Étude structurale et fonctionnelle de la dimérisation du récepteur 5-HT 4 de la sérotonine

Serotonin 5-HT4 receptors (5-HT4R) are members of G protein-coupled receptors (GPCRs). Given the recent recognition that many GPCRs can dimerize, the present study was undertaken to determine whether 5-HT4R can form dimers and what are the molecular determinants involved in the dimerization process. Using co-immunoprecipitation and bioluminescence resonance energy transfer, we showed that the human 5-HT4R isoforms can form constitutive homodimers and heterodimers. In addition, we showed heterodimer formation between the 5-HT4(d)R and the beta2-adrenergic receptor. The 5-HT4(d)R dimer is sensible to dithiothreitol (reducing agent). Mutation of two cysteines in the 3 and 4 transmembrane domains, inhibits 5-HT4 receptor dimerization and brings about reticulum endoplasmic retention. Specific 5-HT4 receptor bivalent ligand do not have special affinity or efficacy for the 5-HT4 receptor but are able to stabilize receptor dimerization.Les récepteurs 5-HT4 de la sérotonine sont des récepteurs couplés aux protéines G (RCPG). Les RCPGs peuvent se dimériser, ce qui influence leurs propriétés pharmacologiques. Les récepteurs 5-HT4 étant impliqués dans d'importants processus physiologiques, nous avons étudié leur processus de dimérisation. Nous avons montré par co-immunoprécipitation et Bioluminescence Resonnance Energy Transfert, que les isoformes 5-HT4 s'homodimérisent et s'hétérodimérisent constitutivement, entre elles et avec le récepteur b2-adrénergique. Le dimère de récepteur 5-HT4 est sensible au dithiothréitol (agent réducteur de ponts disulfures). La mutation de deux cystéines localisées dans les domaines transmembranaires 3 et 4, inhibe la dimérisation du récepteur 5-HT4 et entraîne sa rétention dans le réticulum endoplasmique. Des ligands bivalents spécifiques du récepteur 5-HT4 ne présentent pas d'affinité ou d'efficacité particulière pour le récepteur 5-HT4 mais peuvent stabiliser les dimères de récepteurs.CHATENAY M.-PARIS 11-BU Pharma. (920192101) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Role of Epac in brain and heart.

International audienceEpacs (exchange proteins directly activated by cAMP) are guanine-nucleotide-exchange factors for the Ras-like small GTPases Rap1 and Rap2. Epacs were discovered in 1998 as new sensors for the second messenger cAMP acting in parallel to PKA (protein kinase A). As cAMP regulates many important physiological functions in brain and heart, the existence of Epacs raises many questions regarding their role in these tissues. The present review focuses on the biological roles and signalling pathways of Epacs in neurons and cardiac myocytes. We discuss the potential involvement of Epacs in the manifestation of cardiac and central diseases such as cardiac hypertrophy and memory disorders

New perspectives in cAMP-signaling modulation.

International audienceCyclic adenosine 3',5'-monophosphate (cAMP) mediates the biological effects of various hormones and neurotransmitters. Stimulation of cardiac β-adrenergic receptors (β-AR) via catecholamines leads to activation of adenylyl cyclases and increases cAMP production to enhance myocardial function. Because many other receptors signaling through cAMP generation exist in cardiac myocytes, a central question is how different hormones induce distinct cellular responses through the same second messenger. A large body of evidence suggests that the localization and compartmentalization of β-AR/cAMP signaling affects the net outcome of biological functions. Spatiotemporal dynamics of cAMP action is achieved by various proteins, including protein kinase A (PKA), phosphodiesterases, and scaffolding proteins such as A-kinase-anchoring proteins. In addition, the discovery of the cAMP target Epac (exchange proteins directly activated by cAMP), which functions in a PKA-independent manner, represents a novel mechanism for governing cAMP-signaling specificity. Aberrant cAMP signaling through dysregulation of β-AR/cAMP compartmentalization may contribute to cardiac remodeling and heart failure

Rap-linked cAMP signaling Epac proteins: compartmentation, functioning and disease implications.

International audienceEpac proteins respond to the second messenger cyclic AMP (cAMP) and are activated by Gs coupled receptors. They act as specific guanine nucleotide exchange factors (GEFs) for the small G proteins, Rap1 and Rap2 of the Ras family. A plethora of studies using 8-pCPT-2'-O-Me-cAMP, an Epac agonist, has revealed the importance of these multi-domain proteins in the control of key cellular functions such as cell division, migration, growth and secretion. Epac and protein kinase A (PKA) may act independently but are often associated with the same biological process, in which they fulfill either synergistic or opposite effects. In addition, compelling evidence is now accumulating about the formation of molecular complexes in distinct cellular compartments that influence Epac signaling and cellular function. Epac is spatially and temporally regulated by scaffold protein and its effectors are interconnected with other signaling pathways. Pathophysiological changes in Epac signaling may underlie certain diseases

The deubiquitinases USP33 and USP20 coordinate β2 adrenergic receptor recycling and resensitization

Agonist-induced ubiquitination of the β2 adrenergic receptor (β2AR) functions as an important post-translational modification to sort internalized receptors to the lysosomes for degradation. We now show that this ubiquitination is reversed by two deubiquitinating enzymes, ubiquitin-specific proteases (USPs) 20 and 33, thus, inhibiting lysosomal trafficking when concomitantly promoting receptor recycling from the late-endosomal compartments as well as resensitization of recycled receptors at the cell surface. Dissociation of constitutively bound endogenously expressed USPs 20 and 33 from the β2AR immediately after agonist stimulation and reassociation on prolonged agonist treatment allows receptors to first become ubiquitinated and then deubiquitinated, thus, providing a ‘trip switch' between degradative and recycling pathways at the late-endosomal compartments. Thus, USPs 20 and 33 serve as novel regulators that dictate both post-endocytic sorting as well as the intensity and extent of β2AR signalling from the cell surface

Constitutive dimerization of human serotonin 5-HT 4 receptors in living cells

International audienceSerotonin 5-HT 4 receptor isoforms are G proteincoupled receptors (GPCRs) with distinct pharmacological properties and may represent a valuable target for the treatment of many human disorders. Here, we have explored the process of dimerization of human 5-HT 4 receptor (h5-HT 4 R) by means of co-immunoprecipitation and bioluminescence resonance energy transfer (BRET). Constitutive h5-HT 4(d) R dimer was observed in living cells and membrane preparation of CHO and HEK293 cells. 5-HT 4 R ligands did not influence the constitutive energy transfer of the h5-HT 4(d) R splice variant in intact cells and isolated plasma membranes. In addition, we found that h5-HT 4(d) R and h5-HT 4(g) R which structurally differ in the length of their C-terminal tails were able to form constitutive heterodimers independently of their activation state. Finally, we found that coexpression of h5-HT 4 R and b 2-adrenergic receptor (b 2 AR) led to their heterodimerization. Given the large number of h5-HT 4 R isoforms which are coexpressed in a same tissue, our results points out the complexity by which this 5-HTR sub-type mediates its biological effects

Two transmembrane Cys residues are involved in 5-HT 4 Receptor dimerization

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Design and Synthesis of Specific Probes for Human 5-HT 4 Receptor Dimerization Studies

International audienceRecently, human 5-HT 4 receptors have been demonstrated to form constitutive dimers in living cells. To evaluate the role of dimerization on the 5-HT 4 receptor function, we investigated the conception and the synthesis of bivalent molecules able to influence the dimerization process. Their conception is based on a model of the 5-HT 4 receptor dimer derived from protein/protein docking experiments. These bivalent ligands are constituted by two ML10302 units, a specific 5-HT 4 ligand, linked through a spacer of different sizes and natures. These synthesized bivalent ligands were evaluated in binding assays and cyclic AMP production on the 5-HT 4(e/g) receptor isoform stably transfected in C6 glial cells. Our data showed that bivalent ligands conserved a similar affinity compared to the basal ML10302 unit. Nevertheless, according to the nature and the size of the spacer, the pharmacological profile of ML10302 is more or less conserved. In view of the interest of bivalent ligands for investigating the GPCR dimerization process, these 5-HT 4 specific bivalent ligands constitute valuable pharmacological tools for the study of 5-HT 4 receptor dimerization

Deterioration of Physical Activity Level and Metabolic Risk Factors After Early-Stage Breast Cancer Diagnosis

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