Nociceptive responses in melatonin MT2 receptor knockout mice compared to MT1 and double MT1/MT2 receptor knockout mice

Melatonin, a neurohormone that binds to two G protein-coupled receptors MT₁ and MT_2, is involved in pain regulation, but the distinct role of each receptor has yet to be defined. We characterized the nociceptive responses of mice with genetic inactivation of melatonin MT₁ (MT₁^−/−), or MT₂ (MT₂^−/−), or both MT₁/MT₂ (MT₁^−/−/MT₂^−/−) receptors in the hot plate test (HPT), and the formalin test (FT). In HPT and FT, MT₁^−/− display no differences compared to their wild-type littermates (CTL), whereas both MT₂^−/− and MT₁^−/−/MT₂^−/− mice showed a reduced thermal sensitivity and a decreased tonic nocifensive behavior during phase 2 of the FT in the light phase. The MT₂ partial agonist UCM924 induced an antinociceptive effect in MT₁^−/− but not in MT₂^−/− and MT₁^−/−/MT₂^−/− mice. Also, the competitive opioid antagonist naloxone had no effects in CTL, whereas it induced a decrease of nociceptive thresholds in MT₂^−/− mice. Our results show that the genetic inactivation of melatonin MT₂, but not MT₁ receptors, produces a distinct effect on nociceptive threshold, suggesting that the melatonin MT₂ receptor subtype is selectively involved in the regulation of pain responses.     

Genetic deletion of MT1 and MT2 melatonin receptors differentially abrogates the development and expression of methamphetamine‐induced locomotor sensitization during the day and the night in C3H/HeN mice

Abstract: This study explored the role of the melatonin receptors in methamphetamine (METH)‐induced locomotor sensitization during the light and dark phases in C3H/HeN mice with genetic deletion of the MT₁ and/or MT₂ melatonin receptors. Six daily treatments with METH (1.2 mg/kg, i.p.) in a novel environment during the light phase led to the development of locomotor sensitization in wild‐type (WT), MT₁KO and MT₂KO mice. Following four full days of abstinence, METH challenge (1.2 mg/kg, i.p.) triggered the expression of locomotor sensitization in METH‐pretreated but not in vehicle (VEH)‐pretreated mice. In MT₁/MT₂KO mice, the development of sensitization during the light phase was significantly reduced and the expression of sensitization was completely abrogated upon METH challenge. During the dark phase the development of locomotor sensitization in METH‐pretreated WT, MT₁KO and MT₂KO mice was statistically different from VEH‐treated controls. However, WT and MT₂KO, but not MT₁KO mice receiving repeated VEH pretreatments during the dark phase expressed a sensitized response to METH challenge that is of an identical magnitude to that observed upon 6 days of METH pretreatment. We conclude that exposure to a novel environment during the dark phase, but not during the light phase, facilitated the expression of sensitization to a METH challenge in a manner dependent on MT₁ melatonin receptor activation by endogenous melatonin. We suggest that MT₁ and MT₂ melatonin receptors are potential targets for pharmacotherapeutic intervention in METH abusers.     

Protein interactome mining defines melatonin MT1 receptors as integral component of presynaptic protein complexes of neurons

In mammals, the hormone melatonin is mainly produced by the pineal gland with nocturnal peak levels. Its peripheral and central actions rely either on its intrinsic antioxidant properties or on binding to melatonin MT₁ and MT₂ receptors, belonging to the G protein‐coupled receptor (GPCR) super‐family. Melatonin has been reported to be involved in many functions of the central nervous system such as circadian rhythm regulation, neurotransmission, synaptic plasticity, memory, sleep, and also in Alzheimer's disease and depression. However, little is known about the subcellular localization of melatonin receptors and the molecular aspects involved in neuronal functions of melatonin. Identification of protein complexes associated with GPCRs has been shown to be a valid approach to improve our understanding of their function. By combining proteomic and genomic approaches we built an interactome of MT₁ and MT₂ receptors, which comprises 378 individual proteins. Among the proteins interacting with MT₁, but not with MT₂, we identified several presynaptic proteins, suggesting a potential role of MT₁ in neurotransmission. Presynaptic localization of MT₁ receptors in the hypothalamus, striatum, and cortex was confirmed by subcellular fractionation experiments and immunofluorescence microscopy. MT₁ physically interacts with the voltage‐gated calcium channel Ca_v2.2 and inhibits Ca_v2.2‐promoted Ca²⁺ entry in an agonist‐independent manner. In conclusion, we show that MT₁ is part of the presynaptic protein network and negatively regulates Ca_v2.2 activity, providing a first hint for potential synaptic functions of MT₁.     

Anatomical and cellular localization of melatonin MT1 and MT2 receptors in the adult rat brain

The involvement of melatonin in mammalian brain pathophysiology has received growing interest, but information about the anatomical distribution of its two G‐protein‐coupled receptors, MT₁ and MT₂, remains elusive. In this study, using specific antibodies, we examined the precise distribution of both melatonin receptors immunoreactivity across the adult rat brain using light, confocal, and electron microscopy. Our results demonstrate a selective MT₁ and MT₂ localization on neuronal cell bodies and dendrites in numerous regions of the rat telencephalon, diencephalon, and mesencephalon. Confocal and ultrastructural examination confirmed the somatodendritic nature of MT₁ and MT₂ receptors, both being localized on neuronal membranes. Overall, striking differences were observed in the anatomical distribution pattern of MT₁ and MT₂ proteins, and the labeling often appeared complementary in regions displaying both receptors. Somadendrites labeled for MT₁ were observed for instance in the retrosplenial cortex, the dentate gyrus of the hippocampus, the islands of Calleja, the medial habenula, the suprachiasmatic nucleus, the superior colliculus, the substantia nigra pars compacta, the dorsal raphe nucleus, and the pars tuberalis of the pituitary gland. Somadendrites endowed with MT₂ receptors were mostly observed in the CA3 field of the hippocampus, the reticular thalamic nucleus, the supraoptic nucleus, the inferior colliculus, the substantia nigra pars reticulata, and the ventrolateral periaqueductal gray. Together, these data provide the first detailed neurocytological mapping of melatonin receptors in the adult rat brain, an essential prerequisite for a better understanding of melatonin distinct receptor function and neurophysiology.     

Copper chelators promote nonamyloidogenic processing of AβPP via MT1/2/CREB‐dependent signaling pathways in AβPP/PS1 transgenic mice

Copper is essential for the generation of reactive oxygen species (ROS), which are induced by amyloid‐β (Aβ) aggregation; thus, the homeostasis of copper is believed to be a therapeutic target for Alzheimer’s disease (AD). Although clinical trials of copper chelators show promise when applied in AD, the underlying mechanism is not fully understood. Here, we reported that copper chelators promoted nonamyloidogenic processing of AβPP through MT_1/2/CREB‐dependent signaling pathways. First, we found that the formation of Aβ plaques in the cortex was significantly reduced, and learning deficits were significantly improved in AβPP/PS1 transgenic mice by copper chelator tetrathiomolybdate (TM) administration. Second, TM and another copper chelator, bathocuproine sulfonate (BCS), promoted nonamyloidogenic processing of AβPP via inducing the expression of ADAM10 and the secretion of sAβPPα. Third, the inducible ADAM10 production caused by copper chelators can be blocked by a melatonin receptor (MT_1/2) antagonist (luzindole) and a MT₂ inhibitor (4‐P‐PDOT), suggesting that the expression of ADAM10 depends on the activation of MT_1/2 signaling pathways. Fourth, three of the MT_1/2‐downstream signaling pathways, Gq/PLC/MEK/ERK/CREB, Gs/cAMP/PKA/ERK/CREB and Gs/cAMP/PKA/CREB, were responsible for copper chelator‐induced ADAM10 production. Based on these results, we conclude that copper chelators regulate the balance between amyloidogenic and nonamyloidogenic processing of AβPP via promoting ADAM10 expression through MT_1/2/CREB‐dependent signaling pathways.     

Functional MT1 and MT2 melatonin receptors in mammals

Melatonin, dubbed the hormone of darkness, is known to regulate a wide variety of physiological processes in mammals. This review describes well-defined functional responses mediated through activation of high-affinity MT₁ and MT₂ proteinteoupled receptors viewed as potential targets for drug discovery. MT₁ melatonin receptors modulate neuronal firing, arterial vasoconstriction, cell proliferation in cancer cells, and reproductive and metabolic functions. Ativation of MT₂ melatonin receptors phase shift circadian rhythms of neuronal firing in the suprachiasmatic nucleus, inhibit dopamine release in retina, induce vasodilation and inhibition of leukocyte rolling in arterial beds, and enhance immune responses. The melatonin-mediated responses elicited by activation of MT₁ and MT₂ native melatonin receptors are dependent on circadian time, duration and mode of exposure to endogenous or exogenous melatonin, and functional receptor sensitivity. Together, these studies underscore the importance of carefully linking each melatonin receptor type to specific functional responses in target tissues to facilitate the design and development of novel therapeutic agent.     

Dysfunction of serotonergic activity and emotional responses across the light-dark cycle in mice lacking melatonin MT2 receptors

Melatonin (MLT) levels fluctuate according to the external light/dark cycle in both diurnal and nocturnal mammals. We previously demonstrated that melatonin MT₂ receptor knockout (MT₂^−/−) mice show a decreased nonrapid eye movement sleep over 24 hours and increased wakefulness during the inactive (light) phase. Here, we investigated the role of MT₂ receptors in physiological light/dark cycle fluctuations in the activity of dorsal raphe nucleus (DRN) serotonin (5-HT) neurons and anxiety- and depression-like behavior. We found that the 5-HT burst-firing activity was tonically reduced across the whole 24 hours in MT₂^−/− mice compared with MT₂^+/+ mice.  Importantly, the physiological changes in the spontaneous firing activity of DRN 5-HT neurons during the light/dark cycle were nullified in MT₂^−/− mice, with a higher DRN 5-HT neural firing activity during the light phase in MT₂^−/− than in MT₂^+/+ mice. The role of MT₂ receptors over DRN 5-HT neurons was confirmed by acute pharmacological studies in which the selective MT₂ receptors agonist UCM1014 dose dependently inhibited DRN 5-HT activity, mostly during the dark phase. Compared with MT₂^+/+, MT₂^−/− mice displayed an anxiety-like phenotype in the novelty-suppressed feeding and in the light/dark box tests; while anxiety levels in the light/dark box test were lower during the dark than during the light phase in MT₂^+/+ mice, the opposite was seen in MT₂^−/− mice. No differences between MT₂^+/+ and MT₂^−/− mice were observed for depression-like behavior in the forced swim and in the sucrose preference tests. These results suggest that MT₂ receptor genetic inactivation impacts 5-HT neurotransmission and interferes with anxiety levels by perturbing the physiologic light/dark pattern.     

BRET-based effector membrane translocation assay monitors GPCR-promoted and endocytosis-mediated Gq activation at early endosomes

G protein–coupled receptors (GPCRs) are gatekeepers of cellular homeostasis and the targets of a large proportion of drugs. In addition to their signaling activity at the plasma membrane, it has been proposed that their actions may result from translocation and activation of G proteins at endomembranes—namely endosomes. This could have a significant impact on our understanding of how signals from GPCR-targeting drugs are propagated within the cell. However, little is known about the mechanisms that drive G protein movement and activation in subcellular compartments. Using bioluminescence resonance energy transfer (BRET)–based effector membrane translocation assays, we dissected the mechanisms underlying endosomal G_q trafficking and activity following activation of G_q-coupled receptors, including the angiotensin II type 1, bradykinin B₂, oxytocin, thromboxane A₂ alpha isoform, and muscarinic acetylcholine M₃ receptors. Our data reveal that GPCR-promoted activation of G_q at the plasma membrane induces its translocation to endosomes independently of β-arrestin engagement and receptor endocytosis. In contrast, G_q activity at endosomes was found to rely on both receptor endocytosis-dependent and -independent mechanisms. In addition to shedding light on the molecular processes controlling subcellular G_q signaling, our study provides a set of tools that will be generally applicable to the study of G protein translocation and activation at endosomes and other subcellular organelles, as well as the contribution of signal propagation to drug action.

G protein–coupled receptors (GPCRs) act as signaling hubs that direct molecular events to maintain cellular homeostasis. Historically, signal transduction has been thought to take place exclusively at the plasma membrane—where receptors activate heterotrimeric G proteins and β-arrestins (βARRs) desensitize them (1). This canonical view of receptor signaling has been challenged in recent years by observations that GPCRs can activate G proteins at endomembranes such as early endosomes, the Golgi apparatus, mitochondria, and the nucleus and that βARRs modulate signal amplitude and duration (2). These findings are important in the context of pharmacology and drug discovery because they will redefine how we understand cell signaling and may impact drug development in the future. Our understanding of the mechanisms controlling endomembrane signaling has been limited by the lack of tools required to quantitatively measure not only the presence of G protein but also their signaling at a subcellular level.Studies aimed at better understanding G protein trafficking have largely focused on G_s, which is known to leave the plasma membrane upon activation, become cytoplasmic, and sample multiple endomembrane compartments (3, 4). Mechanistic insight into this phenomenon has revealed that G_s becomes reactivated at endosomes following translocation (5). However, less is known about the activation status of other G protein families found in subcellular organelles. In particular, G_q has been shown to transit from the plasma membrane to endosomes upon receptor activation and as a consequence of activating mutations (6–8). Yet, the contribution of βARR and receptor endocytosis to G protein trafficking and activation at endosomes has not been documented. In the absence of tools that directly measure G protein activation, current evidence for endosomal G_q activity has been limited to amplified downstream signaling events.Nanobodies have been used as crystallization chaperones and as live cell imaging tools to visualize the distribution of active-state proteins (9). These tools have been particularly effective for G_s-coupled receptors in the absence of tools directly measuring the activity of the G protein in organelles (i.e., known effector regulators of G_s signaling). In particular, nanobodies or engineered G proteins that bind the active conformation of the receptor and nanobodies that stabilize the nucleotide-free state of G_s were used to suggest that receptor-G_s complexes were active at endomembranes (5, 10–12). In order to unearth more direct evidence of activation and a better understanding of the mechanisms for the other G protein families, tools that directly monitor the molecular events that immediately follow G protein activation would offer much-needed insight.Enhanced bystander bioluminescence resonance energy transfer (ebBRET) was developed as a way to provide more sensitive and robust measurements of protein trafficking and activity in living cells. This technique relies on the natural association of the luciferase and green fluorescent protein (GFP) that both come from Renilla reniformis (Rluc and rGFP, respectively). These two proteins do not interact spontaneously unless they are concentrated in the same compartment and thus can be used to monitor protein movement when the energy acceptor is anchored to a specific subcellular compartment (13). To measure the activity of G proteins at endosomes, we engineered an ebBRET-based effector membrane translocation assay (EMTA) for subcellular organelles. In addition, we developed organelle-targeted inhibitors to block G protein activity in a compartment-specific manner. Using these tools, we demonstrate the direct activation of G_q/11 by angiotensin II type 1 (AT₁R), bradykinin B₂ (B₂R), oxytocin (OXTR), thromboxane A₂ alpha isoform (TPαR), and muscarinic acetylcholine M₃ (M₃R) receptors in endosomes. Using cells devoid of βARRs (14), our results show that in contrast to the GPCRs, for which trafficking to endosomes is βARR-dependent, the trafficking of G_q to this compartment was not. These findings indicate that G_q translocation can occur through nonconventional receptor endocytosis–independent pathways. Interestingly, activation of G_q in the endosomal compartment was two-tiered, displaying a component that was βARR-independent and another that was further promoted by βARR, supporting the notion that the formation of a receptor–G_q complex in endosomes can promote G protein activation. Moreover, we show that G_q signaling at the plasma membrane not only exhibits a faster onset than the signaling that takes places in endosomes, but it is also functionally different. We anticipate that our methodological advances and mechanistic understanding of endosomal G_q signaling will be of general interest to the study of other GPCRs and to the study of G protein signaling in other compartments.     

Adenosine: trigger and mediator of cardioprotection

Adenosine, a purine nucleoside, is ubiquitous in the body, and is a critical component of ATP. Its concentration jumps 100-fold during periods of oxygen depletion and ischemia. There are four adenosine receptors: A₁ and A₃ coupled to G_i/o and the high-affinity A_2A and low-affinity A_2B coupled to G_s. Adenosine is one of three autacoids released by ischemic tissue which are important triggers of ischemic preconditioning (IPC). It is the A₁ and to some extent A₃ receptors which participate in the intracellular signaling that triggers cardioprotection. Unlike bradykinin and opioids, the other two autacoids, adenosine is not dependent on opening of mitochondrial K_ATP channels or release of reactive oxygen species (ROS), but rather activates phospholipase C and/or protein kinase C (PKC) directly. Another signaling cascade at reperfusion involves activated PKC which initiates binding to and activation of an A₂ adenosine receptor that we believe is the A_2B. Although the latter is the low-affinity receptor, its interaction with PKC increases its affinity and makes it responsive to the accumulated tissue adenosine. A_2B agonists, but not adenosine or A₁ agonists, infused at reperfusion can initiate this second signaling cascade and mimic preconditioning’s protection. The same A_2B receptors are critical for postconditioning’s protection. Thus adenosine is both an important trigger and a mediator of cardioprotection. Returned for 1. revision: 17 September 2007 1. Revision received: 4 October 2007 Returned for 2. revision: 11 October 2007 2. Revision received: 16 October 2007     

Selective recruitment of G protein-coupled receptor kinases (GRKs) controls signaling of the insulin-like growth factor 1 receptor

β-Arrestins are multifunctional proteins that play central roles in G protein-coupled receptor (GPCR) trafficking and signaling. β-Arrestin1 is also recruited to the insulin-like growth factor-1 receptor (IGF-1R), a receptor tyrosine kinase (RTK), mediating receptor degradation and signaling. Because GPCR phosphorylation by GPCR-kinases (GRKs) governs interactions of the receptors with β-arrestins, we investigated the regulatory roles of the four widely expressed GRKs on IGF-1R signaling/degradation. By suppressing GRK expression with siRNA, we demonstrated that lowering GRK5/6 abolishes IGF1-mediated ERK and AKT activation, whereas GRK2 inhibition increases ERK activation and partially inhibits AKT signaling. Conversely, β-arrestin-mediated ERK signaling is enhanced by overexpression of GRK6 and diminished by GRK2. Similarly, we demonstrated opposing effects of GRK2 and -6 on IGF-1R degradation: GRK2 decreases whereas GRK6 enhances ligand-induced degradation. GRK2 and GRK6 coimmunoprecipitate with IGF-1R and increase IGF-1R serine phosphorylation, promoting β-arrestin1 association. Using immunoprecipitation, confocal microscopy, and FRET analysis, we demonstrated β-arrestin/IGF-1R association to be transient for GRK2 and stable for GRK6. Using bioinformatic studies we identified serines 1248 and 1291 as the major serine phosphorylation sites of the IGF-1R, and subsequent mutation analysis demonstrated clear effects on IGF-1R signaling and degradation, mirroring alterations by GRKs. Targeted mutation of S1248 recapitulates GRK2 modulation, whereas S1291 mutation resembles GRK6 effects on IGF-1R signaling/degradation, consistent with GRK isoform-specific serine phosphorylation. This study demonstrates distinct roles for GRK isoforms in IGF-1R signaling through β-arrestin binding with divergent functional outcomes.     

A G protein γ subunit-like domain shared between RGS11 and other RGS proteins specifies binding to Gβ5 subunits

Regulators of G protein signaling (RGS) proteins act as GTPase-activating proteins (GAPs) toward the α subunits of heterotrimeric, signal-transducing G proteins. RGS11 contains a G protein γ subunit-like (GGL) domain between its Dishevelled/Egl-10/Pleckstrin and RGS domains. GGL domains are also found in RGS6, RGS7, RGS9, and the Caenorhabditis elegans protein EGL-10. Coexpression of RGS11 with different G_β subunits reveals specific interaction between RGS11 and G_β5. The expression of mRNA for RGS11 and G_β5 in human tissues overlaps. The G_β5/RGS11 heterodimer acts as a GAP on G_αo, apparently selectively. RGS proteins that contain GGL domains appear to act as GAPs for G_α proteins and form complexes with specific G_β subunits, adding to the combinatorial complexity of G protein-mediated signaling pathways.     

Molecular and cellular pharmacological properties of 5‐methoxycarbonylamino‐N‐acetyltryptamine (MCA‐NAT): a nonspecific MT3 ligand

Abstract: 5‐Methoxycarbonylamino‐N‐acetyltryptamine (MCA‐NAT) has been initially described as a ligand at non MT₁, non MT₂ melatonin binding site (MT3) selective versus MT₁ and MT₂, two membrane melatonin receptors. MCA‐NAT activity has been reported by others in different models, in vivo, particularly in the intra‐ocular pressure (IOP) models in rabbits and monkeys. Its activity was systematically linked to either MT3 or to a new, yet unknown, melatonin receptor. In this article, the melatonin receptor pharmacology of MCA‐NAT is described. MCA‐NAT has micromolar range affinities at the melatonin receptors MT₁ and MT₂, while in functional studies, MCA‐NAT proved to be a powerful MT₁/MT₂ partial agonist in the sub‐micromolar range. These data strongly suggest that MCA‐NAT actions might be mediated by these receptors in vivo. Finally, as described by others, we show that MCA‐NAT is unable to elicit any type of receptor‐like functional responses from Chinese hamster ovary cells over‐expressing quinone reductase 2, the MT3.     

A role for heterotrimeric GTP-binding proteins and ERK1/2 in insulin-mediated, nitric-oxide-dependent, cyclic GMP production in human umbilical vein endothelial cells

Aims/hypothesis Insulin is known to stimulate endothelial nitric oxide synthesis, although much remains unknown about the intracellular mechanisms involved. This study aims to examine, in human endothelial cells, the specific contribution of heterotrimeric G_i proteins and extracellular signal-regulated protein kinases 1/2 (ERK1/2) in insulin signalling upstream of nitric-oxide-dependent cyclic GMP production.Methods Human umbilical vein endothelial cells were treated with 1 nmol/l insulin in the presence or absence of inhibitors of tyrosine kinases (erbstatin), G_i proteins (pertussis toxin) or ERK1/2 (PD098059 or U0126), and nitric oxide production was examined by quantification of intracellular cyclic GMP. Activation/phosphorylation of ERK1/2 by insulin was examined by immunoblotting with specific antibodies, and direct association of the insulin receptor with G_i proteins was examined by immunoprecipitation.Results Treatment of cells with a physiological concentration of insulin (1 nmol/l) for 5 min increased nitric-oxide-dependent cyclic GMP accumulation by 3.3-fold, and this was significantly inhibited by erbstatin. Insulin-stimulated cyclic GMP production was significantly reduced by pertussis toxin and by the inhibitors of ERK1/2, PD098059 and U0126. Immunoblotting indicated that insulin stimulated the phosphorylation of ERK1/2 after 5 min and 1 h, and that this was completely abolished by pertussis toxin, but insensitive to the nitric oxide synthase inhibitor l-NAME. No direct interaction of the insulin receptor with could be demonstrated by immunoprecipitation.Conclusions/interpretation This study demonstrates, for the first time, that nitric oxide production induced by physiologically relevant concentrations of insulin, is mediated by the post-receptor activation of a pertussis-sensitive GTP-binding protein and subsequent downstream activation of the ERK1/2 cascade.     

M3-muscarinic receptor promotes insulin release via receptor phosphorylation/arrestin-dependent activation of protein kinase D1

The activity of G protein-coupled receptors is regulated via hyper-phosphorylation following agonist stimulation. Despite the universal nature of this regulatory process, the physiological impact of receptor phosphorylation remains poorly studied. To address this question, we have generated a knock-in mouse strain that expresses a phosphorylation-deficient mutant of the M(3)-muscarinic receptor, a prototypical G(q/11)-coupled receptor. This mutant mouse strain was used here to investigate the role of M(3)-muscarinic receptor phosphorylation in the regulation of insulin secretion from pancreatic islets. Importantly, the phosphorylation deficient receptor coupled to G(q/11)-signaling pathways but was uncoupled from phosphorylation-dependent processes, such as receptor internalization and β-arrestin recruitment. The knock-in mice showed impaired glucose tolerance and insulin secretion, indicating that M(3)-muscarinic receptors expressed on pancreatic islets regulate glucose homeostasis via receptor phosphorylation-/arrestin-dependent signaling. The mechanism centers on the activation of protein kinase D1, which operates downstream of the recruitment of β-arrestin to the phosphorylated M(3)-muscarinic receptor. In conclusion, our findings support the unique concept that M(3)-muscarinic receptor-mediated augmentation of sustained insulin release is largely independent of G protein-coupling but involves phosphorylation-/arrestin-dependent coupling of the receptor to protein kinase D1.     

β-肾上腺素受体介导的G蛋白及β-arrestin依赖性通路

β-肾上腺素受体所介导的经典G蛋白通路在心功能的调节中发挥着重要的作用,参与调节心肌收缩以及心肌细胞肥大等过程。近年来的研究发现,β-arrestin与β-肾上腺素受体的脱敏、内化、再循环以及降解密切相关,并可以通过其桥梁作用,使受体与多条信号通路之间建立联系。此外,β-arrestin还可以进入细胞核或者通过NF-kB/ERK进而参与表达水平的调控。鉴于β-arrestin与β-肾上腺素受体的密切关系及其本身作用的多样性,对它的深入研究将有助于新一代G蛋白偶联受体药物的研发。     

Melatonin‐mediated inhibition of Cav3.2 T‐type Ca2+ channels induces sensory neuronal hypoexcitability through the novel protein kinase C‐eta isoform

Recent studies implicate melatonin in the antinociceptive activity of sensory neurons. However, the underlying mechanisms are still largely unknown. Here, we identify a critical role of melatonin in functionally regulating Cav3.2 T‐type Ca²⁺ channels (T‐type channel) in trigeminal ganglion (TG) neurons. Melatonin inhibited T‐type channels in small TG neurons via the melatonin receptor 2 (MT₂ receptor) and a pertussis toxin‐sensitive G‐protein pathway. Immunoprecipitation analyses revealed that the intracellular subunit of the MT₂ receptor coprecipitated with Gα_o. Both shRNA‐mediated knockdown of Gα_o and intracellular application of QEHA peptide abolished the inhibitory effects of melatonin. Protein kinase C (PKC) antagonists abolished the melatonin‐induced T‐type channel response, whereas inhibition of conventional PKC isoforms elicited no effect. Furthermore, application of melatonin increased membrane abundance of PKC‐eta (PKC_η) while antagonism of PKC_η or shRNA targeting PKC_η prevented the melatonin‐mediated effects. In a heterologous expression system, activation of MT₂ receptor strongly inhibited Cav3.2 T‐type channel currents but had no effect on Cav3.1 and Cav3.3 current amplitudes. The selective Cav3.2 response was PKC_η dependent and was accompanied by a negative shift in the steady‐state inactivation curve. Furthermore, melatonin decreased the action potential firing rate of small TG neurons and attenuated the mechanical hypersensitivity in a mouse model of complete Freund's adjuvant‐induced inflammatory pain. These actions were inhibited by T‐type channel blockade. Together, our results demonstrated that melatonin inhibits Cav3.2 T‐type channel activity through the MT₂ receptor coupled to novel G_βγ‐mediated PKC_η signaling, subsequently decreasing the membrane excitability of TG neurons and pain hypersensitivity in mice.     

Elucidation of G-protein and β-arrestin functional selectivity at the dopamine D2 receptor

The neuromodulator dopamine signals through the dopamine D2 receptor (D₂R) to modulate central nervous system functions through diverse signal transduction pathways. D₂R is a prominent target for drug treatments in disorders where dopamine function is aberrant, such as schizophrenia. D₂R signals through distinct G-protein and β-arrestin pathways, and drugs that are functionally selective for these pathways could have improved therapeutic potential. How D₂R signals through the two pathways is still not well defined, and efforts to elucidate these pathways have been hampered by the lack of adequate tools for assessing the contribution of each pathway independently. To address this, Evolutionary Trace was used to produce D₂R mutants with strongly biased signal transduction for either the G-protein or β-arrestin interactions. These mutants were used to resolve the role of G proteins and β-arrestins in D₂R signaling assays. The results show that D₂R interactions with the two downstream effectors are dissociable and that G-protein signaling accounts for D₂R canonical MAP kinase signaling cascade activation, whereas β-arrestin only activates elements of this cascade under certain conditions. Nevertheless, when expressed in mice in GABAergic medium spiny neurons of the striatum, the β-arrestin–biased D₂R caused a significant potentiation of amphetamine-induced locomotion, whereas the G protein-biased D₂R had minimal effects. The mutant receptors generated here provide a molecular tool set that should enable a better definition of the individual roles of G-protein and β-arrestin signaling pathways in D₂R pharmacology, neurobiology, and associated pathologies.G protein-coupled receptors (GPCRs) are the largest receptor family and transmit the physiological effects of numerous biologically active molecules. GPCR signal transduction cascades account for diverse genomic, biochemical, cellular, and behavioral responses including cell fate determination, developmental reprogramming, olfactory, taste and light sensation, as well as complex behaviors mediated by neuromodulators (1). The diversity of responses to a particular hormone or neuromodulator is dictated not only by its cognate receptor but also by the ability of that receptor to engage distinct signaling pathways. For a number of GPCRs, their propensity to activate distinct G proteins can elicit diverse responses depending on the cellular environment (2). However, an even more subtle but intriguing mode of signaling has been attributed to the ability of a receptor to activate signaling pathways independent of G-protein activation, through the scaffolding of signaling complexes by β-arrestin, a component of the GPCR desensitization and internalization machinery (3). These two signaling modes harbor distinct functional properties, and in instances the same ligand can act as an agonist for one pathway but antagonist at the other. The selective or biased activation of a given pathway is commonly referred to as “functional selectivity” and can be easily demonstrated in heterologous systems especially when biased small molecule ligands are available (4). Biased GPCR ligands may have high therapeutic potential as these receptors represent the largest targets of drugs on the market. However, determining the functional contributions of G-protein and β-arrestin signaling pathways to the biological actions of an endogenous ligand acting upon its receptor still remains a challenging undertaking.Dopamine (DA) is a neuromodulator that is known to regulate movement, reward, cognition, emotion, and affect. The dopamine D2 receptor (D₂R) is a prominent GPCR that mediates the actions of DA. All typical antipsychotics, such as haloperidol, are potent D₂R blockers (5), whereas atypical antipsychotics, such as aripiprazole and clozapine, have unique pharmacology, exhibiting weak partial agonist activity at D₂R or reduced antagonist efficacy, respectively (6). Previous studies have demonstrated the ability of D₂Rs to engage different signal transduction pathways depending on the cellular complement of G proteins as well as their ability to regulate different physiological processes (7–9). β-arrestin 2 knockout mice provided robust behavioral and biochemical evidence for a critical D₂R/β-arrestin signaling pathway in the striatum (10). Furthermore, neuronal selective deletion of GSK3β, a putative D₂R/β-arrestin 2 effector, could reproduce the pharmacological blockade of D₂Rs with antipsychotics (11). Although these studies suggest that D₂Rs, like many other GPCRs, use pleiotropic signaling pathways to mediate their effects, the brain DA system is uniquely complex, as diverse responses may also rely upon many other determinants. One well-documented variable is the mode of stimulation of DA receptors, which is a function of the tonic or phasic release of DA (12). The expression profile of D₂R is also complex, being expressed not only in DA synthesizing neurons of the substantia nigra and ventral tegmental area where they function as presynaptic autoreceptors but also in GABAergic medium spiny neurons (MSNs), cholinergic interneurons of the striatum, and cortical neurons (13), where they function as postsynaptic receptors. Thus, understanding the contributions of functional selectivity at D₂R in intact biological systems is a challenge that cannot be elucidated in heterologous systems alone. To develop tools where this challenge can begin to be addressed, the Evolutionary Trace (ET) (14) approach was used to engineer D₂R mutants that selectively interact with either G proteins or β-arrestins, designated ^[Gprot]D₂R and ^[βarr]D₂R, respectively. These mutants show separation of G-protein and β-arrestin interactions, and expression of these mutants in vivo in the mouse striatum provides proof-of-concept for their biological activity and discrete functions.