Structural and biological characterization of three novel mastoparan peptides from the venom of the neotropical social wasp Protopolybia exigua (Saussure).

The venom of the Neotropical social wasp Protopolybia exigua(Saussure) was fractionated by RP-HPLC resulting in the elution of 20 fractions. The homogeneity of the preparations were checked out by using ESI-MS analysis and the fractions 15, 17 and 19 (eluted at the most hydrophobic conditions) were enough pure to be sequenced by Edman degradation chemistry, resulting in the following sequences: Protopolybia MPI I-N-W-L-K-L-G-K-K-V-S-A-I-L-NH2 Protopolybia-MP II I-N-W-K-A-I-I-E-A-A-K-Q-A-L-NH2 Protopolybia-MP III I-N-W-L-K-L-G-K-A-V-I-D-A-L-NH2 All the peptides were manually synthesized on-solid phase and functionally characterized. Protopolybia-MP I is a hemolytic mastoparan, probably acting on mast cells by assembling in plasma membrane, resulting in pore formation; meanwhile, the peptides Protopolybia-MP II and -MP III were characterized as a non-hemolytic mast cell degranulator toxins, which apparently act by virtue of their binding to G-protein receptor, activating the mast cell degranulation.     

Expression of G-protein Alpha Subunit Genes in the Vestibular Periphery of Rattus norvegieus and their Chromosomal Mapping

Objective—Heterotrimeric G-proteins play an important role in mediating signals transduced across the cell membrane by membrane-bound receptors. The precise role of G-proteins and their coupled receptors in the physiology of the vestibular neuroepithelium is not well understood. The purpose of this study was to better define the role of these proteins by examining their expression in the rat vestibular periphery and characterizing their chromosomal location.

Material and Methods—To characterize G-protein α subunit gene expression in the target tissue of interest, we performed polymerase chain reaction (PCR) using degenerate G-protein primers corresponding to conserved regions in the G-protein α subunit coding sequence on a normalized rat vestibular cDNA library. PCR amplicons were cloned and 50 clones were randomly selected and sequenced. Radiation hybrid (RH) mapping was used to determine the chromosomal location of Gα_olf and two previously identified G-protein α subunits—Gα_i2 and Gα_i2(vest)—in the rat genome.

Results—The following G-protein α subunits were identified in the normalized cDNA library: Gα_olf, Gα_s, Gα_o and Gα_s2. Gα_olf maps to chromosome 18 between markers D18Mit17b and D18Mgh2. Gα_i2 maps to chromosome 8 between markers D8Rat65 and D8Mgh2. Gα_i2(vest) maps to chromosome 1 between markers D1Rat132 and D1Rat202. These chromosomal locations in the rat genome are syntenic to chromosomal regions in which the homologous G-protein α subunit genes have been localized in the human and mouse genomes, further validating RH mapping as an effective and accurate tool. We were unable to RH map the location of Gα_o due to its extensive homology with the hamster gene.

Conclusion—The characterization of G-protein alpha subunit gene expression in the vestibular periphery and the chromosomal localization of these genes in the rat revealed that a diverse group of these second messengers are expressed.     

Localization of ocular albinism-1 gene product GPR143 in the rat central nervous system

l-3,4-Dihydroxyphenylalanine (DOPA) has been believed to be a precursor of dopamine, and itself being an inert amino acid. Previously, we have proposed DOPA as a neurotransmitter candidate in the central nervous system (CNS). Recent findings have suggested DOPA as an endogenous agonist of a G-protein coupled receptor, ocular albinism 1 gene product (OA1), which is highly expressed in the retinal pigmental epithelium. However, whether OA1 functions as a receptor for DOPA in vivo, and whether this receptor–ligand interaction is responsible for a wide variety of DOPA actions have not been determined yet. To gain insight into the functional implication of OA1, we perform immunohistochemical examination with anti-OA1 antibody to localize OA1 in the adult rat brain. We observed OA1 immunoreactive cells in the hippocampus, cerebral cortex, cerebellum cortex, striatum, substantia nigra, hypothalamic median eminence and supraoptic nucleus, nucleus tractus solitarii and caudal ventrolateral medulla and rostral ventrolateral medulla, medial habenular nucleus and olfactory bulb. This study reveals, for the first time, the unique distribution pattern of OA1-immunoreactive neurons and/or cells in the rat CNS.     

The evolving small-molecule fluorescent-conjugate toolbox for Class A GPCRs

The past decade has witnessed fluorescently tagged drug molecules gaining significant attraction in their use as pharmacological tools with which to visualize and interrogate receptor targets at the single-cell level. Additionally, one can generate detailed pharmacological information, such as affinity measurements, down to almost single-molecule detection limits. The now accepted utilization of fluorescence-based readouts in high-throughput/high-content screening provides further evidence that fluorescent molecules offer a safer and more adaptable substitute to radioligands in molecular pharmacology and drug discovery. One such drug-target family that has received considerable attention are the GPCRs; this review therefore summarizes the most recent developments in the area of fluorescent ligand design for this important drug target. We assess recently reported fluorescent conjugates by adopting a receptor-family-based approach, highlighting some of the strengths and weaknesses of the individual molecules and their subsequent use. This review adds further strength to the arguments that fluorescent ligand design and synthesis requires careful planning and execution; providing examples illustrating that selection of the correct fluorescent dye, linker length/composition and geographic attachment point to the drug scaffold can all influence the ultimate selectivity and potency of the final conjugate when compared with its unlabelled precursor. When optimized appropriately, the resultant fluorescent conjugates have been successfully employed in an array of assay formats, including flow cytometry, fluorescence microscopy, FRET and scanning confocal microscopy. It is clear that fluorescently labelled GPCR ligands remain a developing and dynamic research arena.Linked ArticlesThis article is part of a themed section on Molecular Pharmacology of GPCRs. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-5     

Differential coupling of m-cholinoceptors to Gi/Go-proteins in failing human myocardium

Muscarinic acetylcholine receptors (mAChRs) mediate their main cardiac effects via pertussis toxin-sensitive G-proteins. Physiological effects differ considerably between atrium and ventricle, and it is unknown to which extent these differences derive from selective receptor-G-protein coupling or further downstream events. We have characterized specific coupling between mAChRs and Gi/Go-protein isoforms in atrial and ventricular myocardium by agonist-dependent photoaffinity labeling with [(32)P]azidoanilido GTP (aaGTP) and immunoprecipitation in sarcolemmal membranes from terminally failing human hearts. The total amount of mAChRs, as determined by specific binding of [(3)H]QNB, was significantly higher in right-atrial (RA +/- SEM, 959 +/- 68 fmol/mg, n = 4) than in left-ventricular membranes (LV, 582 +/- 53 fmol/mg, n = 6). Standardized immunoblots revealed that Gialpha-2 was the predominant subtype in both regions. A 40-kDa splice variant of Goalpha (Goalpha-1 and/or Goalpha-3) was almost exclusively detectable in RA. Levels of Gialpha-3 and a 39-kDa splice variant of Goalpha (Goalpha-2) were also higher in RA. Basal aaGTP binding was higher in RA than in LV for all Gialpha/Goalpha subtypes. The carbachol (10 micromol/l)-induced increase in aaGTP binding was significantly higher in RA than in LV for Goalpha-1/3 (336 +/- 95% of LV, n = 4) and for Gialpha-3 (211 +/- 83%), lower for Gialpha-2 (42 +/- 5%), and was similar in both regions for Goalpha-2 (130 +/- 62%). The differential coupling of mAChRs in human RA and LV suggests that the initiation of different physiological responses to mAChR stimulation starts with signal sorting at the receptor-G-protein level.     

干预G蛋白βγ亚基介导的信号转导途径对心肌细胞肥大的影响

目的 研究G蛋白βγ亚基在心肌细胞肥大中的作用.方法 采用细菌内同源重组方法制备重组腺病毒载体rAd-βARKct 50 MOI(multiplicity of infection)感染经去甲肾上腺素处理肥大乳鼠心肌细胞,荧光倒置显微镜下观察心肌细胞形态及绿色荧光蛋白的表达,EMAIL图像分析软件测定心肌细胞的表面积,RT-PCR检测βARKct、α-MHC和β-MHC的mRNA表达水平.结果 经测序鉴定证实rAd-β ARKct构建成功,对心肌细胞的感染率40%～50%,与去甲肾上腺素组相比,重组腺病毒组α-MHC定量比值显著上升,β-MHC定量比值显著降低.结论 编码βARKct的重组腺病毒表达载体可以体外感染乳鼠心肌细胞并在心肌细胞中表达βARKct;抑制Gβγ蛋白的功能,可显著逆转去甲肾上腺素介导的肌球蛋白重链表型的改变(α-MHC/β-MHC),逆转心肌细胞肥大.     

雌激素受体GPR30心血管保护作用研究进展

自从2005年研究发现G蛋白偶联受体30(G-protein coupled receptor 30,GPR30)可介导雌激素在体内的快速反应以来,GPR30对于心血管系统的调节和保护作用已成为研究热点。研究发现,GPR30激活后可作用于内皮细胞、血管平滑肌细胞和肾素-血管紧张素系统,产生舒张血管、降低血压等效应,其还具有抗炎、防止心肌再灌注损伤、改善血脂代谢等心血管保护作用。但目前有关GPR30研究大多仅限于体外细胞和动物实验,其在人体中的生理和病理作用仍需进一步深入研究。     

G蛋白β3亚单位基因825C/T多态性与中国人群原发性高血压相关性的Meta分析

目的系统评价G蛋白β3亚单位(GNB3)基因825C/T多态性与中国人群原发性高血压(EH)发病风险的关系。方法由两名评价者独立检索PubMed、EMbase、CNKI、CBM和WanFang数据库,收集探讨GNB3基因825C/T多态性与中国人EH相关性的病例-对照研究,检索时限均为建库至2013年9月30日。文献筛选及资料提取后,对纳入文献按NOS进行质量评价,然后采用Stata12.0软件行Meta分析。结果最终纳入30个病例-对照研究,包括EH患者5054例,对照人群5565例。Meta分析结果显示,与对照组相比,GNB3基因825C/T多态性与中国人EH发病风险间无统计学差异[TT vs.CC:(OR=1.13,95%CI:0.89~1.43,P=0.33);TT vs.CT+CC:(OR=1.04,95%CI:0.86~1.26,P=0.70);CT vs.CC:(OR=1.08,95%CI:0.98~1.19,P=0.11);TT+CT vs.CC:(OR=1.11,95%CI:0.96~1.29,P=0.15);T vs.C:(OR=1.06,95%CI:0.95~1.20,P=0.30)]。结论当前证据表明中国人群GNB3基因825C/T多态性与EH发病无关。     

Sushi domains confer distinct trafficking profiles on GABAB receptors

GABA(B) receptors mediate slow inhibitory neurotransmission in the brain and feature during excitatory synaptic plasticity, as well as various neurological conditions. These receptors are obligate heterodimers composed of GABA(B)R1 and R2 subunits. The two predominant R1 isoforms differ by the presence of two complement control protein modules or Sushi domains (SDs) in the N terminus of R1a. By using live imaging, with an α-bungarotoxin-binding site (BBS) and fluorophore-linked bungarotoxin, we studied how R2 stabilizes R1b subunits at the cell surface. Heterodimerization with R2 reduced the rate of internalization of R1b, compared with R1b homomers. However, R1aR2 heteromers exhibited increased cell surface stability compared with R1bR2 receptors in hippocampal neurons, suggesting that for receptors containing the R1a subunit, the SDs play an additional role in the surface stability of GABA(B) receptors. Both SDs were necessary to increase the stability of R1aR2 because single deletions caused the receptors to be internalized at the same rate and extent as R1bR2 receptors. Consistent with these findings, a chimera formed from the metabotropic glutamate receptor (mGluR)2 and the SDs from R1a increased the surface stability of mGluR2. These results suggest a role for SDs in stabilizing cell surface receptors that could impart different pre- and postsynaptic trafficking itineraries on GABA(B) receptors, thereby contributing to their physiological and pathological roles.     

P2Y受体与三叉神经痛之间的关系

腺苷三磷酸(ATP)作为一种重要的神经递质具有广泛的生物学效应。多种伤害性刺激均可引起三叉神经节(TG)神经元释放ATP,ATP激活P2受体可引起疼痛。P2受体又分为P2X和P2Y两类。在受损的TG神经元上,P2Y受体的表达明显增多。本文就ATP与P2Y受体表达间的相关性、P2Y受体在TG中的信号转导机制、P2Y受体对神经损伤性痛觉信号的转导作用等研究进展作一综述。