Biological membranes are densely packed with membrane proteins that occupy approximately half of their volume. In almost all cases, membrane proteins in the native state lack the higher-order symmetry required for their direct study by diffraction methods. Despite many technical difficulties, numerous crystal structures of detergent solubilized membrane proteins have been determined that illustrate their internal organization. Among such proteins, class A G protein-coupled receptors have become amenable to crystallization and high resolution X-ray diffraction analyses. The derived structures of native and engineered receptors not only provide insights into their molecular arrangements but also furnish a framework for designing and testing potential models of transformation from inactive to active receptor signaling states and for initiating rational drug design.

It is now well established that any given ligand for a G-protein-couple receptor (GPCR) does not simply possess a single defined efficacy. Rather, a ligand possesses multiple efficacies, depending on the specific down-stream signal transduction pathway analyzed. This diversity may be based on ligand-specific GPCR conformations and is often referred to as "functional selectivity." It has been known for a century that stereoisomers of catecholamines differ in their potency and, in some systems, also in their efficacy. However, the molecular basis for efficacy differences of GPCR ligand stereoisomers has remained poorly defined. In an elegant study published in this issue of Molecular Pharmacology, Woo et al. (p. 158) show that stereoisomers of the β₂-adrenoceptor selective agonist fenoterol differentially activates G_s- and G_i-proteins in native rat cardiomyocytes. This study is so important because it is the first report to show that even the subtle structural differences within a ligand stereoisomer pair are sufficient to discriminate between GPCR conformations with distinct G-protein coupling properties. The study highlights of how important it is to examine the "more active" (eutomer) and the "less active" (distomer) stereoisomer to understand the mechanisms of action and the cellular effects of GPCR ligands. The study by Woo et al. will ignite a renaissance of the analysis of ligand stereoisomers, using sensitive pharmacological and biophysical assays. The available literature supports the notion that meticulous analysis of ligand stereoisomers is a goldmine for understanding mechanisms of GPCR activation, analysis of signal transduction pathways, development of new therapies for important diseases, and drug safety.

Dopamine D₂ and D₃ receptors are similar subtypes with distinct interactions with arrestins; the D₃ receptor mediates less agonist-induced translocation of arrestins than the D₂ receptor. The goals of this study were to compare nonphosphorylated arrestin-binding determinants in the second intracellular domain (IC2) of the D₂ and D₃ receptors to identify residues that contribute to the differential binding of arrestin to the subtypes. Arrestin3 bound to glutathione transferase (GST) fusion proteins of the D₂ receptor IC2 more avidly than to the D₃ receptor IC2. Mutagenesis of the fusion proteins identified a residue at the C terminus of IC2, Lys149, that was important for the preferential binding of arrestin3 to D₂-IC2; arrestin binding to D₂-IC2-K149C was greatly decreased compared with wild-type D₂-IC2, whereas binding to the reciprocal mutant D₃-IC2-C147K was enhanced compared with wild-type D₃-IC2. Mutating this lysine in the full-length D₂ receptor to cysteine decreased the ability of the D₂ receptor to mediate agonist-induced arrestin3 translocation to the membrane and decreased agonist-induced receptor internalization in human embryonic kidney 293 cells. The reciprocal mutation in the D₃ receptor increased receptor-mediated translocation of arrestin3 without affecting agonist-induced receptor internalization. G protein-coupled receptor crystal structures suggest that Lys149, at the junction of IC2 and the fourth membrane-spanning helix, has intramolecular interactions that contribute to maintaining an inactive receptor state. It is suggested that the preferential agonist-induced binding of arrestin3 to the D₂ receptor over the D₃ receptor is due in part to Lys149, which could be exposed as a result of receptor activation.

Hydrogen sulfide (H₂S) has been proposed as a novel neuromodulator, which plays critical roles in the central nervous system affecting both neurons and glial cells. However, its relationship with neurodegenerative diseases is unexplored. The present study was undertaken to investigate the effects of H₂S on cell injury induced by rotenone, a commonly used toxin in establishing in vivo and in vitro Parkinson's disease (PD) models, in human-derived dopaminergic neuroblastoma cell line (SH-SY5Y). We report here that sodium hydrosulfide (NaHS), an H₂S donor, concentration-dependently suppressed rotenone-induced cellular injury and apoptotic cell death. NaHS also prevented rotenone-induced p38- and c-Jun NH₂-terminal kinase (JNK)-mitogen-activated protein kinase (MAPK) phosphorylation and rotenone-mediated changes in Bcl-2/Bax levels, mitochondrial membrane potential (_m) dissipation, cytochrome c release, caspase-9/3 activation and poly(ADP-ribose) polymerase cleavage. Furthermore, 5-hydroxydecanoate, a selective blocker of mitochondrial ATP-sensitive potassium (mitoK_ATP) channel, attenuated the protective effects of NaHS against rotenone-induced cell apoptosis. Thus, we demonstrated for the first time that H₂S inhibited rotenone-induced cell apoptosis via regulation of mitoK_ATP channel/p38- and JNK-MAPK pathway. Our data suggest that H₂S may have potential therapeutic value for neurodegenerative diseases, such as PD.

The progressive debilitation of motor functions in Parkinson's disease (PD) results from degeneration of dopaminergic neurons within the substantia nigra pars compacta of the midbrain. Long-term inflammatory activation of microglia and astrocytes plays a central role in the progression of PD and is characterized by activation of the nuclear factor-B (NF-B) signaling cascade and subsequent overproduction of inflammatory cytokines and nitric oxide (NO). Suppression of this neuroinflammatory phenotype has received considerable attention as a potential target for chemotherapy, but there are no currently approved drugs that sufficiently address this problem. The data presented here demonstrate the efficacy of a novel anti-inflammatory diindolylmethane class compound, 1,1-bis(3'-indolyl)-1-(p-t-butylphenyl)methane (DIM-C-pPhtBu), in suppressing NF-B-dependent expression of inducible nitric-oxide synthase (NOS2) and NO production in astrocytes exposed to the parkinsonian neurotoxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) through a mechanism distinct from that described for the thiazolidinedione-class compound, rosiglitazone. Chromatin immunoprecipitations revealed that micromolar concentrations of DIM-C-pPhtBu prevented association of the p65 subunit of NF-B with enhancer elements in the Nos2 promoter but had little effect on DNA binding of either peroxisome proliferator-activated receptor- (PPAR-) or the nuclear corepressor NCoR2. Treatment with DIM-C-pPhtBu concomitantly suppressed NO production and protein nitration in MPTP-activated astrocytes and completely protected cocultured primary striatal neurons from astrocyte-dependent apoptosis. These data demonstrate the efficacy of DIM-C-pPhtBu in preventing the activation of NF-B-dependent inflammatory genes in primary astrocytes and suggest that this class of compounds may be effective neuroprotective anti-inflammatory agents in vivo.