Receptors for excitatory amino-acid transmitters on nerve cells fall into two main categories associated with non-selective cationic channels, the NMDA (N-methyl-D-aspartate) and non-NMDA (kainate and quisqualate) receptors. Special properties of NMDA receptors such as their voltage-dependent blockade by Mg2+ (refs 3, 4) and their permeability to Na+, K+ as well as to Ca2+ (refs 5, 6), have led to the suggestion that these receptors are important in plasticity during development and learning. They have been implicated in long-term potentiation (LTP), a model for the study of the cellular mechanisms of learning. We report here that glutamate and NMDA, acting at typical NMDA receptors, stimulate the release of arachidonic acid (as well as 11- and 12-hydroxyeicosatetraenoic acids from striatal neurons probably by stimulation of a Ca2+-dependent phospholipase A2. Kainate and quisqualate, as well as K+-induced depolarization were ineffective. Our results provide direct evidence in favour of the hypothesis, that arachidonic acid derivatives, produced by activation of the postsynaptic cell, could be messengers that cross the synaptic cleft to modify the presynaptic functions known to be altered during LTP. In addition, we suggest that NMDA receptors are the postsynaptic receptors which trigger the synthesis of these putative transynaptic messengers.

The neurotransmitter serotonin (5-HT) plays an important role in the regulation of multiple events in the CNS. We demonstrated recently a coupling between the 5-HT 4 receptor and the heterotrimeric G13-protein resulting in RhoA-dependent neurite retraction and cell rounding (Ponimaskin et al., 2002). In the present study, we identified G12 as an additional G-protein that can be activated by another member of serotonin receptors, the 5-HT 7 receptor. Expression of 5-HT 7 receptor induced constitutive and agonist-dependent activation of a serum response element-mediated gene transcription through G12-mediated activation of small GTPases. In NIH3T3 cells, activation of the 5-HT 7 receptor induced filopodia formation via a Cdc42-mediated pathway correlating with RhoA-dependent cell rounding. In mouse hippocampal neurons, activation of the endogenous 5-HT 7 receptors significantly increased neurite length, whereas stimulation of 5-HT 4 receptors led to a decrease in the length and number of neurites. These data demonstrate distinct roles for 5-HT 7 R/G12 and 5-HT 4 R/G13 signaling pathways in neurite outgrowth and retraction, suggesting that serotonin plays a prominent role in regulating the neuronal cytoarchitecture in addition to its classical role as neurotransmitter.

We are creating families of designer G-protein-coupled receptors (GPCRs) to allow for precise spatiotemporal control of GPCR signaling in vivo. These engineered GPCRs, called receptors activated solely by synthetic ligands (RASSLs), are unresponsive to endogenous ligands but can be activated by nanomolar concentrations of pharmacologically inert, drug-like small molecules. Currently, RASSLs exist for the three major GPCR signaling pathways (Gs, Gi, Gq). These new advances are reviewed here to help facilitate the use of these powerful and diverse tools.

Serotonin (5-hydroxytryptamine, 5-HT) is, without doubt, the neurotransmitter for which the number of receptors is the highest. Fifteen genes encoding functional 5-HT receptors have been cloned in mammalian brain. 5-HT(3) receptors are ionotropic receptors, whereas all the others are metabotropic G-protein-coupled receptors (GPCRs). 5-HT receptor diversity is further increased by post-genomic modifications, such as alternative splicing (up to 10 splice variants for the 5-HT(4) receptor) or by mRNA editing in the case of 5-HT(2C) receptors. The cellular and behavioral implications of 5-HT(2C) receptor editing are of great physiological importance. Signaling of 5-HT receptors involves a great variety of pathways, but only some of these have been demonstrated in neurons. The classical view of neurotransmitter receptors localized within the synaptic cleft cannot be applied to 5-HT receptors, which are mostly (but not exclusively) localized at extra-synaptic locations either pre- or post-synaptically. 5-HT receptors are engaged in pre- or post-synaptic complexes composed of many GPCR-interacting proteins. The functions of these proteins are starting to be revealed. These proteins have been implicated in targeting, trafficking to or from the membrane, desensitization, and fine-tuning of signaling.

Pituitary adenylate cyclase-activating polypeptide (PACAP)-27 and PACAP-38 are neuropeptides of the vasoactive intestinal peptide/secretin/glucagon family. We previously described alternative splicing of the region encoding the third intracellular loop of the PACAP receptor generating six isoforms with differential signal transduction properties (Spengler, D., Waeber, C., Pantaloni, C., Holsboer, F., Bockaert, J., Seeburg, P. H., and Journot, L. (1993) Nature 365, 170 -175). In addition, we demonstrated that the potencies of the two forms of PACAP are similar for adenylate cyclase stimulation, whereas PACAP-38 is more potent than PACAP-27 in phospholipase C activation. In the present work, we document the existence of a new splice variant of the PACAP receptor that was characterized by a 21-aminoacid deletion in the N-terminal extracellular domain. We demonstrate that this domain modulates receptor selectivity with respect to PACAP-27 and -38 binding and controls the relative potencies of the two agonists in phospholipase C stimulation.Pituitary adenylate cyclase-activating polypeptides (PACAP) 1 are recently purified neuropeptides (1, 2) that are named according to their amino acid number, PACAP-27 and PACAP-38. The 27-amino acid form corresponds to the 27 N-terminal amino acids of PACAP-38 and shares 68% identity with vasoactive intestinal peptide (VIP). Two classes of PACAP binding sites were pharmacologically defined. Type I PACAP receptors bind PACAP-27 and -38 about two orders of magnitude more efficiently than VIP, whereas type II PACAP receptors do not discriminate between PACAP-27, -38, and VIP. At present, three PACAP/VIP receptor genes have been identified: PACAP 1 -R corresponds to PACAP type I receptors whereas PACAP/VIP 1 -R and PACAP/VIP 2 -R correspond to type II receptors. No VIP-specific receptor has so far been cloned.PACAP 1 -R is abundantly expressed in the brain, the pituitary and pineal glands, and the adrenal medulla. It mediates the neurotrophic action of PACAP-38 on PC12 cells (3) and sympathetic neuroblasts (4, 5). In the pituitary gland, PACAP-38 modulates the release of several hormones (1) and of interleukin-6 (6) through activation of PACAP 1 -R but also of PACAP/VIP 1 -R and PACAP/VIP 2 -R (7-11). PACAP 1 -R activation also controls proliferation of chromaffin cells (12) as well as catecholamine release by the adrenal medulla (13, 14).We and others (15-19) recently cloned the rat PACAP 1 -R cDNA. In addition, we demonstrated that PACAP 1 -R hnRNA is alternatively spliced and gives rise to six variants (18,20). Two cassettes named "hip" and "hop" are possibly inserted at the end of the third intracellular loop of the receptor. The resulting variants were named PACAP 1 -R s (the shortest form, without cassette), PACAP 1 -R hip, PACAP 1 -R hop1, PACAP 1 -R hop2, PACAP 1 -R hip-hop1, and PACAP 1 -R hip-hop2. They display differential signal transduction properties upon expression into LLC PK1 cells and Xenopus oocytes. The short as well as the hop variants potently activate both AC an...

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). Most of them are scaffolding proteins that contain several structural interaction domains such as Src homology 2 (SH2) or 3 (SH3) domains, post-synapticdensity-95/disc-large/zonula-occludens-1 (PDZ) domains and Drosophila enabled and vasodilator-stimulated phosphoprotein homologous (EVH) domains. These proteins participate in the building of large submembrane protein signaling networks. We have recently isolated a complex of at least 15 proteins interacting with the C-terminal tail of the 5-HT2C receptor, using a proteomic approach combining peptide-affinity chromatography and mass spectrometry. This further supports the interaction of the GPCR C-terminus with large protein networks. Moreover, functional studies have established that the proteins that interact with the GPCR C-terminus are implicated in various GPCR functions that do not involve Gproteins. These functions include trafficking, targeting to specific subcellular compartments, clustering with effectors, fine tuning of G-protein activation and desensitization.

All cell types express a great variety of G proteincoupled receptors (GPCRs) that are coupled to only a limited set of G proteins. This disposition favors cross-talk between transduction pathways. However, GPCRs are organized into functional units. They promote speci¢city and thus avoid unsuitable cross-talk. New methodologies (mostly yeast two-hybrid screens and proteomics) have been used to discover more than 50 GPCR-associated proteins that are involved in building these units. In addition, these protein networks participate in the tra⁄cking, targeting, signaling, ¢ne-tuning and allosteric regulation of GPCRs. To date, proteins that interact with the GPCR C-terminus are the most abundant and are the focus of this review.