Short chain fatty acids (SCFAs), including acetate, propionate, and butyrate, are produced at high concentration by bacteria in the gut and subsequently released in the bloodstream. Basal acetate concentrations in the blood (about 100 M) can further increase to millimolar concentrations following alcohol intake. It was known previously that SCFAs can activate leukocytes, particularly neutrophils. In the present work, we have identified two previously orphan G protein-coupled receptors, GPR41 and GPR43, as receptors for SCFAs. Propionate was the most potent agonist for both GPR41 and GPR43. Acetate was more selective for GPR43, whereas butyrate and isobutyrate were more active on GPR41. The two receptors were coupled to inositol 1,4,5-trisphosphate formation, intracellular Ca 2؉ release, ERK1/2 activation, and inhibition of cAMP accumulation. They exhibited, however, a differential coupling to G proteins; GPR41 coupled exclusively though the Pertussis toxinsensitive G i/o family, whereas GPR43 displayed a dual coupling through G i/o and Pertussis toxin-insensitive G q protein families. The broad expression profile of GPR41 in a number of tissues does not allow us to infer clear hypotheses regarding its biological functions. In contrast, the highly selective expression of GPR43 in leukocytes, particularly polymorphonuclear cells, suggests a role in the recruitment of these cell populations toward sites of bacterial infection. The pharmacology of GPR43 matches indeed the effects of SCFAs on neutrophils, in terms of intracellular Ca 2؉ release and chemotaxis. Such a neutrophil-specific SCFA receptor is potentially involved in the development of a variety of diseases characterized by either excessive or inefficient neutrophil recruitment and activation, such as inflammatory bowel diseases or alcoholism-associated immune depression. GPR43 might therefore constitute a target allowing us to modulate immune responses in these pathological situations.

We have cloned and expressed a novel human Gprotein-coupled receptor closely related to the human P2Y 12 receptor. It corresponds to the orphan receptor called GPR86. GPR86 proved to be a G i -coupled receptor displaying a high affinity for ADP, similar to the P2Y 12 receptor and can therefore be tentatively called P2Y 13 . In 1321N1 cells, the P2Y 13 receptor coupled to the phosphoinositide pathway only when coexpressed with G␣ 16 . Inositol trisphosphate formation was stimulated equipotently by nanomolar concentrations of ADP and 2MeSADP, whereas 2MeSATP and ATP were inactive. In CHO-K1 cells expressing the P2Y 13 receptor, ADP and 2MeSADP had a biphasic effect on the forskolin-stimulated accumulation of cAMP: inhibition at nanomolar concentrations and potentiation at micromolar levels. In the same cells, ADP and 2MeSADP also stimulated the phosphorylation of Erk1 and Erk2, in a pertussis toxinsensitive way. The tissue distribution of P2Y 13 was investigated by reverse transcriptase-polymerase chain reaction, and the predominant signals were obtained in spleen and brain. Although these can be discriminated by tissue distribution and some pharmacological features, the P2Y 12 and P2Y 13 receptors form a subgroup of related P2Y subtypes that is structurally different from the other P2Y subtypes but share coupling to G i and a high affinity for ADP.Adenine and uridine nucleotides induce pharmacological and physiological responses through several G-protein-coupled receptors (P2Y) and ligand-gated cation channels (P2X) (1, 2). The P2Y family encompasses two selective purinoceptors: the human P2Y 1 and P2Y 11 receptors, which are preferentially activated respectively by ADP and ATP (3-5). Nucleotide receptors responsive to both adenine and uracil nucleotides are the P2Y 2 receptor, activated equipotently by ATP and UTP (6, 7) as well as the Xenopus P2Y 8 (8) and turkey tp2y receptor (9), activated equally by all triphosphate nucleotides. There are also pyrimidinoceptors: the chicken P2Y 3 (10) and human P2Y 6 (11-13) receptors activated preferentially by UDP, and the human P2Y 4 receptor (13-15) activated preferentially by UTP. All these P2Y subtypes are coupled to the phosphoinositide pathway. The P2Y 11 and tp2y receptors are additionally coupled respectively to stimulation and inhibition of adenylyl cyclase. Other receptors (P2Y 5 , Ref. 16; P2Y 7 , Ref. 17; P2Y 9 , and P2Y 10 ) have been mistakenly included in the P2Y family (18 -20). Recently, a P2Y 12 subtype has been cloned, which corresponds in fact to the platelet ADP receptor previously called P 2T (21,22). It is coupled to an inhibition of adenylyl cyclase and is specifically expressed in the platelets and the brain. Its primary structure is not related to the other P2Y receptors but rather to that of the UDP-glucose receptor (23). Here we report the cloning and characterization of a novel G-protein-coupled receptor that is structurally related to the P2Y 12 receptor and was recently described as an orphan receptor called GPR86 (24).

GPR7 and GPR8 are two structurally related orphan G protein-coupled receptors, presenting high similarities with opioid and somatostatin receptors. Two peptides, L8 and L8C, derived from a larger precursor, were recently described as natural ligands for GPR8 (Mori, M., Shimomura, Y., Harada, M., Kurihara, M., Kitada, C., Asami, T., Matsumoto, Y., Adachi, Y., Watanabe, T., Sugo, T., and Abe, M. (December, 27, 2001) World Patent Cooperation Treaty, Patent Application WO 01/98494A1). L8 is a 23-amino acid peptide, whereas L8C is the same peptide with a C terminus extension of 7 amino acids, running through a dibasic motif of proteolytic processing. Using as a query the amino acid sequence of the L8 peptide, we have identified in DNA databases a human gene predicted to encode related peptides and its mouse ortholog. By analogy with L8 and L8C, two peptides, named L7 and L7C could result from the processing of a 125-amino acid human precursor through the alternative usage of a dibasic amino acid motif. The activity of these four peptides was investigated on GPR7 and GPR8. In binding assays, L7, L7C, L8, and L8C were found to bind with low nanomolar affinities to the GPR7 and GPR8 receptors expressed in Chinese hamster ovary (CHO)-K1 cells. They inhibited forskolin-stimulated cAMP accumulation through a pertussis toxin-sensitive mechanism. The tissue distribution of prepro-L7 (ppL7) and prepro-L8 (ppL8) was investigated by reverse transcription-PCR. Abundant ppL7 transcripts were found throughout the brain as well as in spinal cord, spleen, testis, and placenta; ppL8 transcripts displayed a more restricted distribution in brain, with high levels in substantia nigra, but were more abundant in peripheral tissues. The ppL7 and ppL8 genes therefore encode the precursors of a class of peptide ligands, active on two receptor subtypes, GPR7 and GPR8. The distinct tissue distribution of the receptor and peptide precursors suggest that each ligand and receptor has partially overlapping but also specific roles in this signaling system. G protein-coupled receptors (GPCRs) 1 constitute one of the largest gene families yet identified (2). Over the last decade, a growing number of GPCRs have been made available by various cloning procedures, among which PCR amplification using degenerate oligonucleotides, and more recently the systematic sequencing of cDNA libraries and genomes, have played prominent roles. In addition to about 160 characterized receptors, about 125 human genes encode proteins obviously belonging to this family of receptors, but their ligands and functions remain to be determined. These so far uncharacterized receptors are referred to as orphan GPCRs, but they are expected to play, by analogy with characterized members of the family, important roles in the regulation of physiological processes. From a structural viewpoint, orphan receptors are widely distributed throughout the GPCR superfamily, suggesting that they respond to a diverse range of ligands. Their similarity with well known receptors sometimes allows th...

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Hepatocyte nuclear factor-6 (HNF-6) contains a single cut domain and a homeodomain characterized by a phenylalanine at position 48 and a methionine at position 50. We describe here two isoforms of HNF-6 which differ by the linker that separates these domains. Both isoforms stimulated transcription. The affinity of HNF-6␣ and HNF-6␤ for DNA differed, depending on the target sequence. Binding of HNF-6 to DNA involved the cut domain and the homeodomain, but the latter was not required for binding to a subset of sites. Mutations of the F48M50 dyad that did not affect DNA binding reduced the transcriptional stimulation of constructs that do not require the homeodomain for DNA binding, but did not affect the stimulation of constructs that do require the homeodomain. Comparative trees of mammalian, Drosophila, and Caenorhabditis elegans proteins showed that HNF-6 defines a new class, which we call ONECUT, of homeodomain proteins. C. elegans proteins of this class bound to HNF-6 DNA targets. Thus, depending on their sequence, these targets determine for HNF-6 at least two modes of DNA binding, which hinge on the homeodomain and on the linker that separates it from the cut domain, and two modes of transcriptional stimulation, which hinge on the homeodomain.The phenotype of multicellular organisms is determined in part by the cell type-specific expression of genes. Since the initial observation that expression of liver-specific genes is controlled at the level of transcription (1), several liver-enriched transcription factors have been identified and extensively studied. These factors contain DNA-binding domains that have been conserved throughout evolution. Based on the structure of their functional domains, the liver-enriched transcription factors were classified into five families (2, 3). These include the CCAAT/enhancer binding proteins and the proline acid-rich factors, which both contain a leucine zipper, the homeodomain proteins of the hepatocyte nuclear factor (HNF)