Philippe Marin

The ‘magic tail’ of G protein-coupled receptors: an anchorage for functional protein networks

All cell types express a great variety of G protein‐coupled 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 specificity 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 trafficking, targeting, signaling, fine‐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.

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): nature and functions

The simplistic idea that seven transmembrane receptors are single monomeric proteins that interact with heterotrimeric G-proteins after agonist binding is definitively out of date. Indeed, GPCRs (G-protein-coupled receptors) are part of multiprotein networks organized around scaffolding proteins. These GIPs (GPCR-interacting proteins) are either transmembrane or cytosolic proteins. Proteomic approaches can be used to get global pictures of these receptosomes. This approach allowed us to identify direct but also indirect binding partners of serotonin receptors. GIPs are involved in a wide range of functions including control of the targeting, trafficking and signalling of GPCRs. One of them, Shank, which is a secondary and tertiary partner of metabotropic and ionotropic glutamate receptors, respectively, can induce the formation of a whole functional glutamate receptosome and the structure to which it is associated, the dendritic spine.

Altering the course of schizophrenia: progress and perspectives

Despite a lack of recent progress in the treatment of schizophrenia, our understanding of its genetic and environmental causes has considerably improved, and their relationship to aberrant patterns of neurodevelopment has become clearer. This raises the possibility that disease-modifying strategies could alter the course to — and of — this debilitating disorder, rather than simply alleviating symptoms. A promising window for course-altering intervention is around the time of the first episode of psychosis, especially in young people at risk of transition to schizophrenia. Indeed, studies performed in both individuals at risk of developing schizophrenia and rodent models for schizophrenia suggest that pre-diagnostic pharmacotherapy and psychosocial or cognitive-behavioural interventions can delay or moderate the emergence of psychosis. Of particular interest are hybrid strategies that both relieve presenting symptoms and reduce the risk of transition to schizophrenia or another psychiatric disorder. This Review aims to provide a broad-based consideration of the challenges and opportunities inherent in efforts to alter the course of schizophrenia.

Calpain product of WT‐CRMP2 reduces the amount of surface NR2B NMDA receptor subunit

The brain is particularly vulnerable to ischaemia; however, neurons can become tolerant to ischaemic insult. This tolerance has been shown to involve activation of NMDA receptors, but its mechanisms have not yet been fully elucidated. Using a preconditioning protocol, we show that neurons surviving to a transient NMDA exposure become resistant to the glutamatergic agonist. Using a proteomic approach, we found that alterations of the protein pattern of NMDA‐resistant neurons are restricted mainly to the five collapsin response mediator proteins (CRMPs). A sustained increase in calpain activity following NMDA treatment is responsible for the production of cleaved CRMPs. Finally, we provide evidence for the involvement of the cleaved form of WT‐CRMP2 in the down‐regulation of NR2B. Our data suggests that, beside their role in neuronal morphogenesis, CRMPs may contribute to neuronal plasticity.

Akt mediates the anti-apoptotic effect of NMDA but not that induced by potassium depolarization in cultured cerebellar granule cells

Apoptosis of cultured cerebellar granule neurons (CGNs) deprived of serum is prevented by K+ depolarization or moderate concentrations of N‐methyl‐d‐aspartate (NMDA). Here, we have examined the role of the serine/threonine kinase Akt in these protective effects. The exposure of mouse CGNs to NMDA or K+ depolarization increased the phosphorylation of Akt, compared with that measured in cells incubated in a physiological K+ concentration. Only the NMDA‐evoked response was reduced by inhibitors of phosphatidylinositol 3‐kinase (wortmannin and LY294002) and mitogen‐activated protein kinase (PD98059 and U0126). Similarly, the capacity of NMDA to inhibit apoptosis of CGNs deprived of serum was greatly reduced by these inhibitors as well as by the transfection of neurons with a catalytically inactive mutant of Akt, whereas the protective effect of K+ depolarization remained unaffected. These findings indicate that K+ depolarization and NMDA activate Akt through different signalling pathways in CGNs. Moreover, Akt mediates the anti‐apoptotic effect of NMDA, but not that evoked by K+ depolarization.

Identification of new proteins in follicular fluid of mature human follicles

Proteins present in human follicular fluid (HFF) have been poorly characterized to date. The purpose of our study was to analyse the protein content and identify new proteins originating from fluid of mature human follicles. A total of six females from infertile couples referred for in vitro fertilization (IVF) were stimulated and 44 follicular fluid samples from mature follicles yielding an oocyte were collected 34–36 h after human chorionic gonadotropin administration. HFF samples were processed for high‐resolution two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE). Comparative analysis of the 2‐D gels revealed up to 600 spots, of which four were selected because of variations in their expression level. Using direct sequencing procedures (Edman degradation) or matrix assisted laser desorption/ionization‐mass spectrometry (MALDI‐MS), these four spots were identified as three new proteins: thioredoxin peroxydase 1 (TDPX1), transthyretin (TTR) and retinol‐binding protein (RBP). The proteins identified here may emerge as potential candidates for specific functions during folliculogenesis and may prove useful as biomedical markers for follicle and/or oocyte maturation.

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).

CRMP2 Tethers Kainate Receptor Activity to Cytoskeleton Dynamics during Neuronal Maturation

The CRMP2 and CRMP4 proteins are strongly expressed in the developing nervous system, mediating neurite outgrowth, neuronal polarity, and axon guidance. In the present study, we demonstrate the interaction of the CRMP2 and CRMP4 proteins with the GluK5 subunit of the kainate (KA) receptor (KAR) and investigated the role of KARs in modulating the development of cultured mouse DRG neurons. We found that KARs modulate neuronal maturation and neurite outgrowth in a bidirectional manner. Accordingly, low concentrations of KA delayed maturation and enhanced neurite outgrowth, whereas maturation was promoted by higher concentrations of KA that attenuated neuritic elongation. The effects of weak KAR activation were prevented by blocking their noncanonical signaling and involved a differential regulation of CRMP2. Whereas the delay in maturation involves PKC-mediated phosphorylation of CRMP2 at T555 leading to a downregulation of membrane Cav2.2, the promotion of neurite outgrowth is achieved by dephosphorylation at T514 at the growth cones, the latter reflecting PKC-driven enhancement of GSK3β phosphorylation at S9. Together, these findings indicate that noncanonical KAR signaling influences neuronal development by modulating CRMP2 activity.

E-cooperative design among mechanical and electrical engineers: implications for communication between professional cultures

This paper looks at the collaborative design activity involved in a design experiment of an electromechanical plunger. Much of the coordination was achieved through Internet-based communication. As mechanical and electrical researchers involved in the design project, we discuss the information exchanges highlighted by our different professional cultures and relate how these exchanges lead us to propose some methodology to improve the efficiency of virtual meetings. Moreover, we show the need for new communication tools, ones dedicated to specific tasks that are not currently supported, especially shared concept formalization among technical experts.