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. Thus, USPs 20 and 33 serve as novel regulators that dictate both post-endocytic sorting as well as the intensity and extent of beta(2)AR signalling from the cell surface.

␤-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 ␤-arrestin, we generated a ␤-arrestin2 mutant that is defective in ubiquitination (␤-arrestin2 0K ), by mutating all of the ubiquitin acceptor lysines to arginines and compared its properties with the wild type and a stably ubiquitinated ␤-arrestin2-ubiquitin (Ub) chimera. In vitro translated ␤-arrestin2 and ␤-arrestin2 0K displayed equivalent binding to recombinant ␤ 2 -adrenergic receptor (␤ 2 AR) reconstituted in vesicles, whereas ␤-arrestin2-Ub bound ϳ4-fold more. In cellular coimmunoprecipitation assays, ␤-arrestin2 0K bound nonreceptor partners, such as AP-2 and c-Raf and scaffolded phosphorylated ERK robustly but displayed weak binding to clathrin. Moreover, ␤-arrestin2 0K was recruited only transiently to activated receptors at the membrane, did not enhance receptor internalization, and decreased the amount of phosphorylated ERK assimilated into isolated ␤ 2 AR complexes. Although the wild type ␤-arrestin2 formed ERK signaling complexes with the ␤ 2 AR at the membrane, a stably ubiquitinated ␤-arrestin2-Ub chimera not only stabilized the ERK signalosomes but also led to their endosomal targeting. Interestingly, in cellular fractionation assays, the ubiquitination state of ␤-arrestin2 favors its distribution in membrane fractions, suggesting that ubiquitination increases the propensity of ␤-arrestin for membrane association. Our findings suggest that although ␤-arrestin ubiquitination is dispensable for ␤-arrestin cytosol to membrane translocation and its "constitutive" interactions with some cytosolic proteins, it nevertheless is a prerequisite both for the formation of tight complexes with 7TMRs in vivo and for membrane compartment interactions that are crucial for downstream endocytic and signaling processes.The multifunctional adaptor proteins ␤-arrestins (␤-arrestin1 and -2) were originally identified as desensitizing molecules that prevent the coupling between seven-transmembrane receptors (7TMRs) 3 and G proteins (1-3). More recently, however, it was found that ␤-arrestin binding to receptors not only stops G protein-mediated second messenger signaling but also engages several novel signaling pathways, including mitogenactivated protein kinase (MAPK) cascades (4, 5). Furthermore, ␤-arrestins have also been shown to bind and regulate cell surface receptors other than 7TMRs, and their signaling has been implicated in regulating the actin cytoskeleton, chemotaxis, antiapoptosis, and metastasis (6).␤-Arrestins serve as endocytic adaptors that bind clathrin and adaptin protein subunit 2 (AP-2) and facilitate receptor internalization via clathrin-coated vesicles (7-9). The differing affinity and trafficking patterns of GFP-␤-arrestins induced by several 7TMRs have led to the classification of receptors into two groups, Class A and Class B (10). Class A receptors (e.g. ␤ 2 -adre...

␤-Arrestins are multifunctional adaptors that mediate the desensitization, internalization, and some signaling functions of seventransmembrane receptors (7TMRs). Agonist-stimulated ubiquitination of ␤-arrestin2 mediated by the E3 ubiquitin ligase Mdm2 is critical for rapid ␤2-adrenergic receptor (␤2AR) internalization. We now report the discovery that the deubiquitinating enzyme ubiquitin-specific protease 33 (USP33) binds ␤-arrestin2 and leads to the deubiquitination of ␤-arrestins. USP33 and Mdm2 function reciprocally and favor respectively the stability or lability of the receptor ␤-arrestin complex, thus regulating the longevity and subcellular localization of receptor signalosomes. Receptors such as the ␤2AR, previously shown to form loose complexes with ␤-arrestin (''class A'') promote a ␤-arrestin conformation conducive for binding to the deubiquitinase, whereas the vasopressin V2R, which forms tight ␤-arrestin complexes (''class B''), promotes a distinct ␤-arrestin conformation that favors dissociation of the enzyme. Thus, USP33-␤-arrestin interaction is a key regulatory step in 7TMR trafficking and signal transmission from the activated receptors to downstream effectors.endocytosis ͉ G protein-coupled receptors ͉ ubiquitination ͉ phosphorylation ͉ ERK1/2

No abstract

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 specific functions of Epac proteins may depend upon their microcellular environments as well as their expression and localization. This review discusses recent data showing the involvement of Epac in vascular cell migration, endothelial permeability, and inflammation through specific signaling pathways. In addition, we present evidence that Epac regulates the activity of various cellular compartments of the cardiac myocyte and influences calcium handling and excitation-contraction coupling. The potential role of Epac in cardiovascular disorders such as cardiac hypertrophy and remodeling is also discussed.

Serotonin 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.

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. Their syntheses were based on Sonogashira-Linstrumelle coupling methods. All compounds retained high-affinity binding to 5-HT(4)R but lost the agonistic character of the monomeric ML10302 compound. Direct evidence for the functional interaction of both pharmacophores of bivalent ligands with the 5-HT(4)R was obtained using a bioluminescence resonance energy transfer (BRET) based assay that monitors conformational changes within 5-HT(4) dimers. Whereas the monovalent ML10302 was inactive in this assay, several bivalent derivatives dose-dependently increased the BRET signal, indicating that both pharmacophores functionally interact with the 5-HT(4) dimer. These bivalent ligands may serve as a new basis for the synthesis of potential drugs for 5-HT(4)-associated disorders.

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. These bivalent ligands are constituted by two ML10302 units, a specific 5-HT4 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-HT4(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-HT4 specific bivalent ligands constitute valuable pharmacological tools for the study of 5-HT4 receptor dimerization.