Electron tomography reveals diverse conformations of integrin alphaIIbbeta3 in the active state.

We used electron tomography to determine the three-dimensional (3D) structure of integrin alphaIIbbeta3 in the active state. We found that we obtained better density maps when we reconstructed a 3D volume for each individual particle in the tilt series rather than to extract the particle-containing subvolumes from a 3D reconstruction of the entire specimen area. The 3D tomographic reconstructions of 100 particles revealed that activated alphaIIbbeta3 adopts many different conformations. An average of all the individual 3D reconstructions nicely accommodated the crystal structure of the alphaVbeta3 headpiece, confirming the locations assigned to the alpha- and beta-subunit in the density map. The most striking finding of our study is the structural flexibility of the lower leg of the beta-subunit as opposed to the conformational stability of the leg of the alpha-subunit. The good fit of the atomic structure of the betaI domain and the hybrid domain in the active state showed that the hybrid domain swings out, and most particles used for tomography are in the active state. Multivariate statistical analysis and classification applied to the set of 3D reconstructions revealed that more than 90% reconstructions are grouped into the classes that show the active state. Our results demonstrate that electron tomography can be used to classify complexes with a flexible structure such as integrins.

Production of recombinant human relaxin 3 in AtT20 cells.

Relaxin 3 has been reported recently as a member of the insulin/IGF/relaxin family. To clarify the function of relaxin 3, we prepared recombinant human relaxin 3 using a mouse adrenocorticotrophic hormone (ACTH)-secreting cell line, AtT20. To detect a mature form of recombinant human relaxin 3, a competitive enzyme immunoassay (EIA) was developed using a monoclonal antibody (mAb; HK4-144-10), which was raised for the N-terminal peptide of human relaxin 3 A-chain. We detected immunoreactive (ir-) relaxin 3 in the culture supernatant of AtT20 cells stably transfected with human relaxin 3 cDNA. After treatment with 5 microM forskolin for 3 days, the concentration of the ir-relaxin 3 in the culture supernatant reached 12 nM. Ir-relaxin 3 was purified from the culture supernatant by a combination of various chromatographies. By analyses of N-terminal amino acid sequence and electrospray ionization mass spectrometry (ESI-MS), we confirmed that the purified material was a mature form of human relaxin 3. The recombinant human relaxin 3 thereby obtained increased intracellular cAMP production in THP-1 cells. Our results demonstrate that the expression of relaxin 3 cDNA in AtT20 cells is a useful tool to produce a bioactive and mature form of relaxin 3.

The Formin family protein, formin homolog overexpressed in spleen, interacts with the insulin-responsive aminopeptidase and profilin IIa.

Insulin stimulates translocation of glucose transporter isoform type 4 (GLUT4) and the insulin-responsive aminopeptidase (IRAP) from an intracellular storage pool to the plasma membrane in muscle and fat cells. A role for the cytoskeleton in insulin action has been postulated, and the insulin signaling pathway has been well investigated; however, the molecular mechanism by which GLUT4/IRAP-containing vesicles move from an interior location to the cell surface in response to insulin is incompletely understood. Here, we have screened for IRAP-binding proteins using a yeast two-hybrid system and have found that the C-terminal domain of FHOS (formin homolog overexpressed in spleen) interacts with the N-terminal cytoplasmic domain of IRAP. FHOS is a member of the Formin/Diaphanous family of proteins that is expressed most abundantly in skeletal muscle. In addition, there are two novel types of FHOS transcripts generated by alternative mRNA splicing. FHOS78 has a 78-bp insertion and it is expressed mainly in skeletal muscle where it may be the most abundant isoform in humans. The ubiquitously expressed FHOS24 has a 24-bp insertion encoding an in-frame stop codon that results in a truncated polypeptide. It is known that some formin family proteins interact with the actin-binding profilin proteins. Both FHOS and FHOS78 bound to profilin IIa via their formin homology 1 domains, but neither bound profilin I or IIb. Overexpression of FHOS and FHOS78 resulted in enhanced insulin-stimulated glucose uptake in L6 cells to similar levels. However, overexpression of FHOS24, lacking the IRAP-binding domain, did not affect insulin-stimulated glucose uptake. These findings suggest that FHOS mediates an interaction between GLUT4/IRAP-containing vesicles and the cytoskeleton and may participate in exocytosis and/or retention of this membrane compartment.

Free fatty acids regulate insulin secretion from pancreatic beta cells through GPR40.

Diabetes, a disease in which carbohydrate and lipid metabolism are regulated improperly by insulin, is a serious worldwide health issue. Insulin is secreted from pancreatic beta cells in response to elevated plasma glucose, with various factors modifying its secretion. Free fatty acids (FFAs) provide an important energy source as nutrients, and they also act as signalling molecules in various cellular processes, including insulin secretion. Although FFAs are thought to promote insulin secretion in an acute phase, this mechanism is not clearly understood. Here we show that a G-protein-coupled receptor, GPR40, which is abundantly expressed in the pancreas, functions as a receptor for long-chain FFAs. Furthermore, we show that long-chain FFAs amplify glucose-stimulated insulin secretion from pancreatic beta cells by activating GPR40. Our results indicate that GPR40 agonists and/or antagonists show potential for the development of new anti-diabetic drugs.

A G protein-coupled receptor responsive to bile acids.

So far some nuclear receptors for bile acids have been identified. However, no cell surface receptor for bile acids has yet been reported. We found that a novel G protein-coupled receptor, TGR5, is responsive to bile acids as a cell-surface receptor. Bile acids specifically induced receptor internalization, the activation of extracellular signal-regulated kinase mitogen-activated protein kinase, the increase of guanosine 5'-O-3-thio-triphosphate binding in membrane fractions, and intracellular cAMP production in Chinese hamster ovary cells expressing TGR5. Our quantitative analyses for TGR5 mRNA showed that it was abundantly expressed in monocytes/macrophages in human and rabbit. Treatment with bile acids was found to suppress the functions of rabbit alveolar macrophages including phagocytosis and lipopolysaccharide-stimulated cytokine productions. We prepared a monocytic cell line expressing TGR5 by transfecting a TGR5 cDNA into THP-1 cells that did not express TGR5 originally. Treatment with bile acids suppressed the cytokine productions in the THP-1 cells expressing TGR5, whereas it did not influence those in the original THP-1 cells, suggesting that TGR5 is implicated in the suppression of macrophage functions by bile acids.

Effect of herpes simplex virus-1 gD or gD-IL-2 DNA vaccine on herpetic keratitis.

PURPOSE: To evaluate the efficacy of DNA immunization using herpes simplex virus (HSV)-1 gD or gD-IL-2 (chimeric gene of gD and human IL-2) with reference to their immune responses and preventive effect on the development of murine herpetic stromal keratitis, based on our previous work. METHODS: Plasmids containing gD (pHSDneol) or gD-IL-2 (pHDLneol) were constructed, and gD or gD-IL-2 peptides were purified. BALB/c mice were immunized by hypodermal injection, subconjunctival injection, or topical eye drop twice at 7-day intervals. Attained anti-HSV immune reaction was estimated, including neutralizing antibody, delayed-type hypersensitivity (DTH) measured by the swelling of the ear pinna, and cytotoxic T lymphocyte (CTL) measured by 51Cr release from MHC-matched target cells by splenic and/or local lymph node cells. The corneas of a group of the immunized mice were challenged with CHR3 strain of HSV-1 (10 microL of 3 x 10(6) PFU/mL) 3 weeks after the last immunization. Clinical signs (stromal and epithelial keratitis) were scored. Results. All gD- or gD-lL-2-immunized mice developed specific neutralizing antibody. Delayed-type hypersensitivity and CTL were obtained in gD DNA- or gD-IL-2 DNA-immunized mice. Chimeric DNA vaccine gD-IL-2 elicited higher DTH and more vigorous CTL activity. The scores of stromal keratitis for all gD- or gD-IL-2-immunized mice were significantly suppressed, although those of epithelial keratitis were not. Conclusion. Immunization with gD or gD-IL-2 was effective against herpetic stromal keratitis. By focusing specifically on high induction of cell-mediated immunity, gD-IL-2 DNA could have possible clinical applications against HSV-1 in the future.

The effect of immunization with herpes simplex virus glycoprotein D fused with interleukin-2 against murine herpetic keratitis.

PURPOSE: To evaluate the preventive effect of vaccination using fusion protein (gD-IL-2) consisting of herpes simplex virus type 1 (HSV-1) glycoprotein D (gD), and human interleukin-2 (IL-2), and plasmid DNA encoding gD-IL-2 against murine herpetic keratitis. METHODS: Plasmid containing gD-IL-2 (pHDLneo1) was constructed, and gD-IL-2 peptide was purified. BALB/c mice were injected hypodermally or subconjunctivally twice with 1 microg/0.1 mL of gD-IL-2 peptide, or subconjunctivally twice with 90 microg/0.05 mL of gD-IL-2 plasmid DNA. Neutralizing antibody titer and delayed-type hypersensitivity (DTH) against HSV-1 were measured. Immunized mice were challenged with CHR3 strain of HSV-1 into the cornea. Clinical manifestations of the epithelial and stromal keratitis were scored. RESULTS: Stromal keratitis was inhibited in gD-IL-2 peptide- or DNA-immunized mice; however, epithelial keratitis was not. It was confirmed that plasmid gD-IL-2 elicited significant virus neutralizing titer in sera and DTH response. CONCLUSION: Vaccination with gD-IL-2 was effective against murine herpetic keratitis.

Identification of a neuropeptide modified with bromine as an endogenous ligand for GPR7.

We isolated a novel gene in a search of the Celera data base and found that it encoded a peptidic ligand for a G protein-coupled receptor, GPR7 (O'Dowd, B. F., Scheideler, M. A., Nguyen, T., Cheng, R., Rasmussen, J. S., Marchese, A., Zastawny, R., Heng, H. H., Tsui, L. C., Shi, X., Asa, S., Puy, L., and George, S. R. (1995) Genomics 28, 84-91; Lee, D. K., Nguyen, T., Porter, C. A., Cheng, R., George, S. R., and O'Dowd, B. F. (1999) Mol. Brain Res. 71, 96-103). The expression of this gene was detected in various tissues in rats, including the lymphoid organs, central nervous system, mammary glands, and uterus. GPR7 mRNA was mainly detected in the central nervous system and uterus. In situ hybridization showed that the gene encoding the GPR7 ligand was expressed in the hypothalamus and hippocampus of rats. To determine the molecular structure of the endogenous GPR7 ligand, we purified it from bovine hypothalamic tissue extracts on the basis of cAMP production-inhibitory activity to cells expressing GPR7. Through structural analyses, we found that the purified endogenous ligand was a peptide with 29 amino acid residues and that it was uniquely modified with bromine. We subsequently determined that the C-6 position of the indole moiety in the N-terminal Trp was brominated. We believe this is the first report on a neuropeptide modified with bromine and have hence named it neuropeptide B. In in vitro assays, bromination did not influence the binding of neuropeptide B to the receptor.

Increased expression of the human Ca2+-activated Cl- channel 1 (CaCC1) gene in the asthmatic airway.

Mucus overproduction is a clinical feature of asthma. Ca2+-activated Cl- channel 1 (CaCC1) has been identified as a protein that is expressed in intestinal epithelia and that plays an important role in fluid and electrolyte transport. Recently, its mouse counterpart, gob-5, was identified as a key molecule in the induction of murine asthma through mucus overproduction. To elucidate the relationship of CaCC1 to human asthma, we examined CaCC1 expression using real-time quantitative polymerase chain reaction analysis in bronchial tissues from patients with asthma and normal control subjects. The expression of CaCC1 was significantly upregulated in patients with bronchial asthma compared with control subjects. In situ hybridization and immunohistochemical analysis demonstrated that CaCC1 is located in the bronchial epithelium, especially in mucus-producing goblet cells. In vitro transfection of a CaCC1 expression vector into the human mucoepidermoid cell line, NCI-H292, increased mucus production and induced the MUC5AC gene. These results suggest that CaCC1 plays a direct role in mucus production and differentiation in goblet cells and may contribute to the pathogenesis of asthma through its mucus-inducing activity.

Topical administration of HSV gD-IL-2 DNA is highly protective against murine herpetic stromal keratitis.

PURPOSE: To evaluate the immunopreventive effect of eyedrops that contain gD-IL-2 DNA (a chimeric gene of the glycoprotein D gene of herpes simplex virus type I (HSV-1) and human interleukin-2 (IL-2) on murine herpetic keratitis. METHODS: A plasmid containing gD-IL-2 (pHDLneo1) was constructed. The eyedrops containing 90 microg/10 microL of the DNA was instilled bilaterally into the conjunctival sacs of BALB/c mice on days 0 and 7. Three weeks after the last administration, neutralizing antibody, delayed-type hypersensitivity (DTH), and 51Cr-release from infected targeted cells by lymphocytes from the cervical lymph nodes and spleen were determined. Immunized mice were challenged with HSV-1, after which the clinical signs of the corneal epithelia and stroma were scored. RESULTS: Specific neutralizing antibody was raised and prominent DTH reaction was elicited from immunized mice. Lymphocytes obtained from the local lymph nodes and spleen vigorously potentiated the cytotoxic activity against the virus-infected cells. Clinically, the development of stromal keratitis was completely inhibited, but prevention or reduction of HSV-1 epithelial lesions was not demonstrated statistically. CONCLUSION: Topical immunization with a DNA vaccine encoding gD-IL-2 totally prevented the development of herpetic stromal keratitis. This procedure is a simple and convenient method for possible clinical application in the future.

[Drug discovery based on the research on orphan receptor/ligand].

The present world-wide leading products are mostly receptor antagonists and agonists and enzyme inhibitors when classified by the mechanism of action. We consider there will be less possibilities of taking the initiative in successful finding of a target for an innovative drug winning a worldwide contest only by taking up known receptors and enzymes. Therefore, we have used the results of current genetic researches to utilize orphan G-protein-coupled receptors (GPERs) whose ligands are not known yet as targets for drug discovery, and have conducted various examinations about how to implement it. The results obtained here can be combined with the conventional methods for drug discovery. Hence, this technology is considered to be the main stream of future drug discovery research.