Blood pH is maintained in a narrow range around pH 7.4 mainly through regulation of respiration and renal acid extrusion. The molecular mechanisms involved in pH homeostasis are not completely understood. Here we show that ovarian cancer G-protein-coupled receptor 1 (OGR1), previously described as a receptor for sphingosylphosphorylcholine, acts as a proton-sensing receptor stimulating inositol phosphate formation. The receptor is inactive at pH 7.8, and fully activated at pH 6.8-site-directed mutagenesis shows that histidines at the extracellular surface are involved in pH sensing. We find that GPR4, a close relative of OGR1, also responds to pH changes, but elicits cyclic AMP formation. It is known that the skeleton participates in pH homeostasis as a buffering organ, and that osteoblasts respond to pH changes in the physiological range, but the pH-sensing mechanism operating in these cells was hitherto not known. We detect expression of OGR1 in osteosarcoma cells and primary human osteoblast precursors, and show that these cells exhibit strong pH-dependent inositol phosphate formation. Immunohistochemistry on rat tissue sections confirms the presence of OGR1 in osteoblasts and osteocytes. We propose that OGR1 and GPR4 are proton-sensing receptors involved in pH homeostasis.

Epstein-Barr virus (EBV)-induced gene 2 (EBI2, aka GPR183) is a G protein-coupled receptor that is required for humoral immune responses and polymorphisms in the receptor have been associated with inflammatory autoimmune diseases1-3. The natural ligand for EBI2 has been unknown. Here we describe identification of 7α, 25-dihydroxycholesterol (5-cholesten-3β, 7α, 25-triol; 7α, 25-OHC) as a potent and selective agonist of EBI2. Functional activation of EBI2 by 7α, 25-OHC and closely related oxysterols was verified by monitoring second messenger readouts and saturable, high affinity radioligand binding. Furthermore we find that 7α, 25-OHC and closely related oxysterols act as chemoattractants for immune cells expressing EBI2 by directing cell migration in vitro and in vivo. A key enzyme required for the generation of 7α, 25-OHC is cholesterol 25-hydroxylase (Ch25h)4. Similar to EBI2 receptor knockout mice, mice deficient in Ch25h fail to position activated B cells within the spleen to the outer follicle and mount a reduced plasma cell response after an immune challenge. This demonstrates that Ch25h generates EBI2 bioactivity in vivo and suggests that the EBI2 − oxysterol signaling pathway plays an important role in the adaptive immune response.

Blood gas and tissue pH regulation depend on the ability of the brain to sense CO2 and/or H+ and alter breathing appropriately, a homeostatic process called central respiratory chemosensitivity. We show that selective expression of the proton-activated receptor GPR4 in chemosensory neurons of the mouse retrotrapezoid nucleus (RTN) is required for CO2-stimulated breathing. Genetic deletion of GPR4 disrupted acidosis-dependent activation of RTN neurons, increased apnea frequency and blunted ventilatory responses to CO2. Reintroduction of GPR4 into RTN neurons restored CO2-dependent RTN neuronal activation and rescued the ventilatory phenotype. Additional elimination of TASK-2, a pH-sensitive K+ channel expressed in RTN neurons, essentially abolished the ventilatory response to CO2. The data identify GPR4 and TASK-2 as distinct, parallel and essential central mediators of respiratory chemosensitivity.

Targeting sphingosine-1-phosphate receptors with the oral immunomodulator drug FTY720 (fingolimod) has demonstrated substantial efficacy in the treatment of multiple sclerosis. The drug is phosphorylated in vivo, and most of the clinical effects of FTY720-phosphate (FTY720P) are thought to be mediated via S1P1 receptors on lymphocytes and endothelial cells, leading to sequestration of lymphocytes in secondary lymphoid organs. FTY720P was described to act as a "functional antagonist" by promoting efficient internalization of S1P1 receptors. We demonstrate here that S1P1 receptors activated by FTY720P retain signaling activity for hours in spite of a quantitative internalization. Structural analogs of FTY720P with shorter alkyl side chains retained potency and efficacy in a functional assay but failed to promote long-lasting receptor internalization and signaling. We show that persistent signaling translates into an increased chemokinetic migration of primary human umbilical vein endothelial cells, which suggests persistent agonism as a crucial parameter in the mechanism of action of FTY720.

Chemical biology approaches have a long history in the exploration of the G-protein-coupled receptor (GPCR) family, which represents the largest and most important group of targets for therapeutics. The analysis of the human genome revealed a significant number of new members with unknown physiological function which are today the focus of many reverse pharmacology drug-discovery programs. As the seven hydrophobic transmembrane segments are a defining common structural feature of these receptors, and as signaling through heterotrimeric G proteins is not demonstrated in all cases, these proteins are also referred to as seven transmembrane (7 TM) or serpentine receptors. This review summarizes important historic milestones of GPCR research, from the beginning, when pharmacology was mainly descriptive, to the age of modern molecular biology, with the cloning of the first receptor and now the availability of the entire human GPCR repertoire at the sequence and protein level. It shows how GPCR-directed drug discovery was initially based on the careful testing of a few specifically made chemical compounds and is today pursued with modern drug-discovery approaches, including combinatorial library design, structural biology, molecular informatics, and advanced screening technologies for the identification of new compounds that activate or inhibit GPCRs specifically. Such compounds, in conjunction with other new technologies, allow us to study the role of receptors in physiology and medicine, and will hopefully result in novel therapies. We also outline how basic research on the signaling and regulatory mechanisms of GPCRs is advancing, leading to the discovery of new GPCR-interacting proteins and thus opening new perspectives for drug development. Practical examples from GPCR expression studies, HTS (high-throughput screening), and the design of monoamine-related GPCR-focused combinatorial libraries illustrate ongoing GPCR chemical biology research. Finally, we outline future progress that may relate today's discoveries to the development of new medicines.

A homology model for the human calcium sensing receptor (hCaR) transmembrane domain utilizing bovine rhodopsin (bRho) structural information was derived and tested by docking the allosteric antagonist, NPS 2143, followed by mutagenesis of predicted contact sites. Mutation of residues Phe-668 (helix II), Arg-680, or Phe-684 (helix III) to Ala (or Val or Leu) and Glu-837 (helix VII) to Ile (or Gln) reduced the inhibitory effects of NPS 2143 on [Ca 2؉ ] i responses. The calcimimetic NPS R-568 increases the potency of Ca 2؉ in functional assays of CaR. Mutations at Phe-668, Phe-684, or Glu-837 attenuated the effects of this compound, but mutations at Arg-680 had no effect. In all cases, mutant CaRs responded normally to Ca 2؉ or phenylalanine, which act at distinct site(s). Discrimination by the Arg-680 mutant is consistent with the structural differences between NPS 2143, which contains an alkyl bridge hydroxyl group, and NPS R-568, which does not. The homology model of the CaR transmembrane domain robustly accounts for binding of both an allosteric antagonist and agonist, which share a common site, and provides a basis for the development of more specific and/or potent allosteric modulators of CaR. These studies suggest that the bRho backbone can be used as a starting point for homology modeling of even distantly related G protein-coupled receptors and provide a rational framework for investigation of the contributions of the transmembrane domain to CaR function.

We have examined the phosphorylation and protein kinase activity of p44 mitogen-activated protein kinase (p44mapk) in growth factor-stimulated hamster fibroblasts using a specific antiserum. The activity of p44mapk was stimulated both by receptor tyrosine kinases and G protein-coupled receptors. Detailed kinetics revealed that alpha-thrombin induces a biphasic activation of p44mapk in CCL39 cells: a rapid phase appearing at 5-10 min was followed by a late and sustained phase still elevated after 4 h. Inactivation of alpha-thrombin with hirudin after 30 sec, which prevented DNA synthesis, did not alter the early p44mapk response but completely abolished the late phase. Pretreatment of the cells with pertussis toxin, which inhibits by more than 95% alpha-thrombin-induced mitogenicity, resulted in the complete loss of late phase activity, while the early peak was partially attenuated. Treatment of CCL39 cells with basic fibroblast growth factor also induced a strong activation of p44mapk. Serotonin, which is not a mitogen by its own, had no effect on late phase p44mapk activity, but synergized with basic fibroblast growth factor to induce late kinase response and DNA synthesis. Both early and late phase activation of p44mapk were accompanied by tyrosine phosphorylation of the enzyme. Together, the results indicate that there is a very close correlation between the ability of a growth factor to induce late and sustained p44mapk activation and its mitogenic potential. Therefore, we propose that sustained p44mapk activation is an obligatory event for growth factor-induced cell cycle progression.

Thiazolidinediones are insulin-sensitizing agents and in clinical use for the treatment of type II diabetes. Under specific experimental conditions, these molecules induce adipogenic differentiation of mesenchymal precursor cells at the expense of osteoblasts in vitro, suggesting possible negative effects on the skeleton. We measured effects of the thiazolidinedione BRL49653 on bone tissue of intact and estrogen-deprived skeletally mature adult female Wistar rats (6-9 months old). Weight gain and decreased plasma triglyceride levels confirmed the effectiveness of the treatment. However, no change in bone mass or fat marrow volume was observed in intact rats treated for 8 weeks with 5, 10, or 20 mg/kg of BRL49653. Study of marrow cultures established at necropsy revealed a higher responsiveness to adipogenic differentiation protocols of cultures established from the 10-mg/kg group compared to vehicle control. In a second study, the effects of thiazolidinedione treatment on the skeleton of estrogen-deprived rats were investigated. Application of 10 mg/kg of BRL49653 for 12 weeks resulted in enhanced bone loss (+31%; pQCT) and increased fat marrow volume (+117%; histomorphometry) compared to vehicle-treated OVX control. Interestingly, osteoblast number was comparable in both cases. Bone resorption parameters were significantly increased in the treatment group (+27% osteoclast number, +30% eroded surface). Enhanced bone loss due to treatment was consistently observed in the tibia, femur, and the lumbar spine. Our data indicate that thiazolidinediones may enhance bone loss induced by estrogen deprivation.