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 β2-adrenoceptor/β-arrestin signaling pathway in endothelial cells, which ultimately traps β-arrestin-interacting partners, such as the Src tyrosine kinase and junctional proteins, under bacterial colonies. Cytoskeletal reorganization mediated by β-arrestin-activated Src stabilizes bacterial adhesion to endothelial cells, whereas β-arrestin-dependent delocalization of junctional proteins results in anatomical gaps used by bacteria to penetrate into tissues. Activation of β-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.

Glioma stem-cells are associated with the brain vasculature. However, the way in which this vascular niche regulates stemcell 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.

G protein-coupled receptor kinases (GRKs) mediate desensitization of agonist-occupied G protein-coupled receptors (GPCRs). Here we report that GRK5 contains a DNA-binding nuclear localization sequence (NLS) and that its nuclear localization is regulated by GPCR activation, results that suggest potential nuclear functions for GRK5. As assessed by fluorescence confocal microscopy, transfected and endogenous GRK5 is present in the nuclei of HEp2 cells. Mutation of basic residues in the catalytic domain of GRK5 (between amino acids 388 and 395) results in the nuclear exclusion of the mutant enzyme (GRK5 ⌬NLS ), demonstrating that GRK5 contains a functional NLS. The nuclear localization of GRK5 is subject to dynamic regulation. Calcium ionophore treatment or activation of Gq-coupled muscarinic-M3 receptors promotes the nuclear export of the kinase in a Ca 2؉ /calmodulin (Ca 2؉ /CaM)-dependent fashion. Ca 2؉ /CaM binding to the N-terminal CaM binding site of GRK5 mediates this effect. Furthermore, GRK5, but not GRK5 ⌬NLS or GRK2, binds specifically and directly to DNA in vitro. Consistent with their presence in the nuclei of transfected cells, all the GRK4, but not GRK2, subfamily members contain putative NLSs. These results suggest that the GRK4 subfamily of GRKs may play a signaling role in the nucleus and that GRK4 and GRK2 subfamily members perform divergent cellular functions.G protein-coupled receptor kinases (GRKs) comprise a family of seven serine/threonine protein kinases that phosphorylate agonist-bound, activated, G protein-coupled receptors (GPCRs). GRK-mediated GPCR phosphorylation initiates ␤-arrestin binding, receptor uncoupling from the G protein, and targeting of the ␤-arrestin-receptor complex to a clathrin-coated pit for internalization. The receptor may then be degraded or returned to the cell surface for a further round of signaling (reviewed in reference 4).Seven mammalian GRKs have been identified, which are divided into three subfamilies on the basis of sequence homology and the regulatory mechanisms controlling their activity (reviewed in reference 24): the GRK1-like subfamily, GRK1 (or rhodopsin kinase) and GRK7; the GRK2-like subfamily, GRK2 (␤-adrenergic kinase) and GRK3 (␤-adrenergic kinase 2); and the GRK4-like subfamily, GRK4, GRK5, and GRK6. Four splice variants of GRK4 (␣, ␤, ␥, and ␦) and three splice variants of GRK6 (A, B, and C) have been identified (24).GRKs are regulated by several mechanisms, including modulation of their subcellular localization, kinase activity, and expression (reviewed in reference 21). In many instances, regulation of GRK activity is subfamily specific. PKC phosphorylation results in activation of GRK2 but inhibition of GRK5 activity (21). Additionally, the three GRK subfamilies display differential affinities for calcium binding proteins. GRK1 binds Ca 2ϩ /recoverin, and GRK4␣, GRK5, and GRK6A, -B and -C bind Ca 2ϩ /calmodulin (Ca 2ϩ /CaM) with high affinity (reviewed in reference 32). In contrast, GRK2 exhibits an approximately 40-fold lower affinity for C...

GABA B receptors are heterodimeric G protein-coupled receptors that mediate slow synaptic inhibition in the central nervous system. The dynamic control of the cell surface stability of GABA B receptors is likely to be of fundamental importance in the modulation of receptor signaling. Presently, however, this process is poorly understood. Here we demonstrate that GABA B receptors are remarkably stable at the plasma membrane showing little basal endocytosis in cultured cortical and hippocampal neurons. In addition, we show that exposure to baclofen, a well characterized GABA B receptor agonist, fails to enhance GABA B receptor endocytosis. Lack of receptor internalization in neurons correlates with an absence of agonist-induced phosphorylation and lack of arrestin recruitment in heterologous systems. We also demonstrate that chronic exposure to baclofen selectively promotes endocytosis-independent GABA B receptor degradation. The effect of baclofen can be attenuated by activation of cAMP-dependent protein kinase or co-stimulation of ␤-adrenergic receptors. Furthermore, we show that increased degradation rates are correlated with reduced receptor phosphorylation at serine 892 in GABA B R2. Our results support a model in which GABA B R2 phosphorylation specifically stabilizes surface GABA B receptors in neurons. We propose that signaling pathways that regulate cAMP levels in neurons may have profound effects on the tonic synaptic inhibition by modulating the availability of GABA B receptors. G protein-coupled receptors (GPCRs)1 mediate responses to a wide variety of stimuli such as light, odorants, hormones, and neurotransmitters, and can be divided into three families (A, B, and C) on the basis of their sequence and structural similarity (1). Termination of GPCR signaling is a key process that defines the overall properties of the response to a particular stimulus and extensive observations have allowed the proposal of a conserved series of events, divided into three distinct stages, that take place during receptor inactivation (2). In the first stage, agonist occupancy triggers GPCR phosphorylation by G protein-coupled receptor kinases (GRKs) causing desensitization over a time scale of seconds (3). In the second, phosphorylation results in the binding to proteins of the arrestin family, which mediate recruitment of GPCRs to clathrin-coated vesicles, subsequent internalization, and sorting to endosomes or lysosomes after seconds or minutes of stimulation (4). Finally, prolonged agonist exposure causes down-regulation, a phenomenon defined as the reduction in the total number of receptors and usually correlated with reduced receptor mRNA (5).GABA B receptors belong to family C and unlike other GPCRs they require the formation of a heterodimer composed of two subunits, namely GABA B R1 and GABA B R2 (6). Both subunits display high homology to metabotropic glutamate, Ca 2ϩ -sensing, vomeronasal, and putative pheromone receptors (7). Recombinant heterodimeric GABA B R1/GABA B R2 receptors reproduce most of the charact...

␤-arrestins (␤arrs) are two highly homologous proteins that uncouple G protein-coupled receptors from their cognate G proteins, serve as adaptor molecules linking G protein-coupled receptors to clathrin-coat components (AP-2 complex and clathrin), and act as scaffolding proteins for ERK1/2 and JNK3 cascades. A striking difference between the two ␤arrs (␤arr1 and ␤arr2) is that ␤arr1 is evenly distributed throughout the cell, whereas ␤arr2 shows an apparent cytoplasmic localization at steady state. Here, we investigate the molecular determinants underlying this differential distribution. ␤arr2 is constitutively excluded from the nucleus by a leptomycin B-sensitive pathway because of the presence of a classical leucine-rich nuclear export signal in its C terminus (L395/L397) that is absent in ␤arr1. In addition, using a nuclear import assay in yeast we showed that ␤arr2 is actively imported into the nucleus, suggesting that ␤arr2 undergoes constitutive nucleocytoplasmic shuttling. In cells expressing ␤arr2, JNK3 is mostly cytosolic. A point mutation of the nuclear export signal (L395A) in ␤arr2, which was sufficient to redistribute ␤arr2 from the cytosol to the nucleus, also caused the nuclear relocalization of JNK3. These data indicate that the nucleocytoplasmic shuttling of ␤arr2 controls the subcellular distribution of JNK3.

Arrestins are important proteins, which regulate the function of serpentine heptahelical receptors and contribute to multiple signaling pathways downstream of receptors. The ubiquitous ␤-arrestins are believed to function exclusively as monomers, although selfassociation is assumed to control the activity of visual arrestin in the retina, where this isoform is particularly abundant. Here the oligomerization status of ␤-arrestins was investigated using different approaches, including co-immunoprecipitation of epitope-tagged ␤-arrestins and resonance energy transfer (BRET and FRET) in living cells. At steady state and at physiological concentrations, ␤-arrestins constitutively form both homo-and hetero-oligomers. Co-expression of ␤-arrestin2 and ␤-arrestin1 prevented ␤-arrestin1 accumulation into the nucleus, suggesting that hetero-oligomerization may have functional consequences. Our data clearly indicate that ␤-arrestins can exist as homo-and hetero-oligomers in living cells and raise the hypothesis that the oligomeric state may regulate their subcellular distribution and functions.Arrestins play a central role in the regulation and signaling of serpentine heptahelical G protein-coupled receptors (GPCRs).5 Arrestin 1 and 4 are restricted to retinal rods and cones where they regulate rhodopsin (1). In contrast, arrestin 2 and 3, also referred to as ␤-arrestin 1 (␤arr1) and 2 (␤arr2), respectively, are ubiquitous and translocate to a large variety of ligand-activated GPCRs. Originally identified as negative regulators of GPCR function, promoting desensitization (2), ␤arrs were subsequently shown to be adaptor proteins connecting GPCRs to the endocytic machinery (3, 4). ␤arrs also serve as signaling scaffolds linking receptors to a growing number of effector pathways (5). For example, ␤arrs act as scaffolds for the activation of ERK and JNK3 (5). In addition, ␤arr2 redistributes the ubiquitin ligase Mdm2 and the kinase JNK3 from the nucleus to the cytoplasm, a property related to the presence of a leucine-rich nuclear export signal (NES) in ␤arr2 (6, 7). This signal is absent from ␤arr1, determining some differences in both subcellular distribution and functional roles between the two isoforms (6 -8).Crystal structures of visual arrestin (9, 10) revealed that this molecule contains two globular domains and an extended COOH-terminal tail locking the molecule into an inactive state. Upon binding to receptors, the arrestin C-tail is released, leading to an open active conformation (11). In crystals, visual arrestin is a tetramer composed of two asymmetric dimers (9, 10). In vitro experiments showed that, in solution, tetramers are in equilibrium with monomers at physiological concentrations (12, 13), and it was proposed that self-association might regulate arrestin activity by limiting availability of active monomeric species (13). The crystal structure of ␤arr1 is very similar to that of visual arrestin, but unlike visual arrestin, full-length ␤arr1 was found to be monomeric (14). In addition, because of their lower ...

G-protein-coupled receptors (GPCRs

␤-Arrestins (␤arr) 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 ␤arr-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 ␤arr-binding site involves tandem repeat 22 in the carboxyl terminus. ␤arr binds FLNA through both its N-and C-terminal domains, indicating the presence of multiple binding sites. We demonstrate that ␤arr 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 ␤arr-ERK2 complex formation by FLNA. In Hep2 cells, stimulation of M1MR or AT1AR results in the colocalization of receptor, ␤arr, FLNA, and active ERK in membrane ruffles. Reduction of endogenous levels of ␤arr or FLNA and a catalytically inactive dominant negative MEK1, which prevents ERK activation, inhibit membrane ruffle formation, indicating the functional requirement for ␤arr, FLNA, and active ERK in this process. Our results indicate that ␤arr and FLNA cooperate to regulate ERK activation and actin cytoskeleton reorganization.Cytoskeletal reorganization is fundamental for cell shape change, signaling, locomotion, and many other important dynamic cellular processes. The MAPK JNK, p38, and ERK play key roles in the regulation of cytoskeletal dynamics (21), as many extracellular signals that promote a change in cell shape converge at MAPK, which in turn phosphorylate downstream targets involved in actin cytoskeleton regulation. For example, ERK can phosphorylate myosin light chain kinase (27, 37), calpain (18), focal adhesion kinase (22), and ribosomal S6 kinase (15), which all play important roles in cytoskeletal reorganization and cell migration.Initially appreciated for their roles in G protein-coupled receptor desensitization and endocytosis, ␤-arrestins are now considered multifunctional adaptor molecules, with over 20 binding partners, including trafficking proteins, nonreceptor tyrosine kinases, guanine nucleotide exchange factors, and MAPK components, identified to date (28, 29). There exist two isoforms of ␤arr, ␤arr1 and ␤arr2. These share a high degree of homology (ϳ80%) and several biological functions, such as their abilities to bind to agonist-occupied GPCRs, preventing G protein-mediated signal transduction, and to bridge receptors with components of the clathrin-dependent endocytic machinery (clathrin and AP-2). With regard to MAPK, ␤arr can act as scaffolds for GPCR-mediated activation of ERK1/2 (12, 33), JNK3 (35), and p38 (44). ␤arr bind multiple components of MAPK cascades to promote efficient activation of MAPK, additionally redirecting them to extranuclear compartments (12,33,35). The ...