Meningococcus Hijacks a ß2-adrenoceptor/ß-Arrestin pathway to cross brain microvasculature endothelium.

Following pilus-mediated adhesion to human brain endothelial cells, meningococcus (N. meningitidis), the bacterium causing cerebrospinal meningitis, initiates signaling cascades, which eventually result in the opening of intercellular junctions, allowing meningeal colonization. The signaling receptor activated by the pathogen remained unknown. We report that N. meningitidis specifically stimulates a biased ß2-adrenoceptor/ß-arrestin signaling pathway in endothelial cells, which ultimately traps ß-arrestin-interacting partners, such as the Src tyrosine kinase and junctional proteins, under bacterial colonies. Cytoskeletal reorganization mediated by ß-arrestin-activated Src stabilizes bacterial adhesion to endothelial cells, whereas ß-arrestin-dependent delocalization of junctional proteins results in anatomical gaps used by bacteria to penetrate into tissues. Activation of ß-adrenoceptor endocytosis with specific agonists prevents signaling events downstream of N. meningitidis adhesion and inhibits bacterial crossing of the endothelial barrier. The identification of the mechanism used for hijacking host cell signaling machineries opens perspectives for treatment and prevention of meningococcal infection.

Pharmacological chaperone-rescued cystic fibrosis CFTR-F508del mutant overcomes PRAF2-gated access to endoplasmic reticulum exit sites.

The endoplasmic reticulum exit of some polytopic plasma membrane proteins (PMPs) is controlled by arginin-based retention motifs. PRAF2, a gatekeeper which recognizes these motifs, was shown to retain the GABAB-receptor GB1 subunit in the ER. We report that PRAF2 can interact on a stoichiometric basis with both wild type and mutant F508del Cystic Fibrosis (CF) Transmembrane Conductance Regulator (CFTR), preventing the access of newly synthesized cargo to ER exit sites. Because of its lower abundance, compared to wild-type CFTR, CFTR-F508del recruitment into COPII vesicles is suppressed by the ER-resident PRAF2. We also demonstrate that some pharmacological chaperones that efficiently rescue CFTR-F508del loss of function in CF patients target CFTR-F508del retention by PRAF2 operating with various mechanisms. Our findings open new therapeutic perspectives for diseases caused by the impaired cell surface trafficking of mutant PMPs, which contain RXR-based retention motifs that might be recognized by PRAF2.

Control of CCR5 Cell-Surface Targeting by the PRAF2 Gatekeeper.

The cell-surface targeting of neo-synthesized G protein-coupled receptors (GPCRs) involves the recruitment of receptors into COPII vesicles budding at endoplasmic reticulum exit sites (ERESs). This process is regulated for some GPCRs by escort proteins, which facilitate their export, or by gatekeepers that retain the receptors in the ER. PRAF2, an ER-resident four trans- membrane domain protein with cytoplasmic extremities, operates as a gatekeeper for the GB1 protomer of the heterodimeric GABAB receptor, interacting with a tandem di-leucine/RXR retention motif in the carboxyterminal tail of GB1. PRAF2 was also reported to interact in a two-hybrid screen with a peptide corresponding to the carboxyterminal tail of the chemokine receptor CCR5 despite the absence of RXR motifs in its sequence. Using a bioluminescence resonance energy transfer (BRET)-based subcellular localization system, we found that PRAF2 inhibits, in a concentration-dependent manner, the plasma membrane export of CCR5. BRET-based proximity assays and Co-IP experiments demonstrated that PRAF2/CCR5 interaction does not require the presence of a receptor carboxyterminal tail and involves instead the transmembrane domains of both proteins. The mutation of the potential di-leucine/RXR motif contained in the third intracellular loop of CCR5 does not affect PRAF2-mediated retention. It instead impairs the cell-surface export of CCR5 by inhibiting CCR5's interaction with its private escort protein, CD4. PRAF2 and CD4 thus display opposite roles on the cell-surface export of CCR5, with PRAF2 inhibiting and CD4 promoting this process, likely operating at the level of CCR5 recruitment into COPII vesicles, which leave the ER.

Beta-arrestins operate an on/off control switch for focal adhesion kinase activity.

Focal adhesion kinase (FAK) regulates key biological processes downstream of G protein-coupled receptors (GPCRs) in normal and cancer cells, but the modes of kinase activation by these receptors remain unclear. We report that after GPCR stimulation, FAK activation is controlled by a sequence of events depending on the scaffolding proteins ß-arrestins and G proteins. Depletion of ß-arrestins results in a marked increase in FAK autophosphorylation and focal adhesion number. We demonstrate that ß-arrestins interact directly with FAK and inhibit its autophosphorylation in resting cells. Both FAK-ß-arrestin interaction and FAK inhibition require the FERM domain of FAK. Following the stimulation of the angiotensin receptor AT1AR and subsequent translocation of the FAK-ß-arrestin complex to the plasma membrane, ß-arrestin interaction with the adaptor AP-2 releases inactive FAK from the inhibitory complex, allowing its activation by receptor-stimulated G proteins and activation of downstream FAK effectors. Release and activation of FAK in response to angiotensin are prevented by an AP-2-binding deficient ß-arrestin and by a specific inhibitor of ß-arrestin/AP-2 interaction; this inhibitor also prevents FAK activation in response to vasopressin. This previously unrecognized mechanism of FAK regulation involving a dual role of ß-arrestins, which inhibit FAK in resting cells while driving its activation at the plasma membrane by GPCR-stimulated G proteins, opens new potential therapeutic perspectives in cancers with up-regulated FAK.

TRPV1 Activation Promotes ß-arrestin2 Interaction with the Ribosomal Biogenesis Machinery in the Nucleolus:Implications for p53 Regulation and Neurite Outgrowth.

Transient receptor potential vanilloids (TRPV1) are non-selective cation channels that sense and transduce inflammatory pain signals. We previously reported that activation of TRPV1 induced the translocation of ß-arrestin2 (ARRB2) from the cytoplasm to the nucleus, raising questions about the functional role of ARRB2 in the nucleus. Here, we determined the ARRB2 nuclear signalosome by conducting a quantitative proteomic analysis of the nucleus-sequestered L395Q ARRB2 mutant, compared to the cytosolic wild-type ARRB2 (WT ARRB2), in a heterologous expression system. We identified clusters of proteins that localize to the nucleolus and are involved in ribosomal biogenesis. Accordingly, L395Q ARRB2 or WT ARRB2 after capsaicin treatment were found to co-localize and interact with the nucleolar marker nucleophosmin (NPM1), treacle protein (TCOF1) and RNA polymerase I (POL I). We further investigated the role of nuclear ARRB2 signaling in regulating neuroplasticity. Using neuroblastoma (neuro2a) cells and dorsal root ganglia (DRG) neurons, we found that L395Q ARRB2 mutant increased POL I activity, inhibited the tumor suppressorp53 (p53) level and caused a decrease in the outgrowth of neurites. Together, our results suggest that the activation of TRPV1 promotes the ARRB2-mediated regulation of ribosomal biogenesis in the nucleolus. The ARRB2-TCOF1-p53 checkpoint signaling pathway might be involved in regulating neurite outgrowth associated with pathological pain conditions.

Targeting PTEN in Colorectal Cancers.

Phosphatase and tensin homolog (PTEN) is a tumour suppressor that represents one of the most common targets for genetic defect in human cancer. PTEN controls an array of physiopathological processes related to cell proliferation, differentiation, DNA/chromosome integrity, apoptosis and invasiveness. PTEN dephosphorylates not only proteins, but also phosphoinositides generated by phosphatidylinositol 3-kinase, thus counteracting the Akt signalling pathway. Interestingly, PTEN can also exert some biological functions independently of its catalytic activity.A feature of colorectal cancers is the relatively low incidence of PTEN mutation or deletion, whereas PTEN downregulation occurs in approximately one third of tumours. PTEN inactivation may be even higher when changes in posttranslational modifications and/or mislocalization of the tumour suppressor are accounted for. Strategies based on pharmacologically-induced restoration of wild-type PTEN function in colon cancer cells could therefore be considered, to impact cell growth, trigger apoptosis, and sensitize tumour cells to therapeutic agents.This review details current knowledge of the mechanisms regulating PTEN expression, activity and function. It also focuses on the use of small molecules targeting positive or negative PTEN regulators and summarizes alternative strategies that could be used to alter PTEN conformation/activity. Finally, we propose an outline of a personalized approach to restore PTEN function in colon cancer cells.

Receptor sequestration in response to ß-arrestin-2 phosphorylation by ERK1/2 governs steady-state levels of GPCR cell-surface expression.

MAPKs are activated in response to G protein-coupled receptor (GPCR) stimulation and play essential roles in regulating cellular processes downstream of these receptors. However, very little is known about the reciprocal effect of MAPK activation on GPCRs. To investigate possible crosstalk between the MAPK and GPCRs, we assessed the effect of ERK1/2 on the activity of several GPCR family members. We found that ERK1/2 activation leads to a reduction in the steady-state cell-surface expression of many GPCRs because of their intracellular sequestration. This subcellular redistribution resulted in a global dampening of cell responsiveness, as illustrated by reduced ligand-mediated G-protein activation and second-messenger generation as well as blunted GPCR kinases and ß-arrestin recruitment. This ERK1/2-mediated regulatory process was observed for GPCRs that can interact with ß-arrestins, such as type-2 vasopressin, type-1 angiotensin, and CXC type-4 chemokine receptors, but not for the prostaglandin F receptor that cannot interact with ß-arrestin, implicating this scaffolding protein in the receptor's subcellular redistribution. Complementation experiments in mouse embryonic fibroblasts lacking ß-arrestins combined with in vitro kinase assays revealed that ß-arrestin-2 phosphorylation on Ser14 and Thr276 is essential for the ERK1/2-promoted GPCR sequestration. This previously unidentified regulatory mechanism was observed after constitutive activation as well as after receptor tyrosine kinase- or GPCR-mediated activation of ERK1/2, suggesting that it is a central node in the tonic regulation of cell responsiveness to GPCR stimulation, acting both as an effector and a negative regulator.

CXCR7 participates in CXCL12-induced CD34+ cell cycling through ß-arrestin-dependent Akt activation.

In addition to its well-known effect on migration and homing of hematopoietic stem/progenitor cells (HSPCs), CXCL12 chemokine also exhibits a cell cycle and survival-promoting factor for human CD34(+) HSPCs. CXCR4 was suggested to be responsible for CXCL12-induced biological effects until the recent discovery of its second receptor, CXCR7. Until now, the participation of CXCR7 in CXCL12-induced HSPC cycling and survival is unknown. We show here that CXCL12 was capable of binding CXCR7 despite its scarce expression at CD34(+) cell surface. Blocking CXCR7 inhibited CXCL12-induced Akt activation as well as the percentage of CD34(+) cells in cycle, colony formation, and survival, demonstrating its participation in CXCL12-induced functional effects in HSPCs. At steady state, CXCR7 and ß-arrestin2 co-localized near the plasma membrane of CD34(+) cells. After CXCL12 treatment, ß-arrestin2 translocated to the nucleus, and this required both CXCR7 and CXCR4. Silencing ß-arrestin expression decreased CXCL12-induced Akt activation in CD34(+) cells. Our results demonstrate for the first time the role of CXCR7, complementary to that played by CXCR4, in the control of HSPC cycling, survival, and colony formation induced by CXCL12. We also provide evidence for the involvement of ß-arrestins as signaling hubs downstream of both CXCL12 receptors in primary human HSPCs.

Targeting spare CC chemokine receptor 5 (CCR5) as a principle to inhibit HIV-1 entry.

CCR5 binds the chemokines CCL3, CCL4, and CCL5 and is the major coreceptor for HIV-1 entry into target cells. Chemokines are supposed to form a natural barrier against human immunodeficiency virus, type 1 (HIV-1) infection. However, we showed that their antiviral activity is limited by CCR5 adopting low-chemokine affinity conformations at the cell surface. Here, we investigated whether a pool of CCR5 that is not stabilized by chemokines could represent a target for inhibiting HIV infection. We exploited the characteristics of the chemokine analog PSC-RANTES (N-α-(n-nonanoyl)-des-Ser(1)-[l-thioprolyl(2), l-cyclohexylglycyl(3)]-RANTES(4-68)), which displays potent anti-HIV-1 activity. We show that native chemokines fail to prevent high-affinity binding of PSC-RANTES, analog-mediated calcium release (in desensitization assays), and analog-mediated CCR5 internalization. These results indicate that a pool of spare CCR5 may bind PSC-RANTES but not native chemokines. Improved recognition of CCR5 by PSC-RANTES may explain why the analog promotes higher amounts of ß-arrestin 2·CCR5 complexes, thereby increasing CCR5 down-regulation and HIV-1 inhibition. Together, these results highlight that spare CCR5, which might permit HIV-1 to escape from chemokines, should be targeted for efficient viral blockade.

Distinct functional outputs of PTEN signalling are controlled by dynamic association with ß-arrestins.

The tumour suppressor PTEN (phosphatase and tensin deleted on chromosome 10) regulates major cellular functions via lipid phosphatase-dependent and -independent mechanisms. Despite its fundamental pathophysiological importance, how PTEN's cellular activity is regulated has only been partially elucidated. We report that the scaffolding proteins ß-arrestins (ß-arrs) are important regulators of PTEN. Downstream of receptor-activated RhoA/ROCK signalling, ß-arrs activate the lipid phosphatase activity of PTEN to negatively regulate Akt and cell proliferation. In contrast, following wound-induced RhoA activation, ß-arrs inhibit the lipid phosphatase-independent anti-migratory effects of PTEN. ß-arrs can thus differentially control distinct functional outputs of PTEN important for cell proliferation and migration.

Arrestins as regulatory hubs in cancer signalling pathways.

Non-visual arrestins were initially appreciated for the roles they play in the negative regulation of G protein-coupled receptors through the processes of desensitisation and endocytosis. The arrestins are also now known as protein scaffolding platforms that act downstream of multiple types of receptors, ensuring relevant transmission of information for an appropriate cellular response. They function as regulatory hubs in several important signalling pathways that are often dysregulated in human cancers. Interestingly, several recent studies have documented changes in expression and localisation of arrestins that occur during cancer progression and that correlate with clinical outcome. Here, we discuss these advances and how changes in expression/localisation may affect functional outputs of arrestins in cancer biology.

Secreted factors from brain endothelial cells maintain glioblastoma stem-like cell expansion through the mTOR pathway.

Glioma stem-cells are associated with the brain vasculature. However, the way in which this vascular niche regulates stem-cell renewal and fate remains unclear. Here, we show that factors emanating from brain endothelial cells positively control the expansion of long-term glioblastoma stem-like cells. We find that both pharmacological inhibition of and RNA interference with the mammalian target of rapamycin (mTOR) pathway reduce their spheroid growth. Conversely, the endothelial secretome is sufficient to promote this mTOR-dependent survival. Thus, interfering with endothelial signals might present opportunities to identify treatments that selectively target malignant stem-cell niches.

Mechanical Activation of the ß2-Adrenergic Receptor by Meningococcus: A Historical and Future Perspective Analysis of How a Bacterial Probe Can Reveal Signalling Pathways in Endothelial Cells, and a Unique Mode of Receptor Activation Involving Its N-Terminal Glycan Chains.

More than 12 years have passed since the seminal observation that meningococcus, a pathogen causing epidemic meningitis in humans, occasionally associated with infectious vasculitis and septic shock, can promote the translocation of ß-arrestins to the cell surface beneath bacterial colonies. The cellular receptor used by the pathogen to induce signalling in host cells and allowing it to open endothelial cell junctions and reach meninges was unknown. The involvement of ß-arrestins, which are scaffolding proteins regulating G protein coupled receptor signalling and function, incited us to specifically investigate this class of receptors. In this perspective article we will summarize the events leading to the discovery that the ß2-adrenergic receptor is the receptor that initiates the signalling cascades induced by meningococcus in host cells. This receptor, however, cannot mediate cell infection on its own. It needs to be pre-associated with an "early" adhesion receptor, CD147, within a hetero-oligomeric complex, stabilized by the cytoskeletal protein α-actinin 4. It then required several years to understand how the pathogen actually activates the signalling receptor. Once bound to the N-terminal glycans of the ß2-adrenergic receptor, meningococcus provides a mechanical stimulation that induces the biased activation of ß-arrestin-mediated signalling pathways. This activating mechanical stimulus can be reproduced in the absence of any pathogen by applying equivalent forces on receptor glycans. Mechanical activation of the ß2-adrenergic receptor might have a physiological role in signalling events promoted in the context of cell-to-cell interaction.

CD4-CCR5 interaction in intracellular compartments contributes to receptor expression at the cell surface.

The association of CD4, a glycoprotein involved in T-cell development and antigen recognition, and CC chemokine receptor 5 (CCR5), a chemotactic G protein-coupled receptor, which regulates trafficking and effector functions of immune cells, forms the main receptor for HIV. We observed that the majority of CCR5 is maintained within the intracellular compartments of primary T lymphocytes and in a monocytic cell line, contrasting with its relatively low density at the cell surface. The CCR5-CD4 association, which occurs in the endoplasmic reticulum, enhanced CCR5 export to the plasma membrane in a concentration-dependent manner, whereas inhibition of endogenous CD4 with small interfering RNAs decreased cell-surface expression of endogenous CCR5. This effect was specific for CCR5, as CD4 did not affect cellular distribution of CXCR4, the other HIV coreceptor. These results reveal a previously unappreciated role of CD4, which contributes to regulating CCR5 export to the plasma membrane.

The RanBP2/RanGAP1-SUMO complex gates ß-arrestin2 nuclear entry to regulate the Mdm2-p53 signaling axis.

Mdm2 antagonizes the tumor suppressor p53. Targeting the Mdm2-p53 interaction represents an attractive approach for the treatment of cancers with functional p53. Investigating mechanisms underlying Mdm2-p53 regulation is therefore important. The scaffold protein ß-arrestin2 (ß-arr2) regulates tumor suppressor p53 by counteracting Mdm2. ß-arr2 nucleocytoplasmic shuttling displaces Mdm2 from the nucleus to the cytoplasm resulting in enhanced p53 signaling. ß-arr2 is constitutively exported from the nucleus, via a nuclear export signal, but mechanisms regulating its nuclear entry are not completely elucidated. ß-arr2 can be SUMOylated, but no information is available on how SUMO may regulate ß-arr2 nucleocytoplasmic shuttling. While we found ß-arr2 SUMOylation to be dispensable for nuclear import, we identified a non-covalent interaction between SUMO and ß-arr2, via a SUMO interaction motif (SIM), that is required for ß-arr2 cytonuclear trafficking. This SIM promotes association of ß-arr2 with the multimolecular RanBP2/RanGAP1-SUMO nucleocytoplasmic transport hub that resides on the cytoplasmic filaments of the nuclear pore complex. Depletion of RanBP2/RanGAP1-SUMO levels result in defective ß-arr2 nuclear entry. Mutation of the SIM inhibits ß-arr2 nuclear import, its ability to delocalize Mdm2 from the nucleus to the cytoplasm and enhanced p53 signaling in lung and breast tumor cell lines. Thus, a ß-arr2 SIM nuclear entry checkpoint, coupled with active ß-arr2 nuclear export, regulates its cytonuclear trafficking function to control the Mdm2-p53 signaling axis.

beta-arrestin 2 oligomerization controls the Mdm2-dependent inhibition of p53.

beta-arrestins (beta-arrs), two ubiquitous proteins involved in serpentine heptahelical receptor regulation and signaling, form constitutive homo- and heterooligomers stabilized by inositol 1,2,3,4,5,6-hexakisphosphate (IP6). Monomeric beta-arrs are believed to interact with receptors after agonist activation, and therefore, beta-arr oligomers have been proposed to represent a resting biologically inactive state. In contrast to this, we report here that the interaction with and subsequent titration out of the nucleus of the protooncogene Mdm2 specifically require beta-arr2 oligomers together with the previously characterized nucleocytoplasmic shuttling of beta-arr2. Mutation of the IP6-binding sites impair oligomerization, reduce interaction with Mdm2, and inhibit p53-dependent antiproliferative effects of beta-arr2, whereas the competence for receptor regulation and signaling is maintained. These observations suggest that the intracellular concentration of beta-arr2 oligomers might control cell survival and proliferation.

Posttranslational Regulation and Conformational Plasticity of PTEN.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a tumor suppressor that is frequently down-modulated in human cancer. PTEN inhibits the phosphatidylinositol 3-phosphate kinase (PI3K)/AKT pathway through its lipid phosphatase activity. Multiple PI3K/AKT-independent actions of PTEN, protein-phosphatase activities and functions within the nucleus have also been described. PTEN, therefore, regulates many cellular processes including cell proliferation, survival, genomic integrity, polarity, migration, and invasion. Even a modest decrease in the functional dose of PTEN may promote cancer development. Understanding the molecular and cellular mechanisms that regulate PTEN protein levels and function, and how these may go awry in cancer contexts, is, therefore, key to fully understanding the role of PTEN in tumorigenesis. Here, we discuss current knowledge on posttranslational control and conformational plasticity of PTEN, as well as therapeutic possibilities toward reestablishment of PTEN tumor-suppressor activity in cancer.

Mechanical GPCR Activation by Traction Forces Exerted on Receptor N-Glycans.

Cells are sensitive to chemical stimulation which is converted into intracellular biochemical signals by the activation of specific receptors. Mechanical stimulations can also induce biochemical responses via the activation of various mechano-sensors. Although principally appreciated for their chemosensory function, G-protein-coupled receptors (GPCRs) may participate in mechano-transduction. They are indirectly activated by the paracrine release of chemical compounds secreted in response to mechanical stimuli, but they might additionally behave as mechano-sensors that are directly stimulated by mechanical forces. Although several studies are consistent with this latter hypothesis, the molecular mechanisms of a potential direct mechanical activation of GPCRs have remained elusive until recently. In particular, investigating the activation of the catecholamine ß2-adrenergic receptor by a pathogen revealed that traction forces directly exerted on the N-terminus of the receptor via N-glycan chains activate specific signaling pathways. These findings open new perspectives in GPCR biology and pharmacology since most GPCRs express N-glycan chains in their N-terminus, which might similarly be involved in the interaction with cell-surface glycan-specific lectins in the context of cell-to-cell mechanical signaling.

An escort for GPCRs: implications for regulation of receptor density at the cell surface.

G-protein-coupled receptors (GPCRs) are dynamically regulated by various mechanisms that tune their response to external stimuli. Modulation of their plasma membrane density, via trafficking between subcellular compartments, constitutes an important process in this context. Substantial information has been accumulated on cellular pathways that remove GPCRs from the cell surface for subsequent degradation or recycling. In comparison, much less is known about the mechanisms controlling trafficking of neo-synthesized GPCRs from intracellular compartments to the cell surface. Although GPCR export to the plasma membrane is commonly considered to mostly implicate the default, unregulated secretory pathway, an increasing number of observations indicate that trafficking to the plasma membrane from the endoplasmic reticulum might be tightly regulated and involve specific protein partners. Moreover, a new paradigm is emerging in some cellular contexts, in which stocks of functional receptors retained within intracellular compartments can be rapidly mobilized to the plasma membrane to maintain sustained physiological responsiveness.

Cooperative regulation of extracellular signal-regulated kinase activation and cell shape change by filamin A and beta-arrestins.

beta-Arrestins (betaarr) are multifunctional adaptor proteins that can act as scaffolds for G protein-coupled receptor activation of mitogen-activated protein kinases (MAPK). Here, we identify the actin-binding and scaffolding protein filamin A (FLNA) as a betaarr-binding partner using Son of sevenless recruitment system screening, a classical yeast two-hybrid system, coimmunoprecipitation analyses, and direct binding in vitro. In FLNA, the betaarr-binding site involves tandem repeat 22 in the carboxyl terminus. betaarr binds FLNA through both its N- and C-terminal domains, indicating the presence of multiple binding sites. We demonstrate that betaarr and FLNA act cooperatively to activate the MAPK extracellular signal-regulated kinase (ERK) downstream of activated muscarinic M1 (M1MR) and angiotensin II type 1a (AT1AR) receptors and provide experimental evidence indicating that this phenomenon is due to the facilitation of betaarr-ERK2 complex formation by FLNA. In Hep2 cells, stimulation of M1MR or AT1AR results in the colocalization of receptor, betaarr, FLNA, and active ERK in membrane ruffles. Reduction of endogenous levels of betaarr or FLNA and a catalytically inactive dominant negative MEK1, which prevents ERK activation, inhibit membrane ruffle formation, indicating the functional requirement for betaarr, FLNA, and active ERK in this process. Our results indicate that betaarr and FLNA cooperate to regulate ERK activation and actin cytoskeleton reorganization.