Targeted disruption of G protein-coupled bile acid receptor 1 (Gpbar1/M-Bar) in mice.

G protein-coupled bile acid receptor 1 (Gpbar1/M-Bar) is a novel G protein-coupled receptor for bile acid. Tissue distribution and cell-type specificity of Gpbar1 mRNA suggest a potential role for the receptor in the endocrine system; however, the precise physiological role of Gpbar1 still remains to be elucidated. To investigate the role of Gpbar1 in vivo, the Gpbar1 gene was disrupted in mice. In homozygous mice, total bile acid pool size was significantly decreased by 21-25% compared with that of the wild-type mice, suggesting that Gpbar1 contributes to bile acid homeostasis. In order to assess the impact of Gpbar1 deficiency in bile acid homeostasis more precisely, Gpbar1 homozygous mice were fed a high-fat diet for 2 months. As a result, female Gpbar1 homozygous mice showed significant fat accumulation with body weight gain compared with that of the wild-type mice. These findings were also observed in heterozygous mice to the same extent. Although the precise mechanism for fat accumulation in female Gpbar1 homozygous mice remains to be addressed, these data indicate that Gpbar1 is a potential new player in energy homeostasis. Thus, Gpbar1-deficient mice are useful in elucidating new physiological roles for Gpbar1.

Identification of membrane-type receptor for bile acids (M-BAR).

Bile acids play an essential role in the solubilization and absorption of dietary fat and lipid-soluble vitamins. Bile acids also modulate the transcription of various genes for enzymes and transport proteins for their own and cholesterol homeostasis through binding to nuclear receptors. Here we report a novel category of bile acid receptor, a membrane-type G protein-coupled receptor (GPCR), BG37. Bile acids induced rapid and dose-dependent elevation of intracellular cAMP levels in BG37-expressing cells, but not in mock-transfected cells, independently of nuclear receptor expression. The rank order of potency of various bile acids for BG37-expressing cells was different from that for the nuclear receptor-mediated response. These observations demonstrate the presence of two independent signaling pathways for bile acids; membrane-type GPCR for rapid signaling and nuclear receptors for delayed signaling. Expression of BG37 was detected in various specific tissues, suggesting its physiological role, although it remains to be further characterized.

A novel binding assay for metabotropic glutamate receptors using [3H] L-quisqualic acid and recombinant receptors.

We established a methodology to analyze radioligand binding to the recombinant type la metabotropic glutamate receptor (mGluRla). A full-length cDNA encoding mGluR1a, which was isolated from a lambda gt 11 cDNA library of human cerebellar origin, was expressed in a baculovirus/Sf9 insect cell system. Membrane fractions with recombinant receptor expression were analyzed for the binding of [3H]L-quisqualic acid (L-QA), which is known to be a potent agonist of mGluRla. Efficient binding of the radioligand to the human receptor was observed in a saturable manner, giving an apparent Kd= 0.091 microM. [3H]L-QA bound to the human mGluR1a was displaced by known ligands such as L-QA, L-Glu, t-ACPD ((+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid) with IC50s = 0.056, 0.97 and 4.0 microM, respectively. MCPG (alpha-methyl-4-carboxyphenylglycine) displaced the radioligand binding with lower potency. Using this binding protocol, we then evaluated the ligand ability of synthetic dipeptides. Among peptides tested, only Glu-containing dipeptides inhibited the radioligand binding, e.g. IC50 of L-Met-L-Glu was 4.3 microM. When phosphatidyl inositol turnover was assayed in mGluR1a-expressing CHO cells, L-Met-L-Glu was partially agonistic. We further expanded this [3H]L-QA binding protocol to type 5a mGluR, another member of group I mGluRs, as well as to AMPA receptor, a member of ionotropic glutamate receptors, since L-QA is also known to be a potent ligand for these receptors. Data shown here will provide a novel system not only to search for ligands for the glutamate receptors, but also to biochemically analyze the interaction modes between glutamate receptors and their ligands.