Stress and anxiety disorders are risk factors for depression and these behaviours are modulated by corticotropin releasing factor (CRFR1) and serotonin (5-HT2R) receptors. However, the potential behavioral and cellular interaction between these two receptors is unclear. Here, we showed that pre-administration of CRF into the prefrontal cortex of mice sensitized 5-HT2R-mediated anxiety behaviours in response to 2,5-dimethoxy-4-iodoamphetamine. In both heterologous cell cultures and mouse cortical neurons, the activation of CRFR1 also sensitized 5-HT2 receptor-mediated inositol phosphate formation. CRFR1-mediated increases in 5-HT2R signaling were dependent upon receptor internalization and receptor recycling via rapid recycling endosomes resulting in increased cell surface 5-HT2R expression. The sensitization of 5-HT2R signaling by CRFR1 required intact PDZ domain binding motifs at the end of the C-terminal tails of both receptor types. These data reveal a novel mechanism by which CRF, a peptide known to be released by stress, sensitized anxiety-related behaviour via sensitization of 5-HT2R signaling.

Oxidative stress has been implicated as a key trigger of neuronal apoptosis in stroke and neurodegenerative conditions such as Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis. The Bcl-2 homology 3 (BH3)-only subfamily of Bcl-2 genes consists of multiple members that can be activated in a cell-type-and stimulus-specific manner to promote cell death. In the present study, we demonstrate that, in cortical neurons, oxidative stress induces the expression of the BH3-only members Bim, Noxa, and Puma. Importantly, we have determined that Puma؊/؊ neurons, but not Bim؊/؊ or Noxa؊/؊ neurons, are remarkably resistant to the induction of apoptosis by multiple oxidative stressors. Furthermore, we have determined that Bcl-2-associated X protein (Bax) is also required for oxidative stress induced cell death and that Puma plays a dominant role in regulating Bax activation. Specifically, we have established that the induction of Puma, but not Bim or Noxa, is necessary and sufficient to induce a conformational change in Bax to its active state, its translocation to the mitochondria and mitochondrial membrane permeabilization. Finally, we demonstrate that whereas both Puma and Bim EL can bind to the antiapoptotic family member Bcl-X L , only Puma was found to associate with Bax. This suggests that in addition to neutralizing antiapoptotic members, Puma may play a dominant role by complexing with Bax and directly promoting its activation. Overall, we have identified Puma as a dominant regulator of oxidative stress induced Bax activation and neuronal apoptosis, and suggest that Puma may be an effective therapeutic target for the treatment of a number of neurodegenerative conditions.

No abstract

beta-Arrestins are important in chemoattractant receptor-induced granule release, a process that may involve Ral-dependent regulation of the actin cytoskeleton. We have identified the Ral GDP dissociation stimulator (Ral-GDS) as a beta-arrestin-binding protein by yeast two-hybrid screening and co-immunoprecipitation from human polymorphonuclear neutrophilic leukocytes (PMNs). Under basal conditions, Ral-GDS is localized to the cytosol and remains inactive in a complex formed with beta-arrestins. In response to formyl-Met-Leu-Phe (fMLP) receptor stimulation, beta-arrestin Ral-GDS protein complexes dissociate and Ral-GDS translocates with beta-arrestin from the cytosol to the plasma membrane, resulting in the Ras-independent activation of the Ral effector pathway required for cytoskeletal rearrangement. The subsequent re-association of beta-arrestin Ral-GDS complexes is associated with the inactivation of Ral signalling. Thus, beta-arrestins regulate multiple steps in the Ral-dependent processes that result in chemoattractant-induced cytoskeletal reorganization.

Metabotropic glutamate receptors (mGluRs) constitute a unique subclass of G protein-coupled receptors (GPCRs) that bear little sequence homology to other members of the GPCR superfamily. The mGluR subtypes that are coupled to the hydrolysis of phosphoinositide contribute to both synaptic plasticity and glutamatemediated excitotoxicity in neurons. In the present study, the expression of mGluR1a in HEK 293 cells led to agonist-independent cell death. Since G protein-coupled receptor kinases (GRKs) desensitize a diverse variety of GPCRs, we explored whether GRKs contributed to the regulation of both constitutive and agonist-stimulated mGluR1a activity and thereby may prevent mGluR1a-mediated excitotoxicity associated with mGluR1a overactivation. We find that the co-expression of mGluR1a with GRK2 and GRK5, but not GRK4 and GRK6, reduced both constitutive and agonist-stimulated mGluR1a activity. Agonist-stimulated mGluR1a phosphorylation was enhanced by the co-expression of GRK2 and was blocked by two different GRK2 dominant-negative mutants. Furthermore, GRK2-dependent mGluR1a desensitization protected against mGluR1a-mediated cell death, at least in part by blocking mGluR1a-stimulated apoptosis. Our data indicate that as with other members of the GPCR superfamily, a member of the structurally distinct mGluR family (mGluR1a) serves as a substrate for GRK-mediated phosphorylation and that GRK-dependent "feedback" modulation of mGluR1a responsiveness protects against pathophysiological mGluR1a signaling.Glutamate is the major excitatory neurotransmitter in the central nervous system and is essential in the regulation of brain functions and neural cell development (1, 2). Receptors that respond to glutamate are classified into two types, ionotropic and metabotropic. The ionotropic glutamate receptors are cation-specific ion channels that mediate fast excitatory glutamate responses and are subdivided into ␣-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/kainate and NMDA 1 receptors (1, 2). In contrast, metabotropic glutamate receptors (mGluRs) are members of the G protein-coupled receptor (GPCR) superfamily and mediate slower glutamate responses by coupling to various second messenger cascades via heterotrimeric G proteins (1, 2). This property allows mGluRs to translate relatively short neuronal activation into long lasting changes in synaptic activity. As a consequence, mGluR signaling plays an important role in the processes underlying synaptic plasticity (e.g. memory and learning) (3).The mGluR family of receptors constitute a unique subclass of GPCRs that bear no sequence or structural homology to prototypic Class 1 (rhodopsin, ␤ 2 -adrenergic family) and Class 2 (secretin family) GPCRs other than the retention of the seven transmembrane spanning domain topology that is characteristic of a GPCR (4). Furthermore, unlike other GPCR family members, mGluRs couple to heterotrimeric G proteins via the second intracellular loop domain of the receptor rather than the third intracellular loop domain (5). The mGl...

Metabotropic glutamate receptors (mGluRs) are G protein-coupled receptors (GPCRs) that contribute to the regulation of integrative brain functions such as cognition, motor control, and neural development. Metabotropic glutamate receptors are members of a unique class of GPCRs (class III) that include the calcium sensing and gamma-aminobutyric acid type B receptors. Although mGluRs bear little sequence homology to well-characterized members of the GPCR superfamily, both second messenger-dependent protein kinases and G protein-coupled receptor kinases (GRKs) contribute to mGluR desensitization. Therefore, in the present study, we examined whether beta-arrestins, regulators of GPCR desensitization and endocytosis, are required for mGluR1a desensitization and internalization in human embryonic kidney (HEK) 293 cells. Unlike what has been reported for other GPCRs, we find that in response to agonist stimulation, mGluR1a internalization is selectively mediated by beta-arrestin1 in HEK 293 cells. However, even though beta-arrestin1 binds directly to the carboxyl-terminal tail of mGluR1a and redistributes with mGluR1a to endosomes, neither beta-arrestin1 nor beta-arrestin2 seems to contribute to mGluR1a desensitization in HEK 293 cells. We also observed extensive tonic mGluR1a internalization via clathrin-coated vesicles in the absence of agonist. The tonic internalization of mGluR1a is insensitive to antagonist treatment, dominant-negative mutants of GRK2, beta-arrestin1, and dynamin as well as treatments that disrupt caveolae, but is blocked by hypertonic sucrose and concanavalin A treatment. Internalized mGluR1a is colocalized with clathrin, transferrin receptor, beta2-adrenergic receptor, and Rab5 GTPase in endocytic vesicles. Therefore, although mGluR1a internalizes with beta-arrestin in response to agonist, the agonist-independent internalization of mGluR1a involves the beta-arrestin-independent targeting of mGluR1a to clathrin-coated vesicles.

The corticotropin releasing factor (CRF) type 1a receptor, a member of the G protein-coupled receptor (GPCR) subfamily B, is involved in the aetiology of anxiety and depressive disorders. In the present study, we examined the internalization and trafficking of the CRF1a receptor in both human embryonic kidney (HEK)293 cells and primary cortical neurons. We found that CRF1a receptor activation leads to the selective recruitment of b-arrestin2 in both HEK293 cells and neurons. We observed distinct distribution patterns of CRF1a receptor and b-arrestin2 in HEK293 cells and cortical neurons. In HEK293 cells, b-arrestin2-green fluorescent protein (GFP) co-localized with CRF1a receptor in vesicles at the plasma membrane but was dissociated from the receptor in endosomes. In contrast, in primary cortical neurons, b-arrestin2 and CRF1a receptor were internalized in distinct endocytic vesicles. By bioluminescence resonance energy transfer, we demonstrated that b-arrestin2 association with CRF1a receptor was increased in cells transfected with G proteincoupled receptor kinase (GRK)3 and GRK6 and decreased in cells transfected with GRK2 and GRK5. In both HEK293 cells and cortical neurons, internalized CRF1a receptor transited from Rab5-positive early endosomes to Rab4-positive recycling endosomes and was not targeted to lysosomes. However, CRF1a receptor resensitization was blocked by the overexpression of wild-type, but not dominant-negative, Rab5 and Rab4 GTPases. Taken together, our results suggest that b-arrestin trafficking differs between HEK293 cells and neurons, and that CRF1a receptor resensitization is regulated in an atypical manner by Rab GTPases.

Previous studies have demonstrated that the interaction of the angiotensin II type 1A receptor (AT 1A R) carboxyl-terminal tail with Rab5a may modulate Rab5a activity, leading to the homotypic fusion of endocytic vesicles. Therefore, we have investigated whether AT 1A R/Rab5a interactions mediate the retention of AT 1A R⅐␤-arrestin complexes in early endosomes and whether the overexpression of Rab7 and Rab11 GTPases influences AT 1A R lysosomal degradation and plasma membrane recycling. We found that internalized AT 1A R was retained in Rab5a-positive early endosomes and was neither targeted to lysosomes nor recycled back to the cell surface, whereas a mutant defective in Rab5a binding, AT 1A R-(1-349), was targeted to lysosomes for degradation. However, the loss of Rab5a binding to the AT 1A R carboxyl-terminal tail did not promote AT 1A R recycling. Rather, it was the stable binding of ␤-arrestin to the AT 1A R that prevented, at least in part, AT 1A R recycling. The overexpression of wild-type Rab7 and Rab7-Q67L resulted in both increased AT 1A R degradation and AT 1A R targeting to lysosomes. The Rab7 expression-dependent transition of "putative" AT 1A R⅐␤-arrestin complexes to late endosomes was blocked by the expression of dominant-negative Rab5a-S34N. Rab11 overexpression established AT 1A R recycling and promoted the redistribution of AT 1A R⅐␤-arrestin complexes from early to recycling endosomes. Taken together, our data suggest that Rab5, Rab7, and Rab11 work in concert with one another to regulate the intracellular trafficking patterns of the AT 1A R.The angiotensin II type 1A receptor (AT 1A R) 1 is a member of the G protein-coupled receptor (GPCR) superfamily, the largest family of integral membrane receptor proteins. The AT 1A R is coupled via G q to the stimulation of phospholipase C␤, leading to increases in intracellular diacylglycerol and inositol 1,4,5-triphosphate and the release of calcium from intracellular stores (1). Agonist activation of the AT 1A R also leads to the desensitization of AT 1A R second messenger responses and the removal of cell-surface AT 1A R into the intracellular compartment of the cell (2-4). The agonist-stimulated desensitization and endocytosis of many GPCRs is initiated by GPCR kinasemediated phosphorylation, followed by ␤-arrestin binding (5). Both ␤-arrestin-dependent and ␤-arrestin-independent mechanisms of AT 1A