昆虫多巴胺及其受体的研究进展

多巴胺（dopamine, DA）是脊椎动物和无脊椎动物体内一种重要的生物胺, 其参与调控了昆虫的多种生理反应和行为过程, 如学习与记忆、 认知、 性取向、 抉择、 运动以及型变等。多巴胺主要通过结合特异性的G蛋白偶联受体, 即多巴胺受体（dopamine receptors, DARs）来发挥生理作用。本文综述了多巴胺在昆虫中的调控、 分布及所参与的生理功能, 如多巴胺调控昆虫的交配、 发育、 嗅觉以及运动行为等, 特别对DARs的信号转导、 生理功能以及药理学等方面进行了详细评述。昆虫的DARs大致可分为两大类： D1-like DARs和D2-like DARs。D1-like DARs包含有2种亚型, 分别为DOP1和DOP2。DOP1仅能偶联胞内cAMP的上升, 而DOP2不仅可以起胞内cAMP的上升, 还可偶联胞内Ca²⁺的释放。 D2-like DARs仅包含有1种亚型DOP3, 其被激活后引起胞内cAMP的降低。DA通过激活不同的DARs可偶联不同的第二信使系统, 所产生的下游细胞反应则与昆虫的各种行为相关, 而对昆虫DARs的药理学研究将有助于我们开发特异性的杀虫剂用于害虫防治。     

Sphingosine‐1‐phosphate receptors and innate immunity

Sphingosine‐1‐phosphate (S1P) is a signalling lipid that regulates many cellular processes in mammals. One well‐studied role of S1P signalling is to modulate T‐cell trafficking, which has a major impact on adaptive immunity. Compounds that target S1P signalling pathways are of interest for immune system modulation. Recent studies suggest that S1P signalling regulates many more cell types and processes than previously appreciated. This review will summarise current understanding of S1P signalling, focusing on recent novel findings in the roles of S1P receptors in innate immunity.     

The melanosomal/lysosomal protein OA1 has properties of a G protein‐coupled receptor

The protein product of the ocular albinism type 1 gene, named OA1, is a pigment cell‐specific integral membrane glycoprotein, localized to melanosomes and lysosomes and possibly implicated in melanosome biogenesis. Although its function remains unknown, we previously showed that OA1 shares structural similarities with G protein‐coupled receptors (GPCRs). To ascertain the molecular function of OA1 and in particular its nature as a GPCR, we adopted a heterologous expression strategy commonly exploited to demonstrate GPCR‐mediated signaling in mammalian cells. Here we show that when expressed in COS7 cells OA1 displays a considerable and spontaneous capacity to activate heterotrimeric G proteins and the associated signaling cascade. In contrast, OA1 mutants carrying either a missense mutation or a small deletion in the third cytosolic loop lack this ability. Furthermore, OA1 is phosphorylated and interacts with arrestins, well‐established multifunctional adaptors of conformationally active GPCRs. In fact, OA1 colocalizes and coprecipitates with arrestins, which downregulate the signaling of OA1 by specifically reducing its expression levels. These findings indicate that heterologously expressed OA1 exhibits two fundamental properties of GPCRs, being capable to activate heterotrimeric G proteins and to functionally associate with arrestins, and provide proof of principle that OA1 can actually function as a canonical GPCR in mammalian cells.     

Ins and outs of GPCR signaling in primary cilia

Primary cilia are specialized microtubule‐based signaling organelles that convey extracellular signals into a cellular response in most vertebrate cell types. The physiological significance of primary cilia is underscored by the fact that defects in assembly or function of these organelles lead to a range of severe diseases and developmental disorders. In most cell types of the human body, signaling by primary cilia involves different G protein‐coupled receptors (GPCRs), which transmit specific signals to the cell through G proteins to regulate diverse cellular and physiological events. Here, we provide an overview of GPCR signaling in primary cilia, with main focus on the rhodopsin‐like (class A) and the smoothened/frizzled (class F) GPCRs. We describe how such receptors dynamically traffic into and out of the ciliary compartment and how they interact with other classes of ciliary GPCRs, such as class B receptors, to control ciliary function and various physiological and behavioral processes. Finally, we discuss future avenues for developing GPCR‐targeted drug strategies for the treatment of ciliopathies.     

Loop prediction for a GPCR homology model: Algorithms and results

We present loop structure prediction results of the intracellular and extracellular loops of four G‐protein‐coupled receptors (GPCRs): bovine rhodopsin (bRh), the turkey β1‐adrenergic (β1Ar), the human β2‐adrenergic (β2Ar) and the human A2a adenosine receptor (A2Ar) in perturbed environments. We used the protein local optimization program, which builds thousands of loop candidates by sampling rotamer states of the loops' constituent amino acids. The candidate loops are discriminated between with our physics‐based, all‐atom energy function, which is based on the OPLS force field with implicit solvent and several correction terms. For relevant cases, explicit membrane molecules are included to simulate the effect of the membrane on loop structure. We also discuss a new sampling algorithm that divides phase space into different regions, allowing more thorough sampling of long loops that greatly improves results. In the first half of the paper, loop prediction is done with the GPCRs' transmembrane domains fixed in their crystallographic positions, while the loops are built one‐by‐one. Side chains near the loops are also in non‐native conformations. The second half describes a full homology model of β2Ar using β1Ar as a template. No information about the crystal structure of β2Ar was used to build this homology model. We are able to capture the architecture of short loops and the very long second extracellular loop, which is key for ligand binding. We believe this the first successful example of an RMSD validated, physics‐based loop prediction in the context of a GPCR homology model. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

PKD, PKD2, and p38 MAPK mediate Hsp27 serine-82 phosphorylation induced by neurotensin in pancreatic cancer PANC-1 cells

It is widely recognized that Hsp27 is a downstream substrate of the p38 MAPK cascade whereas the role of PKD family members in mediating receptor-stimulated Hsp27 Ser-82 phosphorylation has not been evaluated. Here, we show that neurotensin induced a rapid and striking increase in Hsp27 Ser-82 phosphorylation in PANC-1 cells, which was closely correlated with stimulation of activation loop phosphorylation of PKDs and p38 MAPK Thr180/Tyr182 phosphorylation. Treatment of PANC-1 cells with either the selective PKC inhibitor GF-I or the p38 MAPK inhibitor SB202190 partially reduced neurotensin-induced Hsp27 Ser-82 phosphorylation. However, treatment of the cells with a combination of GF-I and SB202190 virtually abolished neurotensin-induced Hsp27 Ser-82 phosphorylation. Overexpression of PKD in stably transfected PANC-1 cells increased the magnitude and prolonged the duration of Hsp27 Ser-82 phosphorylation in response to neurotensin. Either PKD or PKD2 gene silencing utilizing siRNAs targeting distinct PKD or PKD2 sequences reduced neurotensin-stimulated Hsp27 Ser-82 phosphorylation, but cotransfection of siRNAs targeting both, PKD and PKD2, markedly decreased neurotensin-induced Hsp27 Ser-82 phosphorylation. Knockdown of PKD and PKD2 abolished Hsp27 phosphorylation in cells treated with SB202190. Thus, neurotensin induces Hsp27 Ser-82 phosphorylation through p38 MAPK- and PKC/PKD-dependent pathways in PANC-1 cells. Our results demonstrate, for the first time, that neurotensin induces a striking increase in Hsp27 phosphorylation on Ser-82 in PANC-1 cells through convergent p38 MAPK, PKD, and PKD2 signaling.     

Isobolographic analysis of the opioid-opioid interactions in a tonic and a phasic mouse model of induced nociceptive pain

Background

Opioids have been used for the management of pain and coadministration of two opioids may induce synergism. In a model of tonic pain, the acetic acid writhing test and in a phasic model, the hot plate, the antinociceptive interaction between fentanyl, methadone, morphine, and tramadol was evaluated.

Results

The potency of opioids in the writhing test compared to the hot plate assay was from 2.5 (fentanyl) to 15.5 (morphine) times, respectively. The ED₅₀ was used in a fixed ratio for each of the six pairs of opioid combinations, which, resulted in a synergistic antinociception except for methadone/tramadol and fentanyl/tramadol which were additive, in the hot plate. The opioid antagonists naltrexone, naltrindole and nor-binaltorphimine, suggests that the synergism of morphine combinations are due to the activation of MOR subtypes with partially contribution of DOR and KOR, however fentanyl and methadone combinations are partially due to the activation of MOR and DOR subtypes and KOR lack of participation. The antinociceptive effects of tramadol combinations, are partially due to the activation of MOR, DOR and KOR opioid subtypes.

Conclusion

These results suggets that effectiveness and magnitude of the interactions between opioids are dependent on pain stimulus intensity.     

Group 1 metabotropic glutamate receptors 1 and 5 form a protein complex in mouse hippocampus and cortex

The group 1 metabotropic glutamate receptors 1 and 5 (mGluR1/5) have been implicated in mechanisms of synaptic plasticity and may serve as potential therapeutic targets in autism spectrum disorders. The interactome of group 1 mGluRs has remained largely unresolved. Using a knockout‐controlled interaction proteomics strategy we examined the mGluR5 protein complex in two brain regions, hippocampus and cortex, and identified mGluR1 as its major interactor in addition to the well described Homer proteins. We confirmed the presence of mGluR1/5 complex by (i) reverse immunoprecipitation using an mGluR1 antibody to pulldown mGluR5 from hippocampal tissue, (ii) coexpression in HEK293 cells followed by coimmunoprecipitation to reveal the direct interaction of mGluR1 and 5, and (iii) superresolution microscopy imaging of hippocampal primary neurons to show colocalization of the mGluR1/5 in the synapse.     

家蚕神经肽及其受体的功能和信号转导机制研究进展

神经肽是一类由神经分泌细胞分泌、用于调节生物胞间信号传递的信号分子,其信号分子的膜定位、相应胞内信使的激活以及一系列级联反应的引发,是由存在于细胞表面的特异性受体分子来完成的。神经肽及其受体能够调控昆虫的几乎所有生命活动,在昆虫生长发育中起着关键作用。家蚕Bombyx mori作为鳞翅目昆虫的模式物种,是昆虫生长发育与生理学研究的重要模型。特别是家蚕基因组测序完成后,越来越多的家蚕神经肽及其受体被鉴定,并发现其在家蚕的生长发育、取食消化、蜕皮、滞育、繁殖、吐丝结茧等各种生理活动中都发挥了重要的调节作用。本文综述了家蚕重要神经肽的种类及其对家蚕取食消化、蜕皮变态、生殖发育等的调控作用,探讨了神经肽通过结合特异性受体而激活细胞内ERK、TOR等下游信号通路的分子作用机制,以期为昆虫神经肽及其受体研究提供借鉴和参考,并以此推进家蚕功能基因的研究,促进蚕丝产业的发展。     

G‐protein coupled receptor array technologies: Site directed immobilisation of liposomes containing the H1‐histamine or M2‐muscarinic receptors

This paper describes a novel strategy to create a microarray of G‐protein coupled receptors (GPCRs), an important group of membrane proteins both physiologically and pharmacologically. The H₁‐histamine receptor and the M₂‐muscarinic receptor were both used as model GPCRs in this study. The receptor proteins were embedded in liposomes created from the cellular membrane extracts of Spodoptera frugiperda (Sf9) insect cell culture line with its accompanying baculovirus protein insert used for overexpression of the receptors. Once captured onto a surface these liposomes provide a favourable lipidic environment for the integral membrane proteins. Site directed immobilisation of these liposomes was achieved by introduction of cholesterol‐modified oligonucleotides (oligos). These oligo/cholesterol conjugates incorporate within the lipid bilayer and were captured by the complementary oligo strand exposed on the surface. Sequence specific immobilisation was demonstrated using a quartz crystal microbalance with dissipation (QCM‐D). Confirmatory results were also obtained by monitoring fluorescent ligand binding to GPCRs captured on a spotted oligo microarray using Confocal Laser Scanning Microscopy and the ZeptoREADER microarray imaging system. Sequence specific immobilisation of such biologically important membrane proteins could lead to the development of a heterogeneous self‐sorting liposome array of GPCRs which would underpin a variety of future novel applications.     

Exposure to AC and DC magnetic fields induces changes in 5-HT1B receptor binding parameters in rat brain membranes

The binding properties of the G-protein coupled receptor (GPCR) serotonin 5-HT1B receptor were studied under exposure to AC (50 and 400 Hz) and DC magnetic fields (MF) in rat brain membranes. This was an attempt at replicating the positive findings of Massot et al. In saturation experiments using [3H]5-HT, 1-h exposures at 1.1 mT(rms) 50 Hz caused statistically significant increases in both the K(D) and B(max) binding parameters, from 1.74 +/- 0.3 to 4.51 +/- 0.86 nM and from 1428 +/- 205 to 2137 +/- 399 CPM, respectively, in good agreement with previous results. Exposure of the membranes at 400 Hz 0.675 mT(rms) did not elicit a larger increase in K(D) in spite of a much larger induced current density. DC fields (1.1 and 11 mT) had a lesser effect compared to AC fields at low values of K(Dsham), but decreased the affinity at higher values of K(Dsham). Modeling of the receptor-ligand-G protein interactions using the extended ternary complex model yielded good fits for all our data and that of Massot et al., showing that the AC field may act by decreasing the ability of the G-protein to alter the ligand-receptor affinity. The hypothesis is that the bipolar nature of the AC field explains the different nature of the effects observed with AC and DC exposures. These findings constitute one of the few documented pieces of evidence for cell-free effects of DC and extremely low frequency (ELF) AC MFs in the mT range.     

Effects of apelin-13 in mice model of experimental pain and peripheral nociceptive signaling in rat sensory neurons

Objective: Apelin-13 is an endogenous peptide with potential analgesic action, although the sites of its analgesic effects remain uncertain and the results are even controversial with regard to its pain modulating action. This study evaluated the possible pain-modulating action of peripherally administered apelin-13 using heat-induced, withdrawal latency to the thermal stimuli, acute pain model in mice. Involvement of peripheral mechanisms was tested, by using the intracellular calcium concentrations as a key signal for nociceptive transmission, in cultured rat dorsal root ganglion (DRG) neurons. Methods: DRG neurons were cultured on glass coverslips following enzymatic digestion and mechanical agitation, and loaded with the calcium-sensitive dye Fura-2 acetoxymethyl ester (1?µM). Intracellular calcium responses in individual DRG neurons were quantified by ratiometric calcium imaging technique. Results: Peripheral injection of a single dose of apelin-13 (100?mg/kg and 300?mg/kg) significantly decreases the latency to painful stimuli in a dose and time-dependent manner (p?<?0.01, p?<?0.05, respectively, n?=?8 each). Apelin-13 (0.1?µM and 1?µM) did not produce a significant effect on cytoplasmic Ca²⁺ ([Ca²⁺]_i) responses, evoked by membrane depolarization, in cultured rat DRG neurons. Conclusion: Together these results indicate that apelin-13 can cause increased pain sensitivity after peripheral administration, but this effect does not involve calcium mediated signaling in primary sensory neurons.     

How a small change in retinal leads to G-protein activation: initial events suggested by molecular dynamics calculations

Rhodopsin is the prototypical G-protein coupled receptor, coupling light activation with high efficiency to signaling molecules. The dark-state X-ray structures of the protein provide a starting point for consideration of the relaxation from initial light activation to conformational changes that may lead to signaling. In this study we create an energetically unstable retinal in the light activated state and then use molecular dynamics simulations to examine the types of compensation, relaxation, and conformational changes that occur following the cis-trans light activation. The results suggest that changes occur throughout the protein, with changes in the orientation of Helices 5 and 6, a closer interaction between Ala 169 on Helix 4 and retinal, and a shift in the Schiff base counterion that also reflects changes in sidechain interactions with the retinal. Taken together, the simulation is suggestive of the types of changes that lead from local conformational change to light-activated signaling in this prototypical system.     

Theoretical evidence of a salt bridge disruption as the initiating process for the alpha1d-adrenergic receptor activation: a molecular dynamics and docking study

This study reports the building of the three-dimensional structure of the rat alpha1d-adrenergic receptor through a topology approach based on the structure of the rhodopsin receptor from cryoelectron microscopy. The validity and reliability of the receptor model were assessed through exhaustive molecular dynamics and docking studies. Some interesting ligand-receptor interactions were identified along with significant differences between the binding mode of agonists and antagonists. The importance of the disruption of a salt bridge as a possible initial event leading to receptor activation is discussed on the basis of data from mutagenesis and molecular dynamics studies.     

不同频率的电针对大鼠神经源性痛的治疗作用

目的：探讨不同频率的电针能否减轻大鼠神经源性痛。方法：将大鼠右侧L5／L6脊神经结扎,用引起50％抬足的机械刺激阈值评价机械性痛觉超敏,用大鼠5min内从5℃冷权上的抬足次数反映冷诱发的持续性疼痛。用韩氏穴位神经刺激仪给与2Hz或100Hz电刺激。结果：①2Hz和100Hz电针均一轻痛觉超敏,2Hz起效较早。②两种频率电针均能减轻冷诱发的持续性疼痛,但2Hz持续的时间长,多次电针后2Hz的镇痛效果可持续长达48h。③针刺而不通电也能显著减轻冷诱发的持续性痛。结论：电针能减轻神经源性痛,且低频（2Hz）电针的镇痛效果优于高频（100Hz）电针。     

Quantitative expression profiling of G-protein-coupled receptors (GPCRs) in metastatic melanoma: the constitutively active orphan GPCR GPR18 as novel drug target

G-protein-coupled receptors (GPCRs) have been implicated in the tumorigenesis and metastasis of human cancers and are considered amongst the most desirable targets for drug development. Utilizing a robust quantitative PCR array, we quantified expression of 94 human GPCRs, including 75 orphan GPCRs and 19 chemokine receptors, and 36 chemokine ligands, in 40 melanoma metastases from different individuals and benign nevi. Inter-metastatic site comparison revealed that orphan GPR174 and CCL28 are statistically significantly overexpressed in subcutaneous metastases, while P2RY5 is overexpressed in brain metastases. Comparison between metastases (all three metastatic sites) and benign nevi revealed that 16 genes, including six orphan receptors (GPR18, GPR34, GPR119, GPR160, GPR183 and P2RY10) and chemokine receptors CCR5, CXCR4, and CXCR6, were statistically significantly differentially expressed. Subsequent functional experiments in yeast and melanoma cells indicate that GPR18, the most abundantly overexpressed orphan GPCR in all melanoma metastases, is constitutively active and inhibits apoptosis, indicating an important role for GPR18 in tumor cell survival. GPR18 and five other orphan GPCRs with yet unknown biological function may be considered potential novel anticancer targets in metastatic melanoma.