Role of Epac in brain and heart.

Epacs (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.

New perspectives in cAMP-signaling modulation.

Cyclic 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.

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

Epac 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.

Role of the cAMP-binding protein Epac in cardiovascular physiology and pathophysiology.

Exchange proteins directly activated by cyclic AMP (Epac) were discovered 10 years ago as new sensors for the second messenger cyclic AMP (cAMP). Epac family, including Epac1 and Epac2, are guanine nucleotide exchange factors for the Ras-like small GTPases Rap1 and Rap2 and function independently of protein kinase A. Given the importance of cAMP in the cardiovascular system, numerous molecular and cellular studies using specific Epac agonists have analyzed the role and the regulation of Epac proteins in cardiovascular physiology and pathophysiology.

The deubiquitinases USP33 and USP20 coordinate beta2 adrenergic receptor recycling and resensitization.

Agonist-induced ubiquitination of the beta(2) adrenergic receptor (beta(2)AR) 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 beta(2)AR 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.

Beta-arrestin-dependent signaling and trafficking of 7-transmembrane receptors is reciprocally regulated by the deubiquitinase USP33 and the E3 ligase Mdm2.

Beta-arrestins are multifunctional adaptors that mediate the desensitization, internalization, and some signaling functions of seven-transmembrane receptors (7TMRs). Agonist-stimulated ubiquitination of beta-arrestin2 mediated by the E3 ubiquitin ligase Mdm2 is critical for rapid beta(2)-adrenergic receptor (beta(2)AR) internalization. We now report the discovery that the deubiquitinating enzyme ubiquitin-specific protease 33 (USP33) binds beta-arrestin2 and leads to the deubiquitination of beta-arrestins.

Design, synthesis, and biological evaluation of new 5-HT4 receptor agonists: application as amyloid cascade modulators and potential therapeutic utility in Alzheimer's disease.

Serotonin 5-HT(4) receptor (5-HT(4)R) agonists are of particular interest for the treatment of Alzheimer's disease because of their ability to ameliorate cognitive deficits and to modulate production of amyloid beta-protein (Abeta). However, despite the range of 5-HT(4)R agonists synthesized to date, potent and selective 5-HT(4)R agonists are still lacking. In the present study, two libraries of molecules based on the scaffold of ML10302, a highly specific and partial 5-HT(4)R agonist, were efficiently prepared by parallel supported synthesis and their binding affinities and agonist activities evaluated.

Functional characterization of the cAMP-binding proteins Epac in cardiac myocytes.

The cyclic AMP (cAMP)-binding proteins, Epac, are guanine nucleotide exchange factors for the Ras-like small GTPases. Since their discovery in 1998 and with the development of specific Epac agonists, many data in the literature have illustrated their critical role in multiple cellular events mediated by the second messenger cAMP. Given the importance of cAMP in cardiovascular physiology and physiopathology, there is a growing interest to delineate the role of these multi-domain Epac in the cardiovascular system.

Synthesis of specific bivalent probes that functionally interact with 5-HT(4) receptor dimers.

G-protein-coupled receptor dimerization directs the design of new drugs that specifically bind to receptor dimers. Here, we generated a targeted series of homobivalent ligands for serotonin 5-HT(4) receptor (5-HT(4)R) dimers composed of two 5-HT(4)R-specific ML10302 units linked by a spacer. The design of spacers was assisted by molecular modeling using our previously described 5-HT(4)R dimer model.

Ubiquitination of beta-arrestin links seven-transmembrane receptor endocytosis and ERK activation.

Beta-arrestin2 and its ubiquitination play crucial roles in both internalization and signaling of seven-transmembrane receptors (7TMRs). To understand the connection between ubiquitination and the endocytic and signaling functions of beta-arrestin, we generated a beta-arrestin2 mutant that is defective in ubiquitination (beta-arrestin2(0K)), by mutating all of the ubiquitin acceptor lysines to arginines and compared its properties with the wild type and a stably ubiquitinated beta-arrestin2-ubiquitin (Ub) chimera. In vitro translated beta-arrestin2 and beta-arrestin2(0K) displayed equivalent binding to recombinant beta(2)-adrenergic receptor (beta(2)AR) reconstituted in vesicles, whereas beta-arrestin2-Ub bound approximately 4-fold more.

Two transmembrane Cys residues are involved in 5-HT4 receptor dimerization.

The 5-HT(4) receptor (5-HT(4)R) belongs to the G-protein-coupled receptor (GPCR) family and is of considerable interest for the development of new drugs to treat gastrointestinal diseases and memory disorders. The 5-HT(4)R exists as a constitutive dimer but its molecular determinants are still unknown. Using co-immunoprecipitation and Bioluminescence Resonance Energy Transfer (BRET) techniques, we show here that 5-HT(4)R homodimerization but not 5-HT(4)R-beta(2) adrenergic receptor (beta(2)AR) heterodimerization is largely decreased under reducing conditions suggesting the participation of disulfide bonds in 5-HT(4)R dimerization.

Design and synthesis of specific probes for human 5-HT4 receptor dimerization studies.

Recently, human 5-HT4 receptors have been demonstrated to form constitutive dimers in living cells. To evaluate the role of dimerization on the 5-HT4 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-HT4 receptor dimer derived from protein/protein docking experiments.

A high-affinity monoclonal antibody with functional activity against the 5-hydroxytryptaminergic (5-HT4) receptor.

Splenocytes from a BALB/c mouse immunised with a synthetic peptide corresponding to the second extracellular loop of the 5-HT4 receptor were fused with SP2/O myeloma cells to produce a monoclonal antibody. The monoclonal antibody was of the IgG2b isotype. The antibody recognised the human 5-HT4(g) (h5-HT4(g)) receptor by immunoblots and by immunofluorescence on chinese hamster ovary (CHO) cells expressing this 5-HT4 receptor isoform.

Constitutive dimerization of human serotonin 5-HT4 receptors in living cells.

Serotonin 5-HT4 receptor isoforms are G protein-coupled 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-HT4 receptor (h5-HT4R) by means of co-immunoprecipitation and bioluminescence resonance energy transfer (BRET). Constitutive h5-HT4(d)R dimer was observed in living cells and membrane preparation of CHO and HEK293 cells.

New insights into the human 5-HT4 receptor binding site: exploration of a hydrophobic pocket.

A body of evidences suggests that a hydrophobic pocket of the human 5-HT(4) receptor contributes to the high affinity of some bulky 5-HT(4) ligands. A thorough study of this pocket was performed using mutagenesis and molecular modeling. Ligand binding or competition studies with selected bulky ligands (RS39604, RS100235, [(3)H]GR113808 and ML11411) and small ligands (5-HT and ML10375) were carried out on wild-type and mutant receptors (W7.

Influence of dietary cholesterol on vitamin d metabolism in formula-fed preterm neonates.

Objectives: Supplementation of preterm formulas with cholesterol could help to mimic the fat composition of human milk. However, this could possibly influence vitamin D 25-hydroxylation because this reaction is catalyzed in part by the mitochondrial cytochrome P-450, the enzyme responsible for the 27-hydroxylation of cholesterol. The purpose of this study was to verify whether the addition of cholesterol to preterm formulas could interfere with vitamin D metabolism in preterm neonates.