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.

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Bursicon is a neurohumoral agent responsible for tanning and hardening of the cuticle and expansion of the wings during the final phase of insect metamorphosis. Although the hormonal activity was described more than 40 years ago, the molecular nature of bursicon has remained elusive. We identify here Drosophila bioactive bursicon as a heterodimer made of two cystine knot polypeptides. This conclusion was reached in part from the unexpected observation that in the genome of the honey bee, the orthologs of the two Drosophila proteins are predicted to be fused in a single open reading frame. The heterodimeric Drosophila protein displays bursicon bioactivity in freshly eclosed neck-ligated flies and is the natural agonist of the orphan G protein-coupled receptor DLGR2.

). 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.

We aimed at understanding molecular events involved in the activation of a member of the G protein-coupled receptor family, the thyrotropin receptor. We have focused on the transmembrane region and in particular on a network of polar interactions between highly conserved residues. Using molecular dynamics simulations and site-directed mutagenesis techniques we have identified residue Asn-7.49, of the NPxxY motif of TM 7, as a molecular switch in the mechanism of thyrotropin receptor (TSHr) activation. Asn-7.49 appears to adopt two different conformations in the inactive and active states. These two states are characterized by specific interactions between this Asn and polar residues in the transmembrane domain. The inactive gauche؉ conformation is maintained by interactions with residues Thr-6.43 and Asp-6.44. Mutation of these residues into Ala increases the constitutive activity of the receptor by factors of ϳ14 and ϳ10 relative to wild type TSHr, respectively. Upon receptor activation Asn-7.49 adopts the trans conformation to interact with Asp-2.50 and a putatively charged residue that remains to be identified. In addition, the conserved Leu-2.46 of the (N/S)LxxxD motif also plays a significant role in restraining the receptor in the inactive state because the L2.46A mutation increases constitutive activity by a factor of ϳ13 relative to wild type TSHr. As residues Leu-2.46, Asp-2.50, and Asn-7.49 are strongly conserved, this molecular mechanism of TSHr activation can be extended to other members of the rhodopsin-like family of G protein-coupled receptors.Genome sequencing projects have identified the G proteincoupled receptor (GPCR) 1 family as one of the largest class of proteins with more than 800 human sequences. These sequences have been classified into five main families, glutamate, rhodopsin, adhesion, frizzled/taste2, and secretin (1). GPCRs are involved in passing chemical signals across the cell membrane. The incoming signal may arrive in the form of neurotransmitters, peptides, divalent cations, proteases, hormones, and sensory stimuli such as photons and gustatory or odorant molecules (2, 3). Atomic level details of a three-dimensional structure of a GPCR are only known for the inactive form of rhodopsin, the light photoreceptor protein of rod cells (4, 5). Rhodopsin is formed by an extracellular N terminus, seven ␣-helices, which cross the cellular membrane (TM 1-TM 7) connected by hydrophilic loops, and a cytoplasmic C terminus containing an ␣-helix (HX 8) parallel to the cell membrane. The overall structure of the helical bundle seems common in the rhodopsin-like GPCR family because of the large number of conserved sequence patterns in these 7 TM segments (6): GN in TM 1, (N/S)LxxxD in TM 2, (D/E)RY in TM 3, CWxP in TM 6, and NPxxY(x) 5,6 F in TM 7 and HX 8, among others. Activation of GPCRs is commonly discussed in terms of the extended ternary complex model, which proposes an equilibrium between inactive and active states (7). Recent advances in the ␤ 2 -adrenergic receptor have shown that ag...

Clinical and experimental evidence point to a possible role of cerebrovascular dysfunction in Alzheimer's disease (AD). The 5xFAD mouse model of AD expresses human amyloid precursor protein and presenilin genes with mutations found in AD patients. It remains unknown whether amyloid deposition driven by these mutations is associated with cerebrovascular changes. 5xFAD and wild type mice (2 to 12months old; M2 to M12) were used. Thinned skull in vivo 2-photon microscopy was used to determine Aβ accumulation on leptomeningeal or superficial cortical vessels over time. Parenchymal microvascular damage was assessed using FITC-microangiography. Collagen-IV and CD31 were used to stain basal lamina and endothelial cells. Methoxy-XO4, Thioflavin-S or 6E10 were used to visualize Aβ accumulation in living mice or in fixed brain tissues. Positioning of reactive IBA1 microglia and GFAP astrocytes at the vasculature was rendered using confocal microscopy. Platelet-derived growth factor receptor beta (PDGFRβ) staining was used to visualize perivascular pericytes. In vivo 2-photon microscopy revealed Methoxy-XO4(+) amyloid perivascular deposits on leptomeningeal and penetrating cortical vessels in 5xFAD mice, typical of cerebral amyloid angiopathy (CAA). Amyloid deposits were visible in vivo at M3 and aggravated over time. Progressive microvascular damage was concomitant to parenchymal Aβ plaque accumulation in 5xFAD mice. Microvascular inflammation in 5xFAD mice presented with sporadic FITC-albumin leakages at M4 becoming more prevalent at M9 and M12. 3D colocalization showed inflammatory IBA1(+) microglia proximal to microvascular FITC-albumin leaks. The number of perivascular PDGFRβ(+) pericytes was significantly decreased at M4 in the fronto-parietal cortices, with a trend decrease observed in the other structures. At M9-M12, PDGFRβ(+) pericytes displayed hypertrophic perivascular ramifications contiguous to reactive microglia. Cerebral amyloid angiopathy and microvascular inflammation occur in 5xFAD mice concomitantly to parenchymal plaque deposition. The prospect of cerebrovascular pharmacology in AD is discussed.

Serotonin 4 receptors (5-HT(4)Rs) were discovered 15 years ago. They are coded by a very complex gene (700Kb, 38 exons) which generates eight carboxy-terminal variants (a, b, c, d, e, f, g, n). Their sequences differ after position L(358). Another variant is characterized by a 14 residue insertion within the extracellular loop 2. Highly selective potent 5-HT(4) receptor antagonists and partial agonists which cross the blood-brain barrier have been synthesized, but a specific full agonist for brain studies is still missing. Based on physiological and behavioral experiments, 5-HT(4)Rs may be targets to treat cognitive deficits, abdominal pain and feeding disorders. One 5-HT(4)R-directed drug (SL65.0155) is already in phase II to treat patients suffering from memory deficits or dementia.