. In addition, transient expression of phosducin in COS-7 cells significantly inhibits G beta gamma-mediated phosphoinositide hydrolysis. This inhibitory effect is completely reversed by pretreatment of cells with dibutyryl cAMP, an activator of PKA. Thus, the binding of G beta gamma to phosducin can be regulated by PKA-phosphorylation in an intact cell model system.

Amino Acid Sequence↗

PubMed1994

A nuclear juvenile hormone-binding protein from larvae of Manduca sexta: a putative receptor for the metamorphic action of juvenile hormone.

A 29-kDa nuclear juvenile hormone (JH)-binding protein from the epidermis of Manduca sexta larvae was purified by using the photoaffinity analog for JH II ([3H]epoxyhomofarnesyldiazoacetate) and partially sequenced. A 1.1-kb cDNA was isolated by using degenerate oligonucleotide primers for PCR based on these sequences. The cDNA encoded a 262-amino acid protein that showed no similarity with other known proteins, except for short stretches of the interphotoreceptor retinoid-binding protein, rhodopsin, and human nuclear protein p68. Recombinant baculovirus containing this cDNA made a 29-kDa protein that was covalently modified by [3H]epoxyhomofarnesyldiazoacetate and specifically bound the natural enantiomer of JH I (Kd = 10.7 nM). This binding was inhibited by the natural JHs but not by methoprene. Immunocytochemical analysis showed localization of this 29-kDa protein to epidermal nuclei. Both mRNA and protein are present during the intermolt periods; during the larval molt, the mRNA disappears but the protein persists. Later when cells become pupally committed, both the mRNA and protein disappear with a transient reappearance near pupal ecdysis. The properties of this protein are consistent with its being the receptor necessary for the antimetamorphic effects of JH.

Amino Acid Sequence↗

PubMed1994

Functionally active targeting domain of the beta-adrenergic receptor kinase: an inhibitor of G beta gamma-mediated stimulation of type II adenylyl cyclase.

The beta-adrenergic receptor kinase (beta ARK) phosphorylates its membrane-associated receptor substrates, such as the beta-adrenergic receptor, triggering events leading to receptor desensitization. beta ARK activity is markedly stimulated by the isoprenylated beta gamma subunit complex of heterotrimeric guanine nucleotide-binding proteins (G beta gamma), which translocates the kinase to the plasma membrane and thereby targets it to its receptor substrate. The amino-terminal two-thirds of beta ARK1 composes the receptor recognition and catalytic domains, while the carboxyl third contains the G beta gamma binding sequences, the targeting domain. We prepared this domain as a recombinant His6 fusion protein from Escherichia coli and found that it had both independent secondary structure and functional activity. We demonstrated the inhibitory properties of this domain against G beta gamma activation of type II adenylyl cyclase both in a reconstituted system utilizing Sf9 insect cell membranes and in a permeabilized 293 human embryonic kidney cell system. Gi alpha-mediated inhibition of adenylyl cyclase was not affected. These data suggest that this His6 fusion protein derived from the carboxyl terminus of beta ARK1 provides a specific probe for defining G beta gamma-mediated processes and for studying the structural features of a G beta gamma-binding domain.

Adenylate Cyclase Toxin↗

PubMed1994

Binding of G protein beta gamma-subunits to pleckstrin homology domains.

Ligand-induced activation of many receptors leads to dissociation of the alpha- and beta gamma-subunit complexes of heterotrimeric G proteins, both of which regulate a variety of effector molecules involved in cellular signaling processes. In one case, a cytosolic enzyme, the beta-adrenergic receptor kinase (beta ARK) binds to the dissociated, prenylated, membrane-anchored beta gamma-subunits of heterotrimeric G proteins (G beta gamma) and is thereby targeted to its membrane-bound receptor substrate. Quite recently, numerous proteins involved in cellular signal transduction have been shown to contain sequences homologous with a "domain" originally identified in the protein "pleckstrin" (pleckstrin homology domain; PH domain) and subsequently found in the G beta gamma interaction region of the beta ARK sequence. Here we demonstrate that glutathione S-transferase-fusion proteins, containing sequences encompassing the PH domain of nine proteins from this group, bind G beta gamma to varying extents. Binding of G beta gamma to these fusion proteins was documented either by a direct binding assay or by ability to block G beta gamma-mediated membrane translocation of beta ARK1. G beta gamma binding to these fusion proteins was inhibited by the alpha subunit of Go (Go alpha), indicating that the binding of G beta gamma to G alpha and the PH domain-containing fusion proteins is mutually exclusive. Studies with a series of truncated PH domains derived from the Ras-guanine-nucleotide-releasing factor indicate that the G beta gamma binding domain includes only the C-terminal portion of the PH domain and sequences just distal to this. Protein-protein interactions between G beta gamma and PH domain-containing proteins may play a significant role in cellular signaling analogous to that previously demonstrated for Src homology 2 and 3 domains.

Amino Acid Sequence↗

PubMed1994

Larva lights: a decade of photoaffinity labeling with juvenile hormone analogues.

The introduction of photoaffinity labeling into the mode of action of insect hormones and pheromones started 12 yr ago with the photoaffinity labeling of juvenile hormone binding proteins (JHBPs) from cockroaches in the laboratory of the late John K. Koeppe. Applying this technique to Manduca sexta led ultimately to a three-laboratory collaborative project that has begun to dissect the molecular basis for JH transport, metabolism, and nuclear binding and gene activation in Lepidoptera. This review provides (1) a history of the first experiments; (2) an idea of the breadth of the technique in the arthropod classes Insecta, Crustacea, and Arachnida; and (3) evidence for the depth of the technique in unearthing key details about three different types of the molecular action of JH in M. sexta.

Affinity Labels↗

PubMed1993

Juvenile hormone epoxide hydrolase. Photoaffinity labeling, purification, and characterization from tobacco hornworm eggs.

Juvenile hormone epoxide hydrolase (JHEH), which may play a pivotal role in regulating insect juvenile hormone (JH) titer along with JH esterase, was identified in tobacco hornworm (Manduca sexta) eggs by using photoaffinity analogs of JHs. The UV light-induced covalent labeling with [3H]epoxyhomofarnesyl diazoacetate, a JHII analog, revealed a membrane-associated 50-kDa protein that was selectively and specifically labeled. This 50-kDa protein was copurified 171-fold with the JHEH activity to homogeneity through DEAE-Sephacel, Mono Q, and hydroxylapatite columns, which led us to conclude that the labeled 50-kDa protein was a JHEH. The steady-state kinetics of the purified microsomal JHEH showed that it followed Michaelis-Menten kinetics with Km values of 0.61, 0.55, and 0.28 microM for JHI, II, and III, respectively, and that JHIII showed a significantly higher Vmax than JHI or JHII. JH acid was also converted to the corresponding diol at a rate 4-fold slower than the corresponding JH. Thus, the differences in the binding of substrate and the rate of turnover by JHEH were affected by the epoxyfarnesoate ester moiety of JH and the difference between the cis-11-methyl group of JHIII versus the cis-11-ethyl group of JHI and II. Purified JHEH showed optimal enzyme activity at pH 7.5-8.5. Interestingly, the presence of recombinant M. sexta JH binding protein (JHBP) dramatically decreased the degradation of JH by JHEH in vitro. Since the cytosolic JHBP in eggs closely resembles the hemolymph JHBP, we suggest that cytosolic JHBP may play a role in protecting JHs from JHEH in vivo. Furthermore, JHEH may play a significant role in the secondary metabolism of JH acid generated by JH esterase.

Affinity Labels↗

PubMed1993

Ligand binding by a recombinant insect juvenile hormone binding protein.

A cDNA for the hemolymph juvenile hormone binding protein (JHBP) of larval Manduca sexta has been isolated, sequenced, and expressed in an insect cell line. A recombinant baculovirus, containing the JHBP cDNA fused to the p10 promoter of Autographa californica nuclear polyhedrosis virus, was constructed. Insect cells (Sf9) infected with this virus secreted recombinant JHBP (rJHBP) into the medium (> 50 micrograms/mL), and cotranslational removal of an 18 amino acid leader sequence was observed. rJHBP was cross-reactive with an antiserum prepared to the hemolymph JHBP and was specifically labeled by [3H]EHDA, a photoaffinity analog of JH II, demonstrating that rJHBP was an isoform of the previously reported 32-kDa JHBP [Lerro, K. A., & Prestwich, G.D. (1990) J. Biol. Chem. 265, 19800-19806]. rJHBP was purified from insect cell medium to homogeneity by ion-exchange and gel-filtration chromatography. The purified rJHBP had a higher affinity (KD = 11 nM for JH I and KD = 42 nM for JH II) than that reported for crude hemolymph JHBP (KD = 80 nM for JH I). The circular dichroism (CD) spectrum of purified rJHBP indicated 34% alpha-helix and 23% beta-sheet. The CD spectra of rJHBP in the presence and absence of JH II were the same, indicating no change in secondary structure induced by ligand binding. Thus, the rJHBP expressed in insect cells binds JHs and is suitable for structural and functional analysis.

Amino Acid Sequence↗

PubMed1992

Biosynthesis and endocrine control of the production of the German cockroach sex pheromone 3,11-dimethylnonacosan-2-one.

The biosynthesis and endocrine regulation of sex pheromone production in the female German cockroach (Blattella germanica) were determined. Radio-TLC and radio-GLC were used to demonstrate the metabolism of 3,11-dimethylnonacosane, a major cuticular lipid component, to the corresponding alkan-2-ol and methyl ketone. [11,12-3H2]-3,11-Dimethylnonacosan-2-ol was efficiently metabolized to the methyl ketone, and radio-GLC showed that the methyl ketone product from both experiments was coeluted with a methyl ketone standard. A comparison of the metabolism of the labeled dimethylalkane and dimethylalkan-2-ol by age and sex showed that both males and females from day 1 through day 9 after adult emergence readily metabolized the alcohol to the corresponding methyl ketone, whereas only females of 5-9 days postemergence efficiently converted the labeled dimethylalkane to the corresponding methyl ketone. Application of the juvenile hormone analog hydroprene induced significant increases in the conversion of the labeled hydrocarbon to the methyl ketone in starved adult females as well as in females fed a protein-free diet, conditions under which endogenous juvenile hormone biosynthesis is nearly undetectable. These data show that the methyl ketone sex pheromone is formed by the hydroxylation and oxidation of the 3,11-dimethylalkane at the 2 position, show that the age- and sex-specific step in this process is the conversion of 3,11-dimethylnonacosane to 3,11-dimethylnonacosan-2-ol, and provide evidence that juvenile hormone regulates sex pheromone production in the German cockroach.

Animals↗

PubMed1992

Binding site mapping of a photoaffinity-labeled juvenile hormone binding protein.

The juvenile hormone binding protein (JHBP) of larval Manduca sexta was labeled by a photoaffinity analog of JH II and purified by preparative IEF and ion-exchange HPLC. The purified [3H]EHDA-labeled JHBP was selectively cleaved by CNBr and by endoproteinases Lys-C and Glu-C. The radioactive peptides were separated by tricine SDS-PAGE and sequenced after blotting to a PVDF membrane. The sequence revealed that Ala184-Asn226 contained a primary binding site of [3H]EHDA. Furthermore, peptide mapping indicated that Asp1-Glu34 also contained a second covalent attachment site of [3H]EHDA. Labeling of the N-terminal region increased when the photolysis was performed at lower temperature. Since Ala184-Asn226 is predicted to be a hydrophobic beta-sheet region, it may participate in the recognition of lipophilic backbone of JH. Five out of six cysteines are located in these two regions, consistent with a model in which the two binding regions connected by disulfide bridges provide a two-sided binding pocket for juvenile hormone.

Affinity Labels↗