Convergence of Melatonin and Serotonin (5-HT) Signaling at MT2/5-HT2C Receptor Heteromers

Inasmuch as the neurohormone melatonin is synthetically derived from serotonin (5-HT), a close interrelationship between both has long been suspected. The present study reveals a hitherto unrecognized cross-talk mediated via physical association of melatonin MT₂ and 5-HT_2C receptors into functional heteromers. This is of particular interest in light of the “synergistic” melatonin agonist/5-HT_2C antagonist profile of the novel antidepressant agomelatine. A suite of co-immunoprecipitation, bioluminescence resonance energy transfer, and pharmacological techniques was exploited to demonstrate formation of functional MT₂ and 5-HT_2C receptor heteromers both in transfected cells and in human cortex and hippocampus. MT₂/5-HT_2C heteromers amplified the 5-HT-mediated G_q/phospholipase C response and triggered melatonin-induced unidirectional transactivation of the 5-HT_2C protomer of MT₂/5-HT_2C heteromers. Pharmacological studies revealed distinct functional properties for agomelatine, which shows “biased signaling.” These observations demonstrate the existence of functionally unique MT₂/5-HT_2C heteromers and suggest that the antidepressant agomelatine has a distinctive profile at these sites potentially involved in its therapeutic effects on major depression and generalized anxiety disorder. Finally, MT₂/5-HT_2C heteromers provide a new strategy for the discovery of novel agents for the treatment of psychiatric disorders.     

Functional expression of 5-HT2A receptor in osteoblastic MC3T3-E1 cells

In the previous study, we reported the gene expression for proteins related to the function of 5-hydroxytryptamine (5-HT, serotonin) and elucidated the expression patterns of 5-HT₂ receptor subtypes in mouse osteoblasts. In the present study, we evaluated the possible involvement of 5-HT receptor subtypes and its inactivation system in MC3T3-E1 cells, an osteoblast cell line. DOI, a 5-HT_2A and 5-HT_2C receptor selective agonist, as well as 5-HT concentration-dependently increased proliferative activities of MC3T3-E1 cells in their premature period. This effect of 5-HT on cell proliferation were inhibited by ketanserin, a 5-HT_2A receptor specific antagonist. Moreover, both DOI-induced cell proliferation and phosphorylation of ERK1 and 2 proteins were inhibited by PD98059 and U0126, selective inhibitors of MEK in a concentration-dependent manner. Furthermore, treatment with fluoxetine, a 5-HT specific re-uptake inhibitor which inactivate the function of extracellular 5-HT, significantly increased the proliferative activities of MC3T3-E1 cells in a concentration-dependent manner. Our data indicate that 5-HT fill the role for proliferation of osteoblast cells in their premature period. Notably, 5-HT_2A receptor may be functionally expressed to regulate mechanisms underlying osteoblast cell proliferation, at least in part, through activation of ERK/MAPK pathways in MC3T3-E1 cells.     

G-protein Receptor Kinase 5 Regulates the Cannabinoid Receptor 2-induced Up-regulation of Serotonin 2A Receptors

We have recently reported that cannabinoid agonists can up-regulate and enhance the activity of serotonin 2A (5-HT_2A) receptors in the prefrontal cortex (PFCx). Increased expression and activity of cortical 5-HT_2A receptors has been associated with neuropsychiatric disorders, such as anxiety and schizophrenia. Here we report that repeated CP55940 exposure selectively up-regulates GRK5 proteins in rat PFCx and in a neuronal cell culture model. We sought to examine the mechanism underlying the regulation of GRK5 and to identify the role of GRK5 in the cannabinoid agonist-induced up-regulation and enhanced activity of 5-HT_2A receptors. Interestingly, we found that cannabinoid agonist-induced up-regulation of GRK5 involves CB₂ receptors, β-arrestin 2, and ERK1/2 signaling because treatment with CB₂ shRNA lentiviral particles, β-arrestin 2 shRNA lentiviral particles, or ERK1/2 inhibitor prevented the cannabinoid agonist-induced up-regulation of GRK5. Most importantly, we found that GRK5 shRNA lentiviral particle treatment prevented the cannabinoid agonist-induced up-regulation and enhanced 5-HT_2A receptor-mediated calcium release. Repeated cannabinoid exposure was also associated with enhanced phosphorylation of CB₂ receptors and increased interaction between β-arrestin 2 and ERK1/2. These latter phenomena were also significantly inhibited by GRK5 shRNA lentiviral treatment. Our results suggest that sustained activation of CB₂ receptors, which up-regulates 5-HT_2A receptor signaling, enhances GRK5 expression; the phosphorylation of CB₂ receptors; and the β-arrestin 2/ERK interactions. These data could provide a rationale for some of the adverse effects associated with repeated cannabinoid agonist exposure.     

Molecular and Pharmacological Characterization of Serotonin 5-HT2α and 5-HT7 Receptors in the Salivary Glands of the Blowfly Calliphora vicina

Secretion in blowfly (Calliphora vicina) salivary glands is stimulated by the biogenic amine serotonin (5-hydroxytryptamine, 5-HT), which activates both inositol 1,4,5-trisphosphate (InsP₃)/Ca²⁺ and cyclic adenosine 3′,5′-monophosphate (cAMP) signalling pathways in the secretory cells. In order to characterize the signal-inducing 5-HT receptors, we cloned two cDNAs (Cv5-ht2α, Cv5-ht7) that share high similarity with mammalian 5-HT₂ and 5-HT₇ receptor genes, respectively. RT-PCR demonstrated that both receptors are expressed in the salivary glands and brain. Stimulation of Cv5-ht2α-transfected mammalian cells with 5-HT elevates cytosolic [Ca²⁺] in a dose-dependent manner (EC₅₀ = 24 nM). In Cv5-ht7-transfected cells, 5-HT produces a dose-dependent increase in [cAMP]_i (EC₅₀ = 4 nM). We studied the pharmacological profile for both receptors. Substances that appear to act as specific ligands of either Cv5-HT_2α or Cv5-HT₇ in the heterologous expression system were also tested in intact blowfly salivary gland preparations. We observed that 5-methoxytryptamine (100 nM) activates only the Cv5-HT_2α receptor, 5-carboxamidotryptamine (300 nM) activates only the Cv5-HT₇ receptor, and clozapine (1 µM) antagonizes the effects of 5-HT via Cv5-HT₇ in blowfly salivary glands, providing means for the selective activation of each of the two 5-HT receptor subtypes. This study represents the first comprehensive molecular and pharmacological characterization of two 5-HT receptors in the blowfly and permits the analysis of the physiological role of these receptors, even when co-expressed in cells, and of the modes of interaction between the Ca²⁺- and cAMP-signalling cascades.     

Serotonin 5-HT3 Receptor-Mediated Vomiting Occurs via the Activation of Ca2+/CaMKII-Dependent ERK1/2 Signaling in the Least Shrew (Cryptotis parva)

Stimulation of 5-HT₃ receptors (5-HT₃Rs) by 2-methylserotonin (2-Me-5-HT), a selective 5-HT₃ receptor agonist, can induce vomiting. However, downstream signaling pathways for the induced emesis remain unknown. The 5-HT₃R channel has high permeability to extracellular calcium (Ca²⁺) and upon stimulation allows increased Ca²⁺ influx. We examined the contribution of Ca²⁺/calmodulin-dependent protein kinase IIα (Ca²⁺/CaMKIIα), interaction of 5-HT₃R with calmodulin, and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling to 2-Me-5-HT-induced emesis in the least shrew. Using fluo-4 AM dye, we found that 2-Me-5-HT augments intracellular Ca²⁺ levels in brainstem slices and that the selective 5-HT₃R antagonist palonosetron, can abolish the induced Ca²⁺ signaling. Pre-treatment of shrews with either: i) amlodipine, an antagonist of L-type Ca²⁺ channels present on the cell membrane; ii) dantrolene, an inhibitor of ryanodine receptors (RyRs) Ca²⁺-release channels located on the endoplasmic reticulum (ER); iii) a combination of their less-effective doses; or iv) inhibitors of CaMKII (KN93) and ERK1/2 (PD98059); dose-dependently suppressed emesis caused by 2-Me-5-HT. Administration of 2-Me-5-HT also significantly: i) enhanced the interaction of 5-HT₃R with calmodulin in the brainstem as revealed by immunoprecipitation, as well as their colocalization in the area postrema (brainstem) and small intestine by immunohistochemistry; and ii) activated CaMKIIα in brainstem and in isolated enterochromaffin cells of the small intestine as shown by Western blot and immunocytochemistry. These effects were suppressed by palonosetron. 2-Me-5-HT also activated ERK1/2 in brainstem, which was abrogated by palonosetron, KN93, PD98059, amlodipine, dantrolene, or a combination of amlodipine plus dantrolene. However, blockade of ER inositol-1, 4, 5-triphosphate receptors by 2-APB, had no significant effect on the discussed behavioral and biochemical parameters. This study demonstrates that Ca²⁺ mobilization via extracellular Ca²⁺ influx through 5-HT₃Rs/L-type Ca²⁺ channels, and intracellular Ca²⁺ release via RyRs on ER, initiate Ca²⁺-dependent sequential activation of CaMKIIα and ERK1/2, which contribute to the 5-HT₃R-mediated, 2-Me-5-HT-evoked emesis.     

5-HT1A receptors transactivate the platelet-derived growth factor receptor type beta in neuronal cells

In the absence of ligand, certain growth factor receptors can be activated via G-protein coupled receptor (GPCR) activation in a process termed transactivation. Serotonin (5-HT) receptors can transactivate platelet-derived growth factor (PDGF) β receptors in smooth muscle cells, but it is not known if similar pathways occur in neuronal cells. Here we show that 5-HT can transiently increase the phosphorylation of PDGFβ receptors through 5-HT_1A receptors in a time- and dose-dependent manner in SH-SY5Y neuroblastoma cells. 5-HT also transactivates PDGFβ receptors in primary cortical neurons. This transactivation pathway is pertussis-toxin sensitive and Src tyrosine kinase-dependent. This pathway is also dependent on phospholipase C activity and intracellular calcium signaling. Several studies involving PDGFβ receptor transactivation by GPCRs have also demonstrated a PDGFβ receptor-dependent increase in the phosphorylation of ERK1/2. Yet in SH-SY5Y cells, 5-HT treatment causes a PDGFβ receptor-independent increase in ERK1/2 phosphorylation. This crosstalk between 5-HT and PDGFβ receptors identifies a potentially important signaling link between the serotonergic system and growth factor signaling in neurons.     

MAGI proteins can differentially regulate the signaling pathways of 5-HT2AR by enhancing receptor trafficking and PLC recruitment

MAGI proteins are Membrane-Associated Guanylate Kinase Inverted proteins that belong to the MAGUK family. They are scaffolding proteins that were shown to mediate the trafficking and signaling of various G protein-coupled receptors (GPCRs). They contain PDZ domains in their structure and many GPCRs interact with these proteins via the PDZ motifs on the carboxyl terminal end of a receptor. In a PDZ overlay assay performed with the carboxyl terminal tail of 5-HT_2AR, we were able to detect all three members of the MAGI subfamily, MAGI-1, MAGI-2 and MAGI-3 as interacting PDZ proteins. The PDZ motif of 5-HT_2AR consists of three amino acids; serine (S), cysteine (C) and valine (V). In this study, we characterize these 5-HT_2AR interactions with MAGI proteins. We first confirm the interaction using co-immunopricipitation and illustrate that the interaction is PDZ motif-dependent in human embryonic kidney (HEK 293) cells. We then assess the effects of overexpression and knockdown of the MAGI proteins on the internalization, trafficking and signaling of 5-HT_2AR. We find that knockdown of either MAGI-1 or MAGI-3 using siRNA results in a significant reduction in the internalization of 5-HT_2AR. As for signaling, we report here that MAGI proteins can modulate the signaling via the two transduction pathways that 5-HT_2AR can activate. We illustrate a significant effect of modulating MAGI proteins expression on 5-HT-stimulated IP formation. We demonstrate an enhancement in 5-HT_2AR-stimulated IP formation upon MAGI proteins overexpression. In addition, we show that knockdown of MAGI proteins with siRNA leads to a significant reduction in 5-HT_2AR-stimulated IP formation. Furthermore, we illustrate a significant increase in 5-HT-stimulated ERK1/2 phosphorylation upon MAGI proteins knockdown. Interestingly, this effect on ERK1/2 activation is PDZ motif-independent. We also suggest two possible mechanisms of regulation for the effect of MAGI proteins on 5-HT_2AR function. One mechanism involves the regulation of cell surface expression since we show that both MAGI-2 and MAGI-3 can enhance receptor trafficking to the plasma membrane when overexpressed in HEK 293 cells. The other mechanism points to regulation of second messengers in the signaling pathways. Specifically, we show that overexpression of any of the three MAGI proteins can enhance the recruitment of PLCβ3 to 5-HT_2AR. In addition, we report a negative effect for knocking down MAGI-3 on β-arrestin recruitment to the receptor and this effect is PDZ motif-independent. Taken together, our findings document distinct roles for the three MAGI proteins in regulating 5-HT_2AR trafficking and signaling and emphasize the importance of studying PDZ proteins and their interactions with GPCRs to regulate their function.     

Neuronal 5-HT metabotropic receptors: fine-tuning of their structure, signaling, and roles in synaptic modulation

Serotonin (5-hydroxytryptamine, 5-HT) is, without doubt, the neurotransmitter for which the number of receptors is the highest. Fifteen genes encoding functional 5-HT receptors have been cloned in mammalian brain. 5-HT₃ receptors are ionotropic receptors, whereas all the others are metabotropic G-protein-coupled receptors (GPCRs). 5-HT receptor diversity is further increased by post-genomic modifications, such as alternative splicing (up to 10 splice variants for the 5-HT₄ receptor) or by mRNA editing in the case of 5-HT_2C receptors. The cellular and behavioral implications of 5-HT_2C receptor editing are of great physiological importance. Signaling of 5-HT receptors involves a great variety of pathways, but only some of these have been demonstrated in neurons. The classical view of neurotransmitter receptors localized within the synaptic cleft cannot be applied to 5-HT receptors, which are mostly (but not exclusively) localized at extra-synaptic locations either pre- or post-synaptically. 5-HT receptors are engaged in pre- or post-synaptic complexes composed of many GPCR-interacting proteins. The functions of these proteins are starting to be revealed. These proteins have been implicated in targeting, trafficking to or from the membrane, desensitization, and fine-tuning of signaling.     

A Dynamic View of Molecular Switch Behavior at Serotonin Receptors: Implications for Functional Selectivity

Functional selectivity is a property of G protein-coupled receptors that allows them to preferentially couple to particular signaling partners upon binding of biased agonists. Publication of the X-ray crystal structure of serotonergic 5-HT_1B and 5-HT_2B receptors in complex with ergotamine, a drug capable of activating G protein coupling and β-arrestin signaling at the 5-HT_1B receptor but clearly favoring β-arrestin over G protein coupling at the 5-HT_2B subtype, has recently provided structural insight into this phenomenon. In particular, these structures highlight the importance of specific residues, also called micro-switches, for differential receptor activation. In our work, we apply classical molecular dynamics simulations and enhanced sampling approaches to analyze the behavior of these micro-switches and their impact on the stabilization of particular receptor conformational states. Our analysis shows that differences in the conformational freedom of helix 6 between both receptors could explain their different G protein-coupling capacity. In particular, as compared to the 5-HT_1B receptor, helix 6 movement in the 5-HT_2B receptor can be constrained by two different mechanisms. On the one hand, an anchoring effect of ergotamine, which shows an increased capacity to interact with the extracellular part of helices 5 and 6 and stabilize them, hinders activation of a hydrophobic connector region at the center of the receptor. On the other hand, this connector region in an inactive conformation is further stabilized by unconserved contacts extending to the intracellular part of the 5-HT_2B receptor, which hamper opening of the G protein binding site. This work highlights the importance of considering receptor capacity to adopt different conformational states from a dynamic perspective in order to underpin the structural basis of functional selectivity.     

Regulation of Amyloid Precursor Protein Processing by Serotonin Signaling

Proteolytic processing of the amyloid precursor protein (APP) by the β- and γ-secretases releases the amyloid-β peptide (Aβ), which deposits in senile plaques and contributes to the etiology of Alzheimer''s disease (AD). The α-secretase cleaves APP in the Aβ peptide sequence to generate soluble APPα (sAPPα). Upregulation of α-secretase activity through the 5-hydroxytryptamine 4 (5-HT₄) receptor has been shown to reduce Aβ production, amyloid plaque load and to improve cognitive impairment in transgenic mouse models of AD. Consequently, activation of 5-HT₄ receptors following agonist stimulation is considered to be a therapeutic strategy for AD treatment; however, the signaling cascade involved in 5-HT₄ receptor-stimulated proteolysis of APP remains to be determined. Here we used chemical and siRNA inhibition to identify the proteins which mediate 5-HT_4d receptor-stimulated α-secretase activity in the SH-SY5Y human neuronal cell line. We show that G protein and Src dependent activation of phospholipase C are required for α-secretase activity, while, unexpectedly, adenylyl cyclase and cAMP are not involved. Further elucidation of the signaling pathway indicates that inositol triphosphate phosphorylation and casein kinase 2 activation is also a prerequisite for α-secretase activity. Our findings provide a novel route to explore the treatment of AD through 5-HT₄ receptor-induced α-secretase activation.     

5-Hydroxytryptamine (5-HT) Cellular Sequestration during Chronic Exposure Delays 5-HT3 Receptor Resensitization due to Its Subsequent Release

The serotonergic synapse is dynamically regulated by serotonin (5-hydroxytryptamine (5-HT)) with elevated levels leading to the down-regulation of the serotonin transporter and a variety of 5-HT receptors, including the 5-HT type-3 (5-HT₃) receptors. We report that recombinantly expressed 5-HT₃ receptor binding sites are reduced by chronic exposure to 5-HT (IC₅₀ of 154.0 ± 45.7 μm, t_½ = 28.6 min). This is confirmed for 5-HT₃ receptor-induced contractions in the guinea pig ileum, which are down-regulated after chronic, but not acute, exposure to 5-HT. The loss of receptor function does not involve endocytosis, and surface receptor levels are unaltered. The rate and extent of down-regulation is potentiated by serotonin transporter function (IC₅₀ of 2.3 ± 1.0 μm, t_½ = 3.4 min). Interestingly, the level of 5-HT uptake correlates with the extent of down-regulation. Using TX-114 extraction, we find that accumulated 5-HT remains soluble and not membrane-bound. This cytoplasmically sequestered 5-HT is readily releasable from both COS-7 cells and the guinea pig ileum. Moreover, the 5-HT level released is sufficient to prevent recovery from receptor desensitization in the guinea pig ileum. Together, these findings suggest the existence of a novel mechanism of down-regulation where the chronic release of sequestered 5-HT prolongs receptor desensitization.     

Induction of Cardiac Fibrosis by β-Blocker in G Protein-independent and G Protein-coupled Receptor Kinase 5/β-Arrestin2-dependent Signaling Pathways

G-protein coupled receptors (GPCRs) have long been known as receptors that activate G protein-dependent cellular signaling pathways. In addition to the G protein-dependent pathways, recent reports have revealed that several ligands called “biased ligands” elicit G protein-independent and β-arrestin-dependent signaling through GPCRs (biased agonism). Several β-blockers are known as biased ligands. All β-blockers inhibit the binding of agonists to the β-adrenergic receptors. In addition to β-blocking action, some β-blockers are reported to induce cellular responses through G protein-independent and β-arrestin-dependent signaling pathways. However, the physiological significance induced by the β-arrestin-dependent pathway remains much to be clarified in vivo. Here, we demonstrate that metoprolol, a β₁-adrenergic receptor-selective blocker, could induce cardiac fibrosis through a G protein-independent and β-arrestin2-dependent pathway. Metoprolol, a β-blocker, increased the expression of fibrotic genes responsible for cardiac fibrosis in cardiomyocytes. Furthermore, metoprolol induced the interaction between β₁-adrenergic receptor and β-arrestin2, but not β-arrestin1. The interaction between β₁-adrenergic receptor and β-arrestin2 by metoprolol was impaired in the G protein-coupled receptor kinase 5 (GRK5)-knockdown cells. Metoprolol-induced cardiac fibrosis led to cardiac dysfunction. However, the metoprolol-induced fibrosis and cardiac dysfunction were not evoked in β-arrestin2- or GRK5-knock-out mice. Thus, metoprolol is a biased ligand that selectively activates a G protein-independent and GRK5/β-arrestin2-dependent pathway, and induces cardiac fibrosis. This study demonstrates the physiological importance of biased agonism, and suggests that G protein-independent and β-arrestin-dependent signaling is a reason for the diversity of the effectiveness of β-blockers.     

Substitution of 5-HT1A Receptor Signaling by a Light-activated G Protein-coupled Receptor

Understanding serotonergic (5-HT) signaling is critical for understanding human physiology, behavior, and neuropsychiatric disease. 5-HT mediates its actions via ionotropic and metabotropic 5-HT receptors. The 5-HT_1A receptor is a metabotropic G protein-coupled receptor linked to the G_i/o signaling pathway and has been specifically implicated in the pathogenesis of depression and anxiety. To understand and precisely control 5-HT_1A signaling, we created a light-activated G protein-coupled receptor that targets into 5-HT_1A receptor domains and substitutes for endogenous 5-HT_1A receptors. To induce 5-HT_1A-like targeting, vertebrate rhodopsin was tagged with the C-terminal domain (CT) of 5-HT_1A (Rh-CT_5-HT1A). Rh-CT_5-HT1A activates G protein-coupled inward rectifying K⁺ channels in response to light and causes membrane hyperpolarization in hippocampal neurons, similar to the agonist-induced responses of the 5-HT_1A receptor. The intracellular distribution of Rh-CT_5-HT1A resembles that of the 5-HT_1A receptor; Rh-CT_5-HT1A localizes to somatodendritic sites and is efficiently trafficked to distal dendritic processes. Additionally, neuronal expression of Rh-CT_5-HT1A, but not Rh, decreases 5-HT_1A agonist sensitivity, suggesting that Rh-CT_5-HT1A and 5-HT_1A receptors compete to interact with the same trafficking machinery. Finally, Rh-CT_5-HT1A is able to rescue 5-HT_1A signaling of 5-HT_1A KO mice in cultured neurons and in slices of the dorsal raphe showing that Rh-CT_5-HT1A is able to functionally compensate for native 5-HT_1A. Thus, as an optogenetic tool, Rh-CT_5-HT1A has the potential to directly correlate in vivo 5-HT_1A signaling with 5-HT neuron activity and behavior in both normal animals and animal models of neuropsychiatric disease.     

GGA3 Interacts with a G Protein-Coupled Receptor and Modulates Its Cell Surface Export

Molecular mechanisms governing the anterograde trafficking of nascent G protein-coupled receptors (GPCRs) are poorly understood. Here, we have studied the regulation of cell surface transport of α₂-adrenergic receptors (α₂-ARs) by GGA3 (Golgi-localized, γ-adaptin ear domain homology, ADP ribosylation factor-binding protein 3), a multidomain clathrin adaptor protein that sorts cargo proteins at the trans-Golgi network (TGN) to the endosome/lysosome pathway. By using an inducible system, we demonstrated that GGA3 knockdown significantly inhibited the cell surface expression of newly synthesized α_2B-AR without altering overall receptor synthesis and internalization. The receptors were arrested in the TGN. Furthermore, GGA3 knockdown attenuated α_2B-AR-mediated signaling, including extracellular signal-regulated kinase 1/2 (ERK1/2) activation and cyclic AMP (cAMP) inhibition. More interestingly, GGA3 physically interacted with α_2B-AR, and the interaction sites were identified as the triple Arg motif in the third intracellular loop of the receptor and the acidic motif EDWE in the VHS domain of GGA3. In contrast, α_2A-AR did not interact with GGA3 and its cell surface export and signaling were not affected by GGA3 knockdown. These data reveal a novel function of GGA3 in export trafficking of a GPCR that is mediated via a specific interaction with the receptor.     

mu-Opioid receptor-mediated ERK activation involves calmodulin-dependent epidermal growth factor receptor transactivation.

Phosphorylation of the MAPK isoform ERK by G protein-coupled receptors involves multiple signaling pathways. One of these pathways entails growth factor receptor transactivation followed by ERK activation. This study demonstrates that a similar signaling pathway is used by the mu-opioid receptor (MOR) expressed in HEK293 cells and involves calmodulin (CaM). Stimulation of MOR resulted in both epidermal growth factor receptor (EGFR) and ERK phosphorylation. Data obtained with inhibitors of EGFR Tyr kinase and membrane metalloproteases support an intermediate role of EGFR activation, involving release of endogenous membrane-bound epidermal growth factor. Previous studies had demonstrated a role for CaM in opioid signaling based on direct CaM binding to MOR. To test whether CaM contributes to EGFR transactivation and ERK phosphorylation by MOR, we compared wild-type MOR with mutant K273A MOR, which binds CaM poorly, but couples normally to G proteins. Stimulation of K273A MOR with [D-Ala(2),MePhe(4),Gly-ol(5)]enkephalin (10-100 nm) resulted in significantly reduced ERK phosphorylation. Furthermore, wild-type MOR stimulated EGFR Tyr phosphorylation 3-fold more than K273A MOR, indicating that direct CaM-MOR interaction plays a key role in the transactivation process. Inhibitors of CaM and protein kinase C also attenuated [D-Ala(2),MePhe(4),Gly-ol(5)]enkephalin-induced EGFR transactivation in wild-type (but not mutant) MOR-expressing cells. This novel pathway of EGFR transactivation may be shared by other G protein-coupled receptors shown to interact with CaM.     

Synergistic Regulation of Glutamatergic Transmission by Serotonin and Norepinephrine Reuptake Inhibitors in Prefrontal Cortical Neurons

The monoamine system in the prefrontal cortex has been implicated in various mental disorders and has been the major target of anxiolytics and antidepressants. Clinical studies show that serotonin and norepinephrine reuptake inhibitors (SNRIs) produce better therapeutic effects than single selective reuptake inhibitors, but the underlying mechanisms are largely unknown. Here, we found that low dose SNRIs, by acting on 5-HT_1A and α₂-adrenergic receptors, synergistically reduced AMPA receptor (AMPAR)-mediated excitatory postsynaptic currents and AMPAR surface expression in prefrontal cortex pyramidal neurons via a mechanism involving Rab5/dynamin-mediated endocytosis of AMPARs. The synergistic effect of SNRIs on AMPARs was blocked by inhibition of activator of G protein signaling 3, a G protein modulator that prevents reassociation of G_i protein α subunit and prolongs the βγ-mediated signaling pathway. Moreover, the depression of AMPAR-mediated excitatory postsynaptic currents by SNRIs required p38 kinase activity, which was increased by 5-HT_1A and α₂-adrenergic receptor co-activation in an activator of G protein signaling 3-dependent manner. These results have revealed a potential mechanism for the synergy between the serotonin and norepinephrine systems in the regulation of glutamatergic transmission in cortical neurons.     

A complex signaling cascade links the serotonin2A receptor to phospholipase A2 activation: the involvement of MAP kinases

Previous studies in our laboratory have shown that in NIH3T3-5HT2A cells, 5-HT-induced AA release is PLA2-coupled and independent of 5-HT2A receptor-mediated PLC activation. Although 5-HT2A receptor-mediated PLC activation is known to be Galphaq-coupled, much less is understood about 5-HT2A receptor-mediated PLA2 activation. Therefore, the studies presented here were aimed at elucidating the signal transduction pathway linking stimulation of the 5-HT2A receptor to PLA2 activation. By employing various selective inhibitors, toxins, and antagonistic peptide constructs, we propose that the 5-HT2A receptor can couple to PLA2 activation through two parallel signaling cascades. Initial experiments were designed to examine the role of pertussis toxin-sensitive G proteins, namely Galphai/o, as well as pertussis toxin-insensitive G proteins, namely Galpha12/13, in 5-HT-induced AA release. Furthermore, inactivation of both Gbetagamma heterodimers and Rho proteins resulted in decreased agonist-induced AA release, without having any effect on PLC-IP accumulation. We also demonstrated 5-HT2A receptor-mediated phosphorylation of ERK1,2 and p38. Moreover, pretreatment with selective ERK1,2 and p38 inhibitors resulted in decreased 5-HT-induced AA release. Taken together, these results suggest that the 5-HT2A receptor expressed in NIH3T3 cells can couple to PLA2 activation though a complex signaling mechanism involving both Galphai/o-associated Gbetagamma-mediated ERK1,2 activation and Galpha12/13-coupled, Rho-mediated p38 activation.     

Functional Selectivity of 6′-Guanidinonaltrindole (6′-GNTI) at κ-Opioid Receptors in Striatal Neurons

There is considerable evidence to suggest that drug actions at the κ-opioid receptor (KOR) may represent a means to control pain perception and modulate reward thresholds. As a G protein-coupled receptor (GPCR), the activation of KOR promotes Gα_i/o protein coupling and the recruitment of β-arrestins. It has become increasingly evident that GPCRs can transduce signals that originate independently via G protein pathways and β-arrestin pathways; the ligand-dependent bifurcation of such signaling is referred to as “functional selectivity” or “signaling bias.” Recently, a KOR agonist, 6′-guanidinonaltrindole (6′-GNTI), was shown to display bias toward the activation of G protein-mediated signaling over β-arrestin2 recruitment. Therefore, we investigated whether such ligand bias was preserved in striatal neurons. Although the reference KOR agonist U69,593 induces the phosphorylation of ERK1/2 and Akt, 6′-GNTI only activates the Akt pathway in striatal neurons. Using pharmacological tools and β-arrestin2 knock-out mice, we show that KOR-mediated ERK1/2 phosphorylation in striatal neurons requires β-arrestin2, whereas Akt activation depends upon G protein signaling. These findings reveal a point of KOR signal bifurcation that can be observed in an endogenous neuronal setting and may prove to be an important indicator when developing biased agonists at the KOR.