We have previously reported that morphine induces apoptosis. However, the underlying molecular mechanisms remain to be elucidated. Toll-like receptor 2 (TLR2), a key immune receptor in the TLR family, modulates cell survival and cell death in various systems. Evidence indicates that β-arrestin 2 acts as a negative regulator of innate immune activation by TLRs. Here, we investigated the roles of TLR2, the downstreaming mediator MyD88, and β-arrestin 2 in morphine-induced apoptosis. We showed that overexpression of TLR2 in HEK293 cells caused a significant increase in apoptosis after morphine treatment. Inhibition of MyD88 by transfecting dominant negative MyD88 or overexpression of β-arrestin 2 by transfecting β-arrestin 2 full length plasmid in TLR2 overexpressing HEK293 cells attenuated morphine-induced apoptosis. Our study thus demonstrates that TLR2 signaling mediates the morphine-induced apoptosis, and β-arrestin 2 is a negative regulator in morphine-induced, TLR2-mediated apoptosis.

β-arrestins, a family of regulatory and scaffold proteins, are well-known negative regulators of G-protein-coupled receptors (GPCRS) including opioid receptors. Recent studies have shown that β-arrestin2 plays a potential role in inhibiting cell death. It has been reported that opioids such as morphine induce cell death at high concentrations (>500 μM for 24 hours), which is similar to morphine plasma concentrations in cancer patients receiving chronic morphine treatment for pain relievers. However, the role of β-arrestin2 in opioid-induced cell death remains to be elucidated. We report here that β-arrestin2 significantly blocks morphine-induced number of cell death in human breast cancer MCF-7 and MDA-MB231 cells. Suppression of endogenous β-arrestin2 by specific RNA interfering (RNAi) and morphine treatment significantly attenuates the levels of phosphorylated Akt compared with inhibition of β-arrestin2 or morphine treatment alone. However, blockade of morphine-induced cell death by β-arrestin2 seems to be dependent on the inhibition of caspase-8, as inhibition of β-arrestin2 and morphine treatment significantly enhanced the levels of cleaved caspase-8. These studies show for the first time that β-arrestin2 blocks morphine-induced cell death through anti-apoptotic Akt and pro-apoptotic caspase-8 pathways. Therefore, targeting β-arrestin2 may be useful for treating side effects of opioids as pain relievers for cancer patients.

The mechanisms by which opioids affect progression of human immunodeficiency virus type 1 (HIV-1) infection are not well-defined. HIV-1 gp120 is important in the apoptotic death of uninfected, bystander T cells. In this study, we show that co-treatment of human peripheral blood mononuclear cells (PBMC) with HIV-1 gp120/morphine synergistically induces apoptosis in PBMC. Co-treatment of murine splenocytes from μ opiate receptor knockout mice with gp120/morphine resulted in decreased apoptosis when compared to splenocytes from wild type mice. Co-treatment of human PBMC or murine splenocytes with gp120/morphine led to decreased expression of β-arrestin 2, a protein required for opioid-mediated signaling. The role of β-arrestin 2 was confirmed in Jurkat lymphocytes, in which 1) over-expression of β-arrestin 2 inhibited gp120/morphine-induced apoptosis and 2) RNA interference of β-arrestin 2 expression enhanced gp120/morphine-induced apoptosis. These data suggest a novel mechanism by which HIV-1 gp120 and opioids induce lymphocyte cell death.

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<p>The following thesis will focus on understanding the downstream behavioral effects of δORmediated β-arrestinsignaling. δORagonists have been implicated as effective targets for a variety of diseases, however detrimental side effects of opioid-targeting agonists limit their clinical use. δORagonists specifically can induce seizures, however the underlying mechanism contributing to this  behavior  is  unknown.  We  review  this  phenomenon  in  more  detail,  highlighting  current agonists known to induce seizures and potential circuits and pathways involved. Our work suggests β-arrestinsignaling  is  involved,  specifically β-arrestin2  mediated  signaling  may  be  largely contributing  to δORagonist-induced  seizure  behavior.  As  it  is  possible  the β-arrestinisoforms have unique roles in seizure behavior, we also analyzed methods in which to provoke β-arrestinisoform bias of δORtargeting compounds. Though the full mechanism relating δORagonists with seizures remains unknown, our work provides foundational detail of this behavior, implicating the importance of β-arrestinisoform signaling through δOR; allowing for future studies to full define this seizure pathway and develop δORsafer agonists.  </p>

Les récepteurs couplés aux protéines G (RCPG) peuvent activer des voies de signalisation indépendantes des protéines G. Cependant, la régulation de ces voies, et en particulier leur désensibilisation, est peu connue. Le récepteur de la sérotonine de type 4 (R-5-HT4) est un RCPG exprimé dans le cerveau et les organes périphériques. Il est impliqué dans des fonctions physiologiques importantes comme la mémoire, l'apprentissage, la prise de nourriture, le contrôle respiratoire et la mobilité gastro-intestinale. Le R-5-HT4 est couplé à la protéine Gs. De plus, il active la voie Src/ERK1/2, indépendamment des protéines G et des β-arrestines.Nous avons montré que GRK5, physiquement associé à la région C-terminale (C-ter) du R-5-HT4 inhibait la voie Src/ERK1/2 couplée au récepteur, mais pas la voie Gs. Ce résultat a été observé dans la lignée de cellules HEK-293 mais aussi dans des neurones de collicules en culture. Cette inhibition nécessite deux séquences d'évènements : l'association de la β-arrestine1 à une région riche en sérines et thréonines, localisée dans le domaine C-ter du récepteur et la phosphorylation par GRK5, de la β-arrestine1 (en sérine 412) liée au récepteur. La β-arrestine1 phosphorylée empêche l'activation de Src, constitutivement liée au récepteur, nécessaire à l'activation d'ERK1/2. Ceci constitue la première démonstration que la phosphorylation d'une β-arrestine par une GRK régule la signalisation indépendante des protéines G. En plus de ces résultats, nous avons démontré que l'activation d'ERK1/2 par le R-5-HT4, indépendante des β-arrestines, implique la libération d'un ligand induite par une métalloprotéase, conduisant à la transactivation d'un autre récepteur. Par une approche protéomique, nous avons également identifiés plusieurs partenaires potentiels du R-5-HT4. L'étude de ces partenaires pourrait apporter un éclairage supplémentaire sur les voies de signalisation du récepteur et leur régulation. / G protein-coupled receptors (GPCRs) have been found to trigger G protein-independent signalling. However, the regulation of G protein-independent pathways, especially their desensitization, is poorly characterized.The 5-Hydroxytryptamine 4 receptor (5-HT4R) is a GPCR widely expressed in the brain and at the periphery. It is implicated in important physiological functions such as memory, cognition, feeding, respiratory control and gastrointestinal motility. 5-HT4R couples to the Gs/cAMP/PKA pathway. Moreover, this receptor can activate a Src/ERK pathway independently of both G proteins and β-arrestins.Here, we show that the G protein-independent 5-HT4R-operated Src/ERK pathway, but not the Gs pathway, is inhibited by GPCR kinase 5 (GRK5), physically associated with the proximal region of receptor C-terminus, in both HEK-293 cells and colliculi neurons. This inhibition requires two sequences of events: the association of β-arrestin1 to a phosphorylated serine/threonine cluster located within the receptor C-terminal domain and the phosphorylation by GRK5 of β-arrestin1 (at Ser 412) bound to the receptor. Phosphorylated β-arrestin1 prevents in turn activation of Src constitutively bound to 5-HT4R, a necessary step in receptor-stimulated ERK signalling. This is the first demonstration that β-arrestin phosphorylation by a GRK regulates G protein-independent signalling.In addition to these results, we also demonstrated that the β-arrestin-independent activation of ERK1/2 by the 5-HT4R involves a metalloprotease-dependant ectodomain shedding and transactivation of another receptor. By a proteomic approach, we also identified several potential partners of the 5-HT4R. Study of these proteins may provide a better understanding of 5-HT4R signalling and his regulation.

As células endoteliais são capazes de converter o estímulo mecânico em sinais intracelulares e produzir fatores vasoativos como o óxido nítrico (oNO). Evidências recentes sugerem que as beta-arrestinas desempenham um papel importante não somente na dessensibilização e internalização de receptores acoplados à proteína G (GPCR) como também na mecanotransdução. Nós testamos a hipótese de que células endoteliais submetidas ao shear stress (SS) produzem oNO por meio da ativação da via de sinalização dependente de beta-arrestina. Para tal, células endoteliais de veia safena (hSVEC) foram transfectadas com siRNA contra as isoformas 1 e 2 da beta-arrestina e, posteriormente, submetidas ao SS (15 dinas/cm2) durante 10 min. Nós encontramos que as SVEC silenciadas para a beta-arrestina 1/2 (70%) exibiram uma menor produção de nitrito no meio de cultura em resposta ao SS (166±17 vs. 326±44% comparado com hSVEC transfectadas com siRNA controle). Além disso, o silenciamento da beta-arrestina 1 e 2 preveniu os níveis de fosforilação da Akt no resíduo de serina 473 e a fosforilação da eNOS no resíduo de serina 1177, enquanto que a fosforilação da ERK 1/2 manteve-se inalterada. Curiosamente, análises de imunoprecipitação mostraram que a beta-arrestina interage com caveolina-1, um mecanossensor do shear stress, mas não é influenciado pelo SS. Além disso, na situação estática, a beta-arrestina encontra-se em uma localização perinuclear e, após o SS, adquiriu um padrão mais difuso no citosol. Coletivamente, esses dados sugerem que a beta-arrestina e a sinalização downstream Akt/ eNOS são necessárias para a produção de oNO induzido por shear stress em células endoteliais vasculares humana / Endothelial cells are capable of converting mechanical stimuli into intracellular signals generating vasoactive factors such as nitric oxide (oNO). Recent evidence suggests that beta-arrestins play a role not only on G protein-coupled receptors (GPCR) desensibilization but also in mechanotransduction. We tested the hypothesis that beta-arrestin and its downstream signaling influence laminar shear stress (SS)-induced oNO production by endothelial cells. Towards this end, human saphenous vein endothelial cells (hSVEC) transfected with siRNA against beta-arrestins isoforms 1 and 2 were subjected to SS (15 dynes/cm2, 10 minutes). We found that the SS-induced production of nitrite in the cell culture medium from down-expressed beta-arrestin 1/ 2 (70%) SVEC decreased (166±17 vs. 326±44% compared to wild-type hSVEC; P < 0.001). The beta-arrestin 1 and 2 down-regulation in SVEC also inhibited the phosphorylation levels of Akt at the serine residue 473 and the phosphorylation levels of eNOS at the serine residue 1177, whereas ERK phosphorylation remained unchanged. Interestingly, immunoprecipitation analysis showed that beta-arrestin interacts with caveolin-1, a shear stress mechanosensor, which is not influenced by SS despite the fact that the static perinuclear localization of beta-arrestins changed to the cytosol upon SS. Collective these data suggest that beta-arrestin and Akt/eNOS downstream signaling are required for shear stress-induced nitric oxide production in human vascular endothelial cells