Chemical inducers and transcriptional markers of oligodendrocyte differentiation.

Oligodendrocytes generate and maintain myelin, which is essential for axonal function and protection of the mammalian central nervous system. To advance our molecular understanding of differentiation by these cells, we screened libraries of pharmacologically active compounds and identified inducers of differentiation of Oli-neu, a stable cell line of mouse oligodendrocyte precursors (OPCs). We identified four broad classes of inducers, namely, forskolin/cAMP (protein kinase A activators), steroids (glucocorticoids and retinoic acid), ErbB2 inhibitors, and nucleoside analogs, and confirmed the activity of these compounds on rat primary oligodendrocyte precursors and mixed cortical cultures. We also analyzed transcriptional responses in the chemically induced mouse and rat OPC differentiation processes and compared these with earlier studies. We confirm the view that ErbB2 is a natural signaling component that is required for OPC proliferation, whereas ErbB2 inhibition or genetic knockdown results in OPC differentiation.

Convergent functional genomics of oligodendrocyte differentiation identifies multiple autoinhibitory signaling circuits.

Inadequate remyelination of brain white matter lesions has been associated with a failure of oligodendrocyte precursors to differentiate into mature, myelin-producing cells. In order to better understand which genes play a critical role in oligodendrocyte differentiation, we performed time-dependent, genome-wide gene expression studies of mouse Oli-neu cells as they differentiate into process-forming and myelin basic protein-producing cells, following treatment with three different agents. Our data indicate that different inducers activate distinct pathways that ultimately converge into the completely differentiated state, where regulated gene sets overlap maximally. In order to also gain insight into the functional role of genes that are regulated in this process, we silenced 88 of these genes using small interfering RNA and identified multiple repressors of spontaneous differentiation of Oli-neu, most of which were confirmed in rat primary oligodendrocyte precursors cells. Among these repressors were CNP, a well-known myelin constituent, and three phosphatases, each known to negatively control mitogen-activated protein kinase cascades. We show that a novel inhibitor for one of the identified genes, dual-specificity phosphatase DUSP10/MKP5, was also capable of inducing oligodendrocyte differentiation in primary oligodendrocyte precursors. Oligodendrocytic differentiation feedback loops may therefore yield pharmacological targets to treat disease related to dysfunctional myelin deposition.

Molecular characterization of a purified 5-HT4 receptor: a structural basis for drug efficacy.

Serotonin 5-HT(4(a)) receptor, a G-protein-coupled receptor (GPCR), was produced as a functional isolated protein using Escherichia coli as an expression system. The isolated receptor was characterized at the molecular level by circular dichroism (CD) and steady-state fluorescence. A specific change in the near-UV CD band associated with the GPCR disulfide bond connecting the third transmembrane domain to the second extracellular loop (e2) was observed upon agonist binding to the purified receptor. This is a direct experimental evidence for a change in the conformation of the e2 loop upon receptor activation. Different variations were obtained depending whether the ligand was an agonist (partial or full) or an inverse agonist. In contrast, antagonist binding did not induce any variation. These observations provide a first direct evidence for the fact that free (or antagonist-occupied), active (partial- or full agonist-occupied) and silent (inverse agonist-occupied) states of the receptor involve different arrangements of the e2 loop. Finally, ligand-induced changes in the fluorescence emission profile of the purified receptor confirmed that the partial agonist stabilized a single, well-defined, conformational state and not a mixture of different states. This result is of particular interest in a pharmacological perspective since it directly demonstrates that the efficacy of a drug is likely due to the stabilization of a ligand-specific state rather than selection of a mixture of different conformational states of the receptor.

New sorting nexin (SNX27) and NHERF specifically interact with the 5-HT4a receptor splice variant: roles in receptor targeting.

The 5-hydroxytryptamine type 4 receptor (5-HT4R) is involved in learning, feeding, respiratory control and gastrointestinal transit. This receptor is one of the G-protein-coupled receptors for which alternative mRNA splicing generates the most variants that differ in their C-terminal extremities. Some 5-HT4R variants (a, e and f) express canonical PDZ ligands at their C-termini. Here, we have examined whether some mouse 5-HT4R variants associate with specific sets of proteins, using a proteomic approach based on peptide-affinity chromatography, two-dimensional electrophoresis and mass spectrometry. We have identified ten proteins that interact specifically with the 5-HT4(a)R and three that only associate with the 5-HT4(e)R. Most of them are PDZ proteins. Among the proteins that associated specifically with the 5-HT4(a)R variant, NHERF greatly modified its subcellular localization. Moreover, NHERF recruited the 5-HT4(a)R to microvilli, where it localized with activated ezrin, consistent with the role of 5-HT4(a)R in cytoskeleton remodelling. The 5-HT4(a)R also interacted with both the constitutive and inducible (upon methamphetamine treatment) forms of the recently cloned sorting nexin 27 (SNX27a and b, respectively). We found that SNX27a redirected part of 5-HT4(a)R to early endosomes. The interaction of the 5-HT4R splice variants with distinct sets of PDZ proteins might specify their cellular localization as well as their signal transduction properties.

A single mutation in the 5-HT4 receptor (5-HT4-R D100(3.32)A) generates a Gs-coupled receptor activated exclusively by synthetic ligands (RASSL).

To better understand G-protein-coupled receptor (GPCRs) signaling, cellular and animal physiology, as well as gene therapy, a new tool has recently been proposed. It consists of GPCR mutants that are insensitive to endogenous ligands but sensitive to synthetic ligands. These GPCRs are called receptor activated solely by synthetic ligands (RASSL). Only two examples of such engineered receptors have been described so far: one G(i)-coupled (opioid receptors) and one G(s)-coupled (beta(2)-adrenergic receptors). Here, we describe the first RASSL related to serotonin receptors (D100(3.32)A G(s)-coupled 5-HT(4) receptor or 5-HT(4)-RASSL). 5-HT(4)-RASSL is generated by a single mutation, is totally insensitive to serotonin (5-HT), and still responds to synthetic ligands. These ligands have affinities in the range of nanomolar concentrations for the mutant receptor and exhibit full efficacy. More interestingly, two synthetic ligands behave as antagonists on the wild type but as agonists on the 5-HT(4)-RASSL.

A 5-HT4 receptor transmembrane network implicated in the activity of inverse agonists but not agonists.

Activation of G protein-coupled receptors is thought to involve disruption of intramolecular interactions that stabilize their inactive conformation. Such disruptions are induced by agonists or by constitutively active mutations. In the present study, novel potent inverse agonists are described to inhibit the constitutive activity of 5-HT(4) receptors. Using these compounds and specific receptor mutations, we investigated the mechanisms by which inverse agonists may reverse the disruption of intramolecular interactions that causes constitutive activation. Two mutations (D100(3.32)A in transmembrane domain (TMD)-III and F275(6.51)A in TMD-VI) were found to completely block inverse agonist effects without impairing their binding properties nor the molecular activation switches induced by agonists. Based on the rhodopsin model, we propose that these mutated receptors are in equilibrium between two states R and R* but are unable to reach a third "silent" state stabilized by inverse agonists. We also found another mutation in TMD-VI (W272(6.48)A) that stabilized this silent state. This mutant remained fully activated by agonists. Molecular modeling indicated that Asp-100, Phe-275, and Trp-272 might constitute a network required for stabilization of the silent state by the described inverse agonists. However, this network is not necessary for agonist activity.

The 5-hydroxytryptamine(4a) receptor is palmitoylated at two different sites, and acylation is critically involved in regulation of receptor constitutive activity.

We have reported recently that the mouse 5-hydroxytryptamine(4a) (5-HT(4(a))) receptor undergoes dynamic palmitoylation (Ponimaskin, E. G., Schmidt, M. F., Heine, M., Bickmeyer, U., and Richter, D. W. (2001) Biochem. J. 353, 627-663). In the present study, conserved cysteine residues 328/329 in the carboxyl terminus of the 5-HT(4(a)) receptor were identified as potential acylation sites. In contrast to other palmitoylated G-protein-coupled receptors, the additional cysteine residue 386 positioned close to the COOH-terminal end of the receptor was also found to be palmitoylated. Using pulse and pulse-chase labeling techniques, we demonstrated that palmitoylation of individual cysteines is a reversible process and that agonist stimulation of the 5-HT(4(a)) receptor independently increases the rate of palmitate turnover for both acylation sites. Analysis of acylation-deficient mutants revealed that non-palmitoylated 5-HT(4(a)) receptors were indistinguishable from the wild type in their ability to interact with G(s), to stimulate the adenylyl cyclase activity and to activate cyclic nucleotide-sensitive cation channels after agonist stimulation. The most distinctive finding of the present study was the ability of palmitoylation to modulate the agonist-independent constitutive 5-HT(4(a)) receptor activity. We demonstrated that mutation of the proximal palmitoylation site (Cys(328) --> Ser/Cys(329) --> Ser) significantly increases the capacity of receptors to convert from the inactive (R) to the active (R*) form in the absence of agonist. In contrast, the rate of isomerization from R to R* for the Cys(386) --> Ser as well as for the triple, non-palmitoylated mutant (Cys(328) --> Ser/Cys(329) --> Ser/Cys(386) -->Ser) was similar to that obtained for the wild type.