Hisato Jingami

Structural basis of glutamate recognition by a dimeric metabotropic glutamate receptor.

The metabotropic glutamate receptors (mGluRs) are key receptors in the modulation of excitatory synaptic transmission in the central nervous system. Here we have determined three different crystal structures of the extracellular ligand-binding region of mGluR1—in a complex with glutamate and in two unliganded forms. They all showed disulphide-linked homodimers, whose ‘active’ and ‘resting’ conformations are modulated through the dimeric interface by a packed α-helical structure. The bi-lobed protomer architectures flexibly change their domain arrangements to form an ‘open’ or ‘closed’ conformation. The structures imply that glutamate binding stabilizes both the ‘active’ dimer and the ‘closed’ protomer in dynamic equilibrium. Movements of the four domains in the dimer are likely to affect the separation of the transmembrane and intracellular regions, and thereby activate the receptor. This scheme in the initial receptor activation could be applied generally to G-protein-coupled neurotransmitter receptors that possess extracellular ligand-binding sites.

Human Obese Gene Expression: Adipocyte-Specific Expression and Regional Differences in the Adipose Tissue

The obese (ob) gene, the mutation of which results in severe hereditary obesity and diabetes in mice, has recently been isolated through positional cloning. In this study, we isolated a full-length human ob complementary DNA (cDNA) clone and examined the tissue distribution of ob gene expression in humans. The nucleotide sequences of the human ob cDNA coding region were 83% identical to those of the mouse and rat ob cDNA coding regions. Analysis of the deduced amino acid sequences revealed that the human ob protein is a 166–amino acid polypeptide with a putative signal sequence and is 84 and 83% homologous to the mouse and rat ob proteins, respectively. Northern blot analysis using the cloned human ob cDNA fragment as a probe identified a single messenger RNA (mRNA) species 4.5 kb in size found abundantly in the adipose tissues obtained from the subcutaneous, omental, retroperitoneal, perilymphatic, and mesenteric fat pads. However, no significant amount of ob mRNA was present in the brain, heart, lung, liver, stomach, pancreas, spleen, small intestine, kidney, prostate, testis, colon, or skeletal muscle. The ob mRNA level in the adipose tissue varied from region to region even in the same individual. Furthermore, in the human adipose tissue, ob gene expression occurred in mature adipocytes rather than in stromal-vascular cells. This study is the first report of the elucidation of ob gene expression in human tissues, thereby leading to better understanding of the physiological and clinical implications of the ob gene.

Structural views of the ligand-binding cores of a metabotropic glutamate receptor complexed with an antagonist and both glutamate and Gd3+

Crystal structures of the extracellular ligand-binding region of the metabotropic glutamate receptor, complexed with an antagonist, (S)-(α)-methyl-4-carboxyphenylglycine, and with both glutamate and Gd3+ ion, have been determined by x-ray crystallographic analyses. The structure of the complex with the antagonist is similar to that of the unliganded resting dimer. The antagonist wedges the protomer to maintain an inactive open form. The glutamate/Gd3+ complex is an exact 2-fold symmetric dimer, where each bi-lobed protomer adopts the closed conformation. The surface of the C-terminal domain contains an acidic patch, whose negative charges are alleviated by the metal cation to stabilize the active dimeric structure. The structural comparison between the active and resting dimers suggests that glutamate binding tends to induce domain closing and a small shift of a helix in the dimer interface. Furthermore, an interprotomer contact including the acidic patch inhibited dimer formation by the two open protomers in the active state. These findings provide a structural basis to describe the link between ligand binding and the dimer interface.

Structures of the extracellular regions of the group II/III metabotropic glutamate receptors

Metabotropic glutamate receptors play major roles in the activation of excitatory synapses in the central nerve system. We determined the crystal structure of the entire extracellular region of the group II receptor and that of the ligand-binding region of the group III receptor. A comparison among groups I, II, and III provides the structural basis that could account for the discrimination of group-specific agonists. Furthermore, the structure of group II includes the cysteine-rich domain, which is tightly linked to the ligand-binding domain by a disulfide bridge, suggesting a potential role in transmitting a ligand-induced conformational change into the downstream transmembrane region. The structure also reveals the lateral interaction between the two cysteine-rich domains, which could stimulate clustering of the dimeric receptors on the cell surface. We propose a general activation mechanism of the dimeric receptor coupled with both ligand-binding and interprotomer rearrangements.

Cloning and sequence analysis of cDNA for rat corticotropin-releasing factor precursor.

DNA complementary to the rat hypothalamic mRNA coding for the corticotropin‐releasing factor precursor (prepro‐CRF) has been cloned by screening a cDNA library with a human genomic DNA probe. Nucleotide sequence analysis of the cloned cDNA has revealed that rat prepro‐CRF consists of 187 amino acid residues including a putative signal peptide. The CRF and putative signal peptide regions are more highly conserved among rat, human and ovine prepro‐CRF than is the cryptic portion.

α,β-Unsaturated Ketone Is a Core Moiety of Natural Ligands for Covalent Binding to Peroxisome Proliferator-activated Receptor γ

Peroxisome proliferator-activated receptor γ (PPARγ) functions in various biological processes, including macrophage and adipocyte differentiation. Several natural lipid metabolites have been shown to activate PPARγ. Here, we report that some PPARγ ligands, including 15-deoxy-Δ12,14-prostaglandin J2, covalently bind to a cysteine residue in the PPARγ ligand binding pocket through a Michael addition reaction by an α,β-unsaturated ketone. Using rhodamine-maleimide as well as mass spectroscopy, we showed that the binding of these ligands is covalent and irreversible. Consistently, mutation at the cysteine residue abolished abilities of these ligands to activate PPARγ, but not of BRL49653, a non-covalent synthetic agonist, indicating that covalent binding of the α,β-unsaturated ketone in the natural ligands was required for their transcriptional activities. Screening of lipid metabolites containing the α,β-unsaturated ketone revealed that several other oxidized metabolites of hydroxyeicosatetraenoic acid, hydroxyeicosadecaenoic acid, and prostaglandins can also function as novel covalent ligands for PPARγ. We propose that PPARγ senses oxidation of fatty acids by recognizing such an α,β-unsaturated ketone as a common moiety.

Expression and Purification of the Extracellular Ligand Binding Region of Metabotropic Glutamate Receptor Subtype 1

Each metabotropic glutamate receptor possesses a large extracellular domain that consists of a sequence homologous to the bacterial periplasmic binding proteins and a cysteine-rich region. Previous experiments have proposed that the extracellular domain is responsible for ligand binding. However, it is currently unknown whether the extracellular ligand binding site can bind ligands without other domains of the receptor. We began by obtaining a sufficient amount of receptor protein on a baculovirus expression system. In addition to the transfer vector that encodes the entire coding region, transfer vectors that encode portions of the extracellular domain were designed. Here, we report a soluble metabotropic glutamate receptor that encodes only the extracellular domain and retains a ligand binding characteristic similar to that of the full-length receptor. The soluble receptor secreted into culture medium showed a dimerized form. Furthermore, we have succeeded in purifying it to homogeneity. Dose-response curves of agonists for the purified soluble receptor were examined. The effective concentration for half-maximal inhibition (IC50) of quisqualate for the soluble receptor was 3.8 × 10−8 m, which was comparable to that for the full-length receptor. The rank order of inhibition of the agonists was quisqualate ≫ ibotenate ≥l-glutamate ≈ (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid. These data demonstrate that a ligand binding event in metabotropic glutamate receptors can be dissociated from the membrane domain.

Structure of the metabotropic glutamate receptor.

In the twelve years since the molecular elucidation of the metabotropic glutamate receptor subtype 1, a class III family of G-protein-coupled receptors has emerged; members of this family include the calcium-sensing receptor, the GABA(B) receptor, some odorant receptors and some taste receptors. Atomic structures of the ligand-binding core of the original metabotropic glutamate receptor 1 obtained using X-ray crystallography provide a foundation for determining the initial receptor activation of this important family of G-protein-coupled receptors.

Activation of Orphan Nuclear Constitutive Androstane Receptor Requires Subnuclear Targeting by Peroxisome Proliferator-activated Receptor γ Coactivator-1α A POSSIBLE LINK BETWEEN XENOBIOTIC RESPONSE AND NUTRITIONAL STATE

In contrast to the classical nuclear receptors, the constitutive androstane receptor (CAR) is transcriptionally active in the absence of ligand. In the course of searching for the mediator of CAR activation, we found that ligand-independent activation of CAR was achieved in cooperation with the peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α). PGC-1β, a PGC-1α homologue, also activated CAR to less of an extent than PGC-1α. Coexpression of the ligand-binding domain of a heterodimerization partner, retinoid X receptor α, enhanced the PGC-1α-mediated activation of CAR, although it had a weak effect on the basal activity of CAR in the absence of PGC-1α. Both the N-terminal region, with the LXXLL motif, and the C-terminal region, with a serine/arginine-rich domain (RS domain), in PGC-1α were required for full activation of CAR. Pull-down experiments using recombinant proteins revealed that CAR directly interacted with both the LXXLL motif and the RS domain. Furthermore, we demonstrated that the RS domain of PGC-1α was required for CAR localization at nuclear speckles. These results indicate that PGC-1α mediates the ligand-independent activation of CAR by means of subnuclear targeting through the RS domain of PGC-1α.

Negative Cooperativity of Glutamate Binding in the Dimeric Metabotropic Glutamate Receptor Subtype 1

Metabotropic glutamate receptor (mGluR) subtype 1 is a Class III G-protein-coupled receptor that is mainly expressed on the post-synaptic membrane of neuronal cells. The receptor has a large N-terminal extracellular ligand binding domain that forms a homodimer, however, the intersubunit communication of ligand binding in the dimer remains unknown. Here, using the intrinsic tryptophan fluorescence change as a probe for ligand binding events, we examined whether allosteric properties exist in the dimeric ligand binding domain of the receptor. The indole ring of the tryptophan 110, which resides on the upper surface of the ligand binding pocket, sensed the ligand binding events. From saturation binding curves, we have determined the apparent dissociation constants (K0.5) of representative agonists and antagonists for this receptor (3.8, 0.46, 40, and 0.89 μm for glutamate, quisqualate, (S)-α-methyl-4-carboxyphenylglycine ((S)-MCPG), and (+)-2-methyl-4-carboxyphenylglycine (LY367385), respectively). Calcium ions functioned as a positive modulator for agonist but not for antagonist binding (K0.5 values were 1.3, 0.21, 59, and 1.2 μm for glutamate, quisqualate, (S)-MCPG, and LY367385, respectively, in the presence of 2.0 mm calcium ion). Moreover, a Hill analysis of the saturation binding curves revealed the strong negative cooperativity of glutamate binding between each subunit in the dimeric ligand binding domain. As far as we know, this is the first direct evidence that the dimeric ligand binding domain of mGluR exhibits intersubunit cooperativity of ligand binding.