Sigma-1 Receptor Enhances Neurite Elongation of Cerebellar Granule Neurons via TrkB Signaling

Sigma-1 receptor (Sig-1R) is an integral membrane protein predominantly expressed in the endoplasmic reticulum. Sig-1R demonstrates a high affinity to various synthetic compounds including well-known psychotherapeutic drugs in the central nervous system (CNS). For that, it is considered as an alternative target for psychotherapeutic drugs. On the cellular level, when Sig-1R is activated, it is known to play a role in neuroprotection and neurite elongation. These effects are suggested to be mediated by its ligand-operated molecular chaperone activity, and/or upregulation of various Ca²⁺ signaling. In addition, recent studies show that Sig-1R activation induces neurite outgrowth via neurotrophin signaling. Here, we tested the hypothesis that Sig-1R activation promotes neurite elongation through activation of tropomyosin receptor kinase (Trk), a family of neurotrophin receptors. We found that 2-(4-morpholinethyl)1-phenylcyclohexanecarboxylate (PRE-084), a selective Sig-1R agonist, significantly promoted neurite outgrowth, and K252a, a Trk inhibitor, attenuated Sig-1R-mediated neurite elongation in cerebellar granule neurons (CGNs). Moreover, we revealed that Sig-1R interacts with TrkB, and PRE-084 treatment enhances phosphorylation of Y515, but not Y706. Thus, our results indicate that Sig-1R activation promotes neurite outgrowth in CGNs through Y515 phosphorylation of TrkB.     

Evidence That Calmodulin Binding to the Dopamine D2 Receptor Enhances Receptor Signaling

The Ca²⁺ sensor calmodulin (CaM) regulates numerous proteins involved in G protein-coupled receptor (GPCR) signaling. CaM binds directly to some GPCRs, including the dopamine D₂ receptor. We confirmed that the third intracellular loop of the D₂ receptor is a direct contact point for CaM binding using coimmunoprecipitation and a polyHis pull-down assay, and we determined that the D₂-like receptor agonist 7-OH-DPAT increased the colocalization of the D₂ receptor and endogenous CaM in both 293 cells and in primary neostriatal cultures. The N-terminal three or four residues of D₂-IC3 were required for the binding of CaM; mutation of three of these residues in the full-length receptor (I210C/K211C/I212C) decreased the coprecipitation of the D₂ receptor and CaM and also significantly decreased D₂ receptor signaling, without altering the coupling of the receptor to G proteins. Taken together, these findings suggest that binding of CaM to the dopamine D₂ receptor enhances D₂ receptor signaling.     

Targeting CB2-GPR55 Receptor Heteromers Modulates Cancer Cell Signaling

The G protein-coupled receptors CB₂ (CB₂R) and GPR55 are overexpressed in cancer cells and human tumors. Because a modulation of GPR55 activity by cannabinoids has been suggested, we analyzed whether this receptor participates in cannabinoid effects on cancer cells. Here we show that CB₂R and GPR55 form heteromers in cancer cells, that these structures possess unique signaling properties, and that modulation of these heteromers can modify the antitumoral activity of cannabinoids in vivo. These findings unveil the existence of previously unknown signaling platforms that help explain the complex behavior of cannabinoids and may constitute new targets for therapeutic intervention in oncology.     

Signaling Mechanisms of the D3 Dopamine Receptor

A substantial body of evidence shows the capacity of the dopamine D₃ receptor to couple functionally to G proteins when expressed in an appropriate milieu in heterologous expression systems. In these systems, activation of D₃ receptors inhibits adenylate cyclase, modulates ion flow through potassium and calcium channels, and activates kinases, most notably mitogen-activated protein kinase. Coupling to G_i/G_o is implicated in many of these effects, but other G proteins may contribute. Studies with chimeric receptors implicate the third intracellular loop in the mediation of agonist-induced signal transduction. Finally, D₃-preferring drugs modulate expression of c-fos in neuronal cultures and brain. Signaling mechanisms of the D₃ receptor in brain, however, remain to be definitively determined.     

Cdk5 Phosphorylates Dopamine D2 Receptor and Attenuates Downstream Signaling

The dopamine D2 receptor (DRD2) is a key receptor that mediates dopamine-associated brain functions such as mood, reward, and emotion. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase whose function has been implicated in the brain reward circuit. In this study, we revealed that the serine 321 residue (S321) in the third intracellular loop of DRD2 (D2i3) is a novel regulatory site of Cdk5. Cdk5-dependent phosphorylation of S321 in the D2i3 was observed in in vitro and cell culture systems. We further observed that the phosphorylation of S321 impaired the agonist-stimulated surface expression of DRD2 and decreased G protein coupling to DRD2. Moreover, the downstream cAMP pathway was affected in the heterologous system and in primary neuronal cultures from p35 knockout embryos likely due to the reduced inhibitory activity of DRD2. These results indicate that Cdk5-mediated phosphorylation of S321 inhibits DRD2 function, providing a novel regulatory mechanism for dopamine signaling.     

Initial D2 Dopamine Receptor Sensitivity Predicts Cocaine Sensitivity and Reward in Rats

The activation of dopamine receptors within the mesolimbic dopamine system is known to be involved in the initiation and maintenance of cocaine use. Expression of the D₂ dopamine receptor subtype has been implicated as both a predisposing factor and consequence of chronic cocaine use. It is unclear whether there is a predictive relationship between D₂ dopamine receptor function and cocaine sensitivity that would enable cocaine abuse. Therefore, we exploited individual differences in behavioral responses to D₂ dopamine receptor stimulation to test its relationship with cocaine-mediated behaviors. Outbred, male Sprague-Dawley rats were initially characterized by their locomotor responsiveness to the D₂ dopamine receptor agonist, quinpirole, in a within-session ascending dose-response regimen (0, 0.1, 0.3 & 1.0 mg/kg, sc). Rats were classified as high or low quinpirole responders (HD₂ and LD₂, respectively) by a median split of their quinpirole-induced locomotor activity. Rats were subsequently tested for differences in the psychostimulant effects of cocaine by measuring changes in cocaine-induced locomotor activity (5 and 15 mg/kg, ip). Rats were also tested for differences in the development of conditioned place preference to a low dose of cocaine (7.5 mg/kg, ip) that does not reliably produce a cocaine conditioned place preference. Finally, rats were tested for acquisition of cocaine self-administration and maintenance responding on fixed ratio 1 and 5 schedules of reinforcement, respectively. Results demonstrate that HD₂ rats have enhanced sensitivity to the locomotor stimulating properties of cocaine, display greater cocaine conditioned place preference, and self-administer more cocaine compared to LD₂ animals. These findings suggest that individual differences in D₂ dopamine receptor sensitivity may be predictive of cocaine sensitivity and reward.     

G Protein-coupled Receptor Kinase-2 Constitutively Regulates D2 Dopamine Receptor Expression and Signaling Independently of Receptor Phosphorylation

We investigated the regulatory effects of GRK2 on D₂ dopamine receptor signaling and found that this kinase inhibits both receptor expression and functional signaling in a phosphorylation-independent manner, apparently through different mechanisms. Overexpression of GRK2 was found to suppress receptor expression at the cell surface and enhance agonist-induced internalization, whereas short interfering RNA knockdown of endogenous GRK2 led to an increase in cell surface receptor expression and decreased agonist-mediated endocytosis. These effects were not due to GRK2-mediated phosphorylation of the D₂ receptor as a phosphorylation-null receptor mutant was regulated similarly, and overexpression of a catalytically inactive mutant of GRK2 produced the same effects. The suppression of receptor expression is correlated with constitutive association of GRK2 with the receptor complex as we found that GRK2 and several of its mutants were able to co-immunoprecipitate with the D₂ receptor. Agonist pretreatment did not enhance the ability of GRK2 to co-immunoprecipitate with the receptor. We also found that overexpression of GRK2 attenuated the functional coupling of the D₂ receptor and that this activity required the kinase activity of GRK2 but did not involve receptor phosphorylation, thus suggesting the involvement of an additional GRK2 substrate. Interestingly, we found that the suppression of functional signaling also required the Gβγ binding activity of GRK2 but did not involve the GRK2 N-terminal RH domain. Our results suggest a novel mechanism by which GRK2 negatively regulates G protein-coupled receptor signaling in a manner that is independent of receptor phosphorylation.     

Mimicry of Dopamine 1 Receptor Signaling with Cell-Penetrating Peptides

International Journal of Peptide Research and Therapeutics - In this study, through the use of protein mimicry, a peptide was developed to activate the dopamine 1 receptor signaling pathway from...     

Novel Dopamine D2 Receptor Signaling through Proteins Interacting with the Third Cytoplasmic Loop

The diverse activities of dopamine D2-like receptors, including D2, D3, and D4 receptors, are mediated by proteins that interact with the third cytoplasmic loop and regulate receptor signaling, receptor trafficking, and apoptosis. Such interacting proteins include calmodulin, the N-methyl-d-aspartate receptor 2B subunit, calcium/calmodulin-dependent protein kinase II, prostate apoptosis response-4, and β-arrestins, which regulate receptor signaling and the pharmacological action through D2 receptor. The gene encoding the D2 receptor gives rise to two isoforms, termed the dopamine D2 receptor long isoform (D2L) and the dopamine D2 receptor short isoform; the latter lacks 29 amino acids of the D2L receptor within the third cytoplasmic loop. In this review, we first focus on novel functions of the hetero-oligomeric D1/D2 and D2/adenosine A_2A receptors. We next discuss novel signaling through proteins interacting with the D2 receptor third cytoplasmic loop and define the function of a novel binding protein, heart-type fatty acid binding protein, which interacts with the D2L third cytoplasmic loop.     

Dynamic Interaction between Sigma-1 Receptor and Kv1.2 Shapes Neuronal and Behavioral Responses to Cocaine

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Highlights? Sig-1R activity enhances sensitivity to cocaine by decreasing accumbal firing ? Cocaine decreases accumbal firing via Sig-1R-dependent increase of Kv1.2 current ? The Sig-1R forms a complex with Kv1.2 channel in the nucleus accumbens shell ? Cocaine triggers a long-lasting upregulation of the Sig-1R-Kv1.2 channel complex     

Sigma-1 Receptor Chaperone at the ER-Mitochondrion Interface Mediates the Mitochondrion-ER-Nucleus Signaling for Cellular Survival

The membrane of the endoplasmic reticulum (ER) of a cell forms contacts directly with mitochondria whereby the contact is referred to as the mitochondrion-associated ER membrane or the MAM. Here we found that the MAM regulates cellular survival via an MAM-residing ER chaperone the sigma-1 receptor (Sig-1R) in that the Sig-1R chaperones the ER stress sensor IRE1 to facilitate inter-organelle signaling for survival. IRE1 is found in this study to be enriched at the MAM in CHO cells. We found that IRE1 is stabilized at the MAM by Sig-1Rs when cells are under ER stress. Sig-1Rs stabilize IRE1 and thus allow for conformationally correct IRE1 to dimerize into the long-lasting, activated endonuclease. The IRE1 at the MAM also responds to reactive oxygen species derived from mitochondria. Therefore, the ER-mitochondrion interface serves as an important subcellular entity in the regulation of cellular survival by enhancing the stress-responding signaling between mitochondria, ER, and nucleus.     

Dopamine D1-D2 Receptor Heteromer-mediated Calcium Release Is Desensitized by D1 Receptor Occupancy with or without Signal Activation: DUAL FUNCTIONAL REGULATION BY G PROTEIN-COUPLED RECEPTOR KINASE 2*

We identified that activation of the G_q-linked dopamine D1-D2 receptor hetero-oligomer generates a PLC-dependent intracellular calcium signal. Confocal FRET between endogenous dopamine D1 and D2 receptors in striatal neurons confirmed a physical interaction between them. Pretreatment with SKF 83959, which selectively activates the D1-D2 receptor heteromer, or SKF 83822, which only activates the D1 receptor homo-oligomer, led to rapid desensitization of the D1-D2 receptor heteromer-mediated calcium signal in both heterologous cells and striatal neurons. This desensitization response was mediated through selective occupancy of the D1 receptor binding pocket. Although SKF 83822 was unable to activate the D1-D2 receptor heteromer, it still permitted desensitization of the calcium signal. This suggested that occupancy of the D1 receptor binding pocket by SKF 83822 resulted in conformational changes sufficient for desensitization without heteromer activation. Bioluminescence resonance energy transfer and co-immunoprecipitation studies indicated an agonist-induced physical association between the D1-D2 receptor heteromeric complex and GRK2. Increased expression of GRK2 led to a decrease in the calcium signal with or without prior exposure to either SKF 83959 or SKF 83822. GRK2 knockdown by siRNA led to an increase in the signal after pretreatment with either agonist. Expression of the catalytically inactive and RGS (regulator of G protein signaling)-mutated GRK2 constructs each led to a partial recovery of the GRK2-attenuated calcium signal. These results indicated that desensitization of the dopamine D1-D2 receptor heteromer-mediated signal can occur by agonist occupancy even without activation and is dually regulated by both the catalytic and RGS domains of GRK2.     

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.     

Adolescent Maturation of Dopamine D1 and D2 Receptor Function and Interactions in Rodents

Adolescence is a developmental period characterized by heightened vulnerability to illicit drug use and the onset of neuropsychiatric disorders. These clinical phenomena likely share common neurobiological substrates, as mesocorticolimbic dopamine systems actively mature during this period. Whereas prior studies have examined age-dependent changes in dopamine receptor binding, there have been fewer functional analyses. The aim of the present study was therefore to determine whether the functional consequences of D1 and D2-like activation are age-dependent. Adolescent and adult rats were given direct D1 and D2 agonists, alone and in combination. Locomotor and stereotypic behaviors were measured, and brains were collected for analysis of mRNA expression for the immediate early genes (IEGs), cfos and arc. Adolescents showed enhanced D2-like receptor control of locomotor and repetitive behaviors, which transitioned to dominant D1-like mechanisms in adulthood. When low doses of agonists were co-administered, adults showed supra-additive behavioral responses to D1/D2 combinations, whereas adolescents did not, which may suggest age differences in D1/D2 synergy. D1/D2-stimulated IEG expression was particularly prominent in the bed nucleus of the stria terminalis (BNST). Given the BNST’s function as an integrator of corticostriatal, hippocampal, and stress-related circuitry, and the importance of neural network dynamics in producing behavior, an exploratory functional network analysis of regional IEG expression was performed. This data-driven analysis demonstrated similar developmental trajectories as those described in humans and suggested that dopaminergic drugs alter forebrain coordinated gene expression age dependently. D1/D2 recruitment of stress nuclei into functional networks was associated with low behavioral output in adolescents. Network analysis presents a novel tool to assess pharmacological action, and highlights critical developmental changes in functional neural circuitry. Immature D1/D2 interactions in adolescents may underlie their unique responses to drugs of abuse and vulnerability to psychopathology. These data highlight the need for age-specific pharmacotherapy design and clinical application in adolescence.     

Beta-Arrestin1 Prevents Preeclampsia by Downregulation of Mechanosensitive AT1-B2 Receptor Heteromers

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CIAP2 Inhibits Anigen Receptor Signaling by Targeting Bcl10 for Degredation

The cellular inhibitor of apoptosis 2 (cIAP2) is a RING-containing protein ubiquitin ligase. In a high percentage of mucosa-associated lymphoid tissue (MALT) lymphomas, cIAP2 is fused to MALT1/paracaspase as a result of the t(11;18)(q21;q21) translocation. The physiological function of cIAP2 in lymphocytes and how this function may be affected by the translocation are not well understood. We have shown that cIAP2 normally inhibits antigen receptor signaling by mediating the ubiquitination and degradation of Bcl10, a critical component for antigenic signaling to NF-κB. The cIAP2-MALT1 fusion protein lacks this E3 activity and is incapable of ubiquitinating Bcl10, likely causing enhanced Bcl10 expression. Furthermore, cIAP2-MALT1 and Bcl10 synergistically activate NF-κB. These results reveal a physiological function of cIAP2, identify Bcl10 upregulation as a unifying molecular mechanism for MALT lymphomas, and define the mechanism and effects of this upregulation in t(11;18)-positive MALT lymphomas.     

Dopamine D1 and D2 Receptor Immunoreactivities in the Arcuate-Median Eminence Complex and their Link to the Tubero-Infundibular Dopamine Neurons

Dopamine D1 and D2 receptor immunohistochemistry and Golgi techniques were used to study the structure of the adult rat arcuate-median eminence complex, and determine the distribution of the dopamine D1 and D2 receptor immunoreactivities therein, particularly in relation to the tubero-infundibular dopamine neurons. Punctate dopamine D1 and D2 receptor immunoreactivities, likely located on nerve terminals, were enriched in the lateral palisade zone built up of nerve terminals, while the densities were low to modest in the medial palisade zone. A codistribution of dopamine D1 receptor or dopamine D2 receptor immunoreactive puncta with tyrosine hydroxylase immunoreactive nerve terminals was demonstrated in the external layer. Dopamine D1 receptor but not dopamine D2 receptor immnunoreactivites nerve cell bodies were found in the ventromedial part of the arcuate nucleus and in the lateral part of the internal layer of the median eminence forming a continuous cell mass presumably representing neuropeptide Y immunoreactive nerve cell bodies. The major arcuate dopamine/ tyrosine hydroxylase nerve cell group was found in the dorsomedial part. A large number of tyrosine hydroxylase immunoreactive nerve cell bodies in this region demonstrated punctate dopamine D1 receptor immunoreactivity but only a few presented dopamine D2 receptor immunoreactivity which were mainly found in a substantial number of tyrosine hydroxylase cell bodies of the ventral periventricular hypothalamic nucleus, also belonging to the tuberoinfundibular dopamine neurons. Structural evidence for projections of the arcuate nerve cells into the median eminence was also obtained. Distal axons formed horizontal axons in the internal layer issuing a variable number of collaterals classified into single or multiple strands located in the external layer increasing our understanding of the dopamine nerve terminal networks in this region. Dopamine D1 and D2 receptors may therefore directly and differentially modulate the activity and/or Dopamine synthesis of substantial numbers of tubero-infundibular dopamine neurons at the somatic and terminal level. The immunohistochemical work also gives support to the view that dopamine D1 receptors and/or dopamine D2 receptors in the lateral palisade zone by mediating dopamine volume transmission may contribute to the inhibition of luteinizing hormone releasing hormone release from nerve terminals in this region.Key words: Dopamine D1 and D2 receptors, tubero-infundibular dopamine neurons, dopamine receptor colocalization, arcuate-median eminence complex, volume transmission, luteinizing hormone releasing hormone     

Dopamine D2 Receptor Stimulation Potentiates PolyQ-Huntingtin-Induced Mouse Striatal Neuron Dysfunctions via Rho/ROCK-II Activation

Background

Huntington''s disease (HD) is a polyglutamine-expanded related neurodegenerative disease. Despite the ubiquitous expression of expanded, polyQ-Huntingtin (ExpHtt) in the brain, striatal neurons present a higher susceptibility to the mutation. A commonly admitted hypothesis is that Dopaminergic inputs participate to this vulnerability. We previously showed that D2 receptor stimulation increased aggregate formation and neuronal death induced by ExpHtt in primary striatal neurons in culture, and chronic D2 antagonist treatment protects striatal dysfunctions induced by ExpHtt in a lentiviral-induced model system in vivo. The present work was designed to elucidate the signalling pathways involved, downstream D2 receptor (D2R) stimulation, in striatal vulnerability to ExpHtt.

Methodology/Principal Findings

Using primary striatal neurons in culture, transfected with a tagged-GFP version of human exon 1 ExpHtt, and siRNAs against D2R or D1R, we confirm that DA potentiates neuronal dysfunctions via D2R but not D1R stimulation. We demonstrate that D2 agonist treatment induces neuritic retraction and growth cone collapse in Htt- and ExpHtt expressing neurons. We then tested a possible involvement of the Rho/ROCK signalling pathway, which plays a key role in the dynamic of the cytoskeleton, in these processes. The pharmacological inhibitors of ROCK (Y27632 and Hydroxyfasudil), as well as siRNAs against ROCK-II, reversed D2-related effects on neuritic retraction and growth cone collapse. We show a coupling between D2 receptor stimulation and Rho activation, as well as hyperphosphorylation of Cofilin, a downstream effector of ROCK-II pathway. Importantly, D2 agonist-mediated potentiation of aggregate formation and neuronal death induced by ExpHtt, was totally reversed by Y27632 and Hydroxyfasudil and ROCK-II siRNAs.

Conclusions/Significance

Our data provide the first demonstration that D2R-induced vulnerability in HD is critically linked to the activation of the Rho/ROCK signalling pathway. The inclusion of Rho/ROCK inhibitors could be an interesting therapeutic option aimed at forestalling the onset of the disease.     

Kinin-Stimulated B1 Receptor Signaling Depends on Receptor Endocytosis Whereas B2 Receptor Signaling Does Not

Kinins are potent pro-inflammatory peptides that act through two G protein-coupled receptor subtypes, B1 (B1R) and B2 (B2R). Kinin-stimulated B2R signaling is often transient, whereas B1R signaling is sustained. This was confirmed by monitoring agonist-stimulated intracellular Ca²⁺ mobilization in A10 smooth muscle cells expressing human wild-type B2R and B1R. We further studied the role of receptor membrane trafficking in receptor-mediated phosphoinositide (PI) hydrolysis in model HEK293 cell lines stably expressing the receptors. Treatment of cells with brefeldin A, to inhibit maturation of de novo synthesized receptors, or hypertonic sucrose, to inhibit receptor endocytosis, showed that the basal cell surface receptor turnover was considerably faster for B1R than for B2R. Inhibition of endocytosis, which stabilized B1R on the cell surface, inhibited B1R signaling, whereas B2R signaling was not perturbed. Signaling by a B1R construct in which the entire C-terminal domain was deleted remained sensitive to inhibition of receptor endocytosis, whereas signaling by a B1R construct in which this domain was substituted with the corresponding domain in B2R was not sensitive. B2R and B1R co-expression, which also appeared to stabilize B1R on the cell surface, presumably by receptor hetero-dimerization, also inhibited B1R signaling, whereas B2R signaling was slightly enhanced. Furthermore, the B2R-specific agonist bradykinin (BK) directed both receptors through a common endocytic pathway, whereas the B1R-specific agonist Lys-desArg⁹-BK was unable to do so. These results suggest that B1R-mediated PI hydrolysis depends on a step in receptor endocytosis, whereas B2R-mediated PI hydrolysis does not. We propose that B1R uses at least part of the endocytic machinery to sustain agonist-promoted signaling.