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.

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...

CCR5 is the major coreceptor for macrophage-tropic human immunodeficiency virus type I (HIV-1). For most G-protein-coupled receptors that have been tested so far, the disulfide bonds linking together the extracellular loops (ECL) are required for maintaining the structural integrity necessary for ligand binding and receptor activation. A natural mutation affecting Cys 20 , which is thought to form a disulfide bond with Cys 269 , has been described in various human populations, although the consequences of this mutation for CCR5 function are not known. Using site-directed mutagenesis, we mutated the four extracellular cysteines of CCR5 singly or in combination to investigate their role in maintaining the structural conformation of the receptor, its ligand binding and signal transduction properties, and its ability to function as a viral coreceptor. Alanine substitution of any single Cys residue reduced surface expression levels by 40 -70%. However, mutation of Cys 101 or Cys 178 , predicted to link ECL1 and ECL2 of the receptor, abolished recognition of CCR5 by a panel of conformation sensitive anti-CCR5 antibodies. The effects of the mutations on receptor expression and conformation were partially temperature-sensitive, with partial restoration of receptor expression and conformation achieved by incubating cells at 32°C. All cysteine mutants were unable to bind detectable levels of MIP-1␤, and did not respond functionally to CCR5 agonists. Surprisingly, all cysteine mutants did support infection by R5 strains of HIV, though at reduced levels. These results indicate that both disulfide bonds of CCR5 are necessary for maintaining the structural integrity of the receptor necessary for ligand binding and signaling. Env binding and the mechanisms of HIV entry appear much less sensitive to alterations of CCR5 conformation.

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Insect ecdysis is a hormonally programmed physiological sequence that enables insects to escape their old cuticle at the end of each developmental stage. The immediate events leading to ecdysis, which are initiated upon release of ecdysis-triggering hormones (ETH) into the bloodstream, include respiratory inflation and sequential stereotypic behaviors that facilitate shedding of the cuticle. Here we report that the Drosophila gene CG5911 encodes two functionally distinct subtypes of G protein-coupled receptors through alternative splicing (CG5911a and CG5911b) that respond preferentially to ecdysis-triggering hormones of flies and moths. These subtypes show differences in ligand sensitivity and specificity, suggesting that they may play separate roles in ETH signaling. At significantly higher concentrations (>100-fold), certain insect and vertebrate peptides also activate these receptors, providing evidence that CG5911 is evolutionarily related to the thyrotropin-releasing hormone and neuromedin U receptors. The ETH signaling system in insects is a vital system that provides opportunities for the construction of models for the molecular basis of stereotypic animal behavior as well as a target for the design of more sophisticated insect-selective pest control strategies.A defining feature of growth and metamorphosis in insects is the periodic shedding of cuticle known as ecdysis (1). This complex process, by which the insect escapes its outer shell and sheds the cuticular lining of the foregut, hindgut, and respiratory system, is mediated by a peptide hormone-signaling cascade that results in a sequence of precisely timed physiological and behavioral events. Initiation of the ecdysis sequence coincides with the appearance of ecdysis-triggering hormones (ETHs) 1 in the bloodstream, which act directly on the central nervous system to elicit patterned motor output characteristic of pre-ecdysis and ecdysis behaviors (2, 3). In the moth Manduca sexta, two peptides called MasPETH and MasETH act in concert to trigger successive phases of the ecdysis behavioral sequence. Remarkably, the temporal features of these motor patterns recorded from the isolated nervous system closely reflect the behavior observed in whole animals. Related peptides in Drosophila melanogaster known as ETH1 and ETH2 trigger ecdysis, although their respective roles in the recruitment of different parts of the ecdysis sequence are less clear. As yet the molecular and cellular targets of ETHs have not been described. Additional signaling molecules operating downstream of ETH within the central nervous system include the eclosion hormone and the crustacean cardioactive peptide (2, 4 -7). Identification of ETH receptors would be of great help in defining the cellular elements involved in ecdysis behaviors. With this objective, we took advantage of the Drosophila genome project to investigate G protein-coupled receptors (GPCRs) that are likely to respond to ETH (8, 9). The search for ETH receptors was narrowed to a small number of candidates on the basi...

Even if the literature on the effects of pupil composition has been extensive, no clear consensus has been reached concerning the significance and magnitude of this effect. The first objective of this article is to estimate the magnitude of the school composition effect in primary schools (6th grade) in French-speaking Belgium. Different indicators of school composition are used: academic, socio-cultural, 'language' and sex composition. Except for sex composition, the results show that the school composition effect explains significant amount of between schools variance even after controlling for pupils' initial performance, socio-cultural background, and non-cognitive dispositions. The second objective is to examine covariance between school composition and several organisational variables and their joint effect on school performance. The second set of analyses is intended to question the conceptual nature of the school composition effect, establishing whether it is direct or indirect.

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AequoScreen, a cellular aequorin-based functional assay, has been optimized for luminescent high-throughput screening (HTS) of G protein-coupled receptor (GPCRs). AequoScreen is a homogeneous assay in which the cells are loaded with the apoaequorin cofactor coelenterazine, diluted in assay buffer, and injected into plates containing the samples to be tested. A flash of light is emitted following the calcium increase resulting from the activation of the GPCR by the sample. Here we have validated a new plate reader, the Hamamatsu Photonics FDSS6000, for HTS in 96- and 384-well plates with CHO-K1 cells stably coexpressing mitochondrial apoaequorin and different GPCRs (AequoScreen cell lines). The acquisition time, plate type, and cell number per well have been optimized to obtain concentration-response curves with 4000 cells/well in 384-well plates and a high signal:background ratio. The FDSS6000 and AequoScreen cell lines allow reading of twenty 96- or 384-well plates in 1 h with Z' values of 0.71 and 0.78, respectively. These results bring new insights to functional assays, and therefore reinforce the interest in aequorin-based assays in a HTS environment.