Carine Bécamel

Neuronal 5-HT metabotropic receptors: fine-tuning of their structure, signaling, and roles in synaptic modulation

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-HT3 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-HT4 receptor) or by mRNA editing in the case of 5-HT2C receptors. The cellular and behavioral implications of 5-HT2C 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.

Synaptic multiprotein complexes associated with 5‐HT2C receptors: a proteomic approach

Membrane‐bound receptors such as tyrosine kinases and ionotropic receptors are associated with large protein networks structured by protein–protein interactions involving multidomain proteins. Although these networks have emerged as a general mechanism of cellular signalling, much less is known about the protein complexes associated with G‐protein‐coupled receptors (GPCRs). Using a proteomic approach based on peptide affinity chromatography followed by mass spectrometry and immunoblotting, we have identified 15 proteins that interact with the C‐ terminal tail of the 5‐hydroxytryptamine 2C (5‐HT2C) receptor, a GPCR. These proteins include several synaptic multidomain proteins containing one or several PDZ domains (PSD95 and the proteins of the tripartite complex Veli3–CASK–Mint1), proteins of the actin/spectrin cytoskeleton and signalling proteins. Coimmunoprecipitation experiments showed that 5‐HT2C receptors interact with PSD95 and the Veli3–CASK–Mint1 complex in vivo. Electron microscopy also indicated a synaptic enrichment of Veli3 and 5‐HT2C receptors and their colocalization in microvilli of choroidal cells. These results indicate that the 5‐HT2C receptor is associated with protein networks that are important for its synaptic localization and its coupling to the signalling machinery.

GPCR interacting proteins (GIPs) in the nervous system: Roles in physiology and pathologies.

G protein-coupled receptors (GPCRs) are key transmembrane recognition molecules for regulatory signals such as light, odors, taste hormones, and neurotransmitters. In addition to activating guanine nucleotide binding proteins (G proteins), GPCRs associate with a variety of GPCR-interacting proteins (GIPs). GIPs contain structural interacting domains that allow the formation of large functional complexes involved in G protein-dependent and -independent signaling. At the cellular level, other functions of GIPs include targeting of GPCRs to subcellular compartments and their trafficking to and from the plasma membrane. Recently, roles of GPCR-GIP interactions in central nervous system physiology and pathologies have been revealed. Here, we highlight the role of GIPs in some important neurological and psychiatric disorders, as well as their potential for the future development of therapeutic drugs.

G protein-coupled receptors: dominant players in cell-cell communication.

The G protein-coupled receptors (GPCRs) are the most numerous and the most diverse type of receptors (1-5% of the complete invertebrate and vertebrate genomes). They transduce messages as different as odorants, nucleotides, nucleosides, peptides, lipids, and proteins. There are at least eight families of GPCRs that show no sequence similarities and that use different domains to bind ligands and activate a similar set of G proteins. Homo- and heterodimerization of GPCRs seem to be the rule, and in some cases an absolute requirement, for activation. There are about 100 orphan GPCRs in the human genome which will be used to find new message molecules. Mutations of GPCRs are responsible for a wide range of genetic diseases. The importance of GPCRs in physiological processes is illustrated by the fact that they are the target of the majority of therapeutical drugs and drugs of abuse.

A proteomic approach based on peptide affinity chromatography, 2-dimensional electrophoresis and mass spectrometry to identify multiprotein complexes interacting with membrane-bound receptors

There is accumulating evidence that membrane-bound receptors interact with many intracellular proteins. Multiprotein complexes associated with ionotropic receptors have been extensively characterized, but the identification of proteins interacting with G protein-coupled receptors (GPCRs) has so far only been achieved in a piecemeal fashion, focusing on one or two protein species. We describe a method based on peptide affinity chromatography, two-dimensional electrophoresis, mass spectrometry and immunoblotting to identify the components of multiprotein complexes interacting directly or indirectly with intracellular domains of GPCRs or, more generally, any other membrane-bound receptor. Using this global approach, we have characterized multiprotein complexes that bind to the carboxy-terminal tail of the 5-hydroxytryptamine type 2C receptor and are important for its subcellular localization in CNS cells (Bécamel et al., EMBO J., 21(10): 2332, 2002).

Constitutively active mutants of 5‐HT4 receptors are they in unique active states?

Somatic mutations leading to constitutively active G‐protein coupled receptors (GPCRs) are responsible for certain human diseases. A consistent structural description of the molecular change underlying the conversion of GPCRs from an inactive R state to an active R* state is lacking. Here, we show that a series of constitutively active 5‐HT4 receptors (mutated or truncated in the C‐terminal and the third intracellular loop) were characterized by an increase in their denaturation rate at 55°C. The thermal denaturation kinetics were monophasic, suggesting that we were measuring mainly the denaturation rate of R*. Analysis of these kinetics revealed that constitutively active C‐terminal domain mutants, were due to a change in the J constant governing the R/R* equilibrium. However, the constitutive activity of the receptor mutated within the third intracellular loop was the result of both a change in the allosteric J constant and a change in the R* conformation.

5-HT Receptor-Associated Protein Networks: New Targets for Drug Discovery in Psychiatric Disorders?

Serotonin (5-HT) is a phylogenetically ancient transmitter implicated in many vital functions in human such as sleep, food intake, reproduction, nociception, regulation of mood and emotions as well as cognitive functions. Correspondingly, dysfunction of serotonergic transmission has been implicated in numerous psychiatric disorders such as anxio-depressive states, psychoses and addiction, and serotonergic systems are targets for a large array of psychoactive compounds including antidepressants, antipsychotics and hallucinogens. 5-HT acts on numerous receptor subtypes (14). Except for 5-HT3 receptors, which are cationic channels, 5-HT receptors belong to the G protein-coupled receptor (GPCR) superfamily and allow an extraordinarily diverse and complex pattern of cellular signalling. Over the past ten years, the majority of metabotropic 5-HT receptors has been found to interact with specific protein partners in addition to the ubiquitous GPCR modulators, GPCR kinases and β-arrestins, mainly by mean of two-hybrid and proteomic screens. These proteins, called GPCR-interacting proteins (GIPs) were found to profoundly influence the targeting, trafficking and signal transduction properties of 5-HT receptors. This article first describes our current knowledge of the nature of GIPs that bind to the different metabotropic 5-HT receptor categories. It then focuses on their impact on receptor functional status at the cellular level and illustrates how GIPs permit G protein-independent signal transduction at G protein-coupled 5-HT receptors. Finally, it reports recent data dealing with the roles of GIPs in 5-HT-related behaviours and highlights the potential of manipulating 5-HT receptor-GIP interactions to design new treatments in psychiatric disorders related to perturbations of serotonergic systems.

Quantitative Phosphoproteomics Unravels Biased Phosphorylation of Serotonin 2A Receptor at Ser280 by Hallucinogenic versus Nonhallucinogenic Agonists

The serotonin 5-HT2A receptor is a primary target of psychedelic hallucinogens such as lysergic acid diethylamine, mescaline, and psilocybin, which reproduce some of the core symptoms of schizophrenia. An incompletely resolved paradox is that only some 5-HT2A receptor agonists exhibit hallucinogenic activity, whereas structurally related agonists with comparable affinity and activity lack such a psychoactive activity. Using a strategy combining stable isotope labeling by amino acids in cell culture with enrichment in phosphorylated peptides by means of hydrophilic interaction liquid chromatography followed by immobilized metal affinity chromatography, we compared the phosphoproteome in HEK-293 cells transiently expressing the 5-HT2A receptor and exposed to either vehicle or the synthetic hallucinogen 1-[2,5-dimethoxy-4-iodophenyl]-2-aminopropane (DOI) or the nonhallucinogenic 5-HT2A agonist lisuride. Among the 5995 identified phosphorylated peptides, 16 sites were differentially phosphorylated upon exposure of cells to DOI versus lisuride. These include a serine (Ser280) located in the third intracellular loop of the 5-HT2A receptor, a region important for its desensitization. The specific phosphorylation of Ser280 by hallucinogens was further validated by quantitative mass spectrometry analysis of immunopurified receptor digests and by Western blotting using a phosphosite specific antibody. The administration of DOI, but not of lisuride, to mice, enhanced the phosphorylation of 5-HT2A receptors at Ser280 in the prefrontal cortex. Moreover, hallucinogens induced a less pronounced desensitization of receptor-operated signaling in HEK-293 cells and neurons than did nonhallucinogenic agonists. The mutation of Ser280 to aspartic acid (to mimic phosphorylation) reduced receptor desensitization by nonhallucinogenic agonists, whereas its mutation to alanine increased the ability of hallucinogens to desensitize the receptor. This study reveals a biased phosphorylation of the 5-HT2A receptor in response to hallucinogenic versus nonhallucinogenic agonists, which underlies their distinct capacity to desensitize the receptor.

Growing Evidence for Heterogeneous Synaptic Localization of 5-HT2A Receptors

The serotonin 2A (5-HT2A) receptor subtype continues to attract attention as a target for numerous psychoactive drugs including psychedelic hallucinogens, antidepressants, anxiolytics, and atypical antipsychotics. 5-HT2A receptors are a principal G protein-coupled receptor subtype mediating the excitatory effects of serotonin. Nonetheless, pre- vs postsynaptic localization of 5HT2A receptors, relative to glutamatergic synapses, has remained controversial. Here, we discuss recent findings highlighting the existence and roles of presynaptic 5-HT2A receptors in regulating glutamatergic transmission and cognition.

Identification of 5-HT2 and 5-HT4 Receptor-Interacting Proteins

G protein-coupled receptors (GPCRs) not only interact with heterotrimeric G proteins but also with accessory proteins, called GPCR-interacting proteins (GIPs). GIPs are implicated in GPCR targeting to specific cellular compartments, in their assembling into large functional complexes called “receptosomes,” in their trafficking to and from the plasma membrane, as well as in the fine-tuning of their signaling properties. Here, we describe “receptosomes“ associated with the C-terminal tails of 5-hydroxytryptamines 5-HT2A, 5-HT2C, as well as 5-HT4a and 5-HT4e receptors. The three last residues of these receptor C-termini are canonical PDZ ligands interacting with type I PDZ domain-containing proteins (5-HT2A, 5-HT2C, 5-HT4A tails) and type II (5-HT4e). The entire C-terminal tails fused to glutathione-S-transferase or synthetic peptides encompassing the last 14 C-terminal residues of the receptors were used as baits to fish out GIPs from mouse brain. Controls were made with mutant bait (mutated in the PDZ ligand). Proteins, which were specifically retained on native PDZ ligand-containing peptides, were separated on two-dimensional gels and identified by MALDI-TOF mass spectrometry or immunoblotting. Ten and seven PDZ domain-containing proteins were found to bind to the 5-HT2C and 5-HT2A receptors, respectively. The sequences of the C-terminal PDZ ligands of 5-HT2C and 5-HT2A receptors are very similar (SSV and SCV, respectively). If some of the PDZ domain-containing proteins associated with these receptors were identical (ARIP-1/MAGI-2, SAP97, PSD-95, Dlgh3/MPP3), others were clearly different. The 5-HT2C but not the 5-HT2A receptor interacted with the SAP102 and Veli3/CASK/Mint1 ternary complex, whereas the 5-HT2A but not the 5-HT2C receptors interacted with CIPP. MUPP1, which was found to interact with the 5-HT2C receptor in a two-hybrid screen, was also fished out by the 5-HT2C and 5-HT2A C-termini. A few other non-PDZ domain-containing proteins were found in these “receptosomes.” Electron microscopy (EM) studies of 5-HT2A and 5-HT2C receptors and some of their interacting proteins led to the proposition of a presynaptic and postsynaptic localization of 5-HT2C receptors and a preferential postsynaptic localization of 5-HT2A receptors. In a similar manner, we identified 10 and 3 proteins that interacted specifically with the 5-HT4a and 5-HT4e receptor splice variants, respectively. Most of them are PDZ proteins. Among them, NHERF recruited 5-HT4a receptors in microvilli, where they localized with activated ezrin, consistent with a role of 5-HT4a receptors in cytoskeleton remodeling. The same variant interacted with both the constitutive and the inducible (upon metham-phetamine treatment) forms of SNX27 (SNX27a and SNX27b, respectively). SNX27a redirected part of the 5-HT4a receptors to early endosomes.