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.

No abstract