溶血磷脂酸受体及其信号转导

溶血磷脂酸(lysophosphatidic acid,LPA)是一种类生长因子的脂类信号分子.在血栓形成过程中被激活的血小板可以产生LPA.自从证明LPA有胞外信号功能以后,许多新的生物活性又被不断发现.LPA最主要的作用是诱导各类细胞增殖.人们已经找到几种LPA受体cDNA克隆.LPA主要通过G蛋白偶联受体影响靶细胞功能,其信号转导系统包括已知的几条信号通路: 激活G_q从而激活磷脂酶C; 激活G_i从而抑制腺苷酸环化酶并激活MAPK级联通路; 激活G_12/13从而激活Rho级联通路等.     

甾体激素受体的结构

现已克隆出糖皮质激素受体、雌激素受体、孕激素受体和1,25-二羟基维生素D₃受体的cDNA,并对受体的结构和功能进行了卓有成效的研究,为进一步阐明甾体激素受体的作用机制打下了基础。本文对这一进展进行了综述。     

中国人γ－干扰素cDNA在大肠杆菌中的高效表达

应用RT-PCR技术从中国人淋巴细胞mRNA反转录产物中克隆了IFN-γcDNA,序列分析证实了分子进化规律对IFN-γcDNA序列存在多态性的推论.在此基础上应用DNA重组技术,将去信号肽中国人IFN-γcDNA克隆到原核表达质粒pBV220 P_RP_L启动子下游,转化大肠杆菌DH5α,通过温度诱导表达,成功地在大肠杆菌中稳定、高效地表达了中国人IFN-γcDNA,其表达水平占全菌可溶性总蛋白的44.4%,初步复性后生物学活性测定结果表明γ-IFN表达量为0.45×10⁷～2.34×10⁷单位/L.     

人GM-CSF受体β链胞膜外区域与GM-CSF结合位点的研究

研究了抗人GM-CSF受体β链胞膜外区域4种人工合成多肽D1(9～114),D2(115～225),D3(226～325)和D4(326～422)4个多肽片段多克隆抗血清和McAb对GM-CSF生物学活性的阻断作用,结果首次发现抗D1多克隆抗血清和抗D1McAb 2A_9对 GM-CSF促进的 DMSO诱导的 HL60细胞、正常人胎儿骨髓细胞以及人GM-CSF依赖的TF-1细胞增殖有显著的阻断效应,抗D2McAb 1C₁₂对GM-CSF诱导的 TF-1细胞增殖也有显著的阻断效应,阻断抑制率均可达 90％以上,而其它抗受体 β链 McAb和无关对照 McAb E₇均无阻断活性.此结果表明:抗 D1 McAb2A₉和抗D2 McAb 1C₁₂所识别的表位则可能为受体β链与 GM-CSF的结合位点依据受体β链二级结构预测,受体与细胞因子结合部位的结构理论,设计和合成了受体β链45～75,56～75和 66～75 3个人工合成多肽片段,用间接ELISA结合试验分析表明,McAb 2A₉识别表位位于第 45～55位氨基酸内.应用受体与细胞因子结合部位的结构理论推测 McAb 1C₁₂识别表位可能位于β链内 216～220位氨基酸.此结果对于深入了解受体β链的结构与功能以及细胞因子与受体的相互作用均有十分重要的意义.     

前列腺素核受体系统信号转导及基因表达调控

脂肪酸和前列腺素等脂代谢的产物不仅通过膜受体起作用,也可以通过与核受体结合来调节基因表达.前列腺素I₂(PGI₂)既可以与G蛋白偶联的细胞表面IP受体起作用,也可以通过核受体过氧化物酶体增殖因子活化受体（PPARs）发挥生物学功能.前列腺素E₂(PGE₂)的受体（EPs）不仅仅在质膜上有,最近在核膜上也发现了EPs受体.前列腺素核受体介导的信号转导途径与膜受体介导的信号途径不同,对于基因转录的调控机制也不同.     

硫霉素生物合成酶基因克隆的研究

利用NTG诱变从硫霉素产生菌中获得了生物合成阻断变株Y₃。通过对Y3变株原生质体形成、再生条件及DNA转化的研究,初步建立了以变株为受体的克隆系统,以pIJ680为载体,从硫霉素产生菌S．Cattleya中鸟枪克隆,获得了能使Y₃变株恢复产生硫霉素的酶基因。根据对Y₃积累的中间产物的分析,认为该酶基因可能与硫霉素生物合成过程中肽的环化作用有关。重组质粒分子大小为9．8kb左右,插入片段大小为4．5kb,分子杂交试验证明插入片段来源于硫霉素产生菌S．Cattleya。     

H2O2诱发人成纤维细胞衰老样变化的基因表达谱

以50 μmol/L H₂O₂作用体外培养的人胚肺二倍体成纤维细胞4次,使之出现不可逆的衰老表型.提取年轻细胞及H₂O₂处理早老细胞的mRNA,以荧光物Cy3标记年轻细胞cDNA,Cy5标记H₂O₂处理的细胞cDNA,并与点有4 096条人类基因的芯片杂交,利用计算机数据处理判断基因是否存在表达差异.结果显示：有123种基因的表达变化较显著,这些基因参与细胞周期进程、细胞代谢及蛋白质修饰、细胞外基质及细胞骨架蛋白的形成和调节、炎症反应、调节受体酪氨酸蛋白激酶和G蛋白耦联受体信号转导.     

全人源化抗结肠癌单链抗体基因的克隆和表达

分离大肠癌患者外周血单个核细胞 (PBMC) ,在体外用灭活的大肠癌细胞再次致敏后 ,经EBV转化 ,然后用有限稀释法克隆分泌抗大肠癌细胞抗体的B细胞 ;多次PCR扩增和克隆其抗体可变区基因 (V_H/V_LcDNA) ,并用编码 (Gly₄Ser)₃ 的互补序列连接成ScFvcDNA(V_H linker V_L) ,经酶切克隆入载体fUSE 5RF ,使之呈现于噬菌体表面。通过用 3轮肿瘤细胞和正常人PBMC淘选后 ,ELISA检测 80%的单克隆噬菌体抗体显示了很强的阳性反应。以上结果初步说明 :联合应用体外致敏、EBV转化、PCR扩增和噬菌体呈现技术制备高亲和力全人源化的抗肿瘤抗体是可行的.     

大肠杆菌等受体tRNALeu的T7 RNA聚合酶转录

用化学方法合成编码 2个大肠杆菌tRNA^Leu(tRNA^Leu₁和tRNA^Leu₂ )的基因和T7启动子 ,分别克隆到pUC1 9载体上 ,并在纯化的T7RNA聚合酶的体外转录系统中转录出不含修饰核苷酸的tRNA^Leu.在T7转录体系中 ,亚精胺对转录有负影响 .在最适转录条件下 ,可以得到有活力的RNA转录物的量是模板DNA的 2 5 0倍左右 .在大肠杆菌亮氨酰 tRNA合成酶的催化下 ,2种经体外转录产生的未修饰等受体tRNA^Leu(tRNA^Leu₁和tRNA^Leu₂ )的亮氨酸接受能力基本相同 ,但只有从体内纯化对应的tRNA^Leu的四分之一左右 ,表明修饰核苷酸在tRNA^Leu氨酰化过程中起着较为重要但非关键的作用 .     

人粒细胞集落刺激因子受体膜外区在昆虫细胞中的表达

以胎盘组织提取的mRNA为模板,RTPCR扩增人粒细胞集落刺激因子(G-CSF)受体膜外区CRH的cDNA片段,克隆于供体质粒pF_ASTB_AC1,与杆状病毒表达载体Bacmid同源重组后,转染昆虫细胞SF9,获得重组杆状病毒并证明了CRH的高效表达。表达产物经G-CSF亲和层析进一步纯化,纯度可达90%以上。受体竞争性结合实验结果表明,该表达产物能特异性结合G-CSF,具有较高的亲和力(Kd=3.8nmol/L)。     

Coupling of D1 and D5 dopamine receptors to multiple G proteins

Dopamine receptors are a subclass of the super family of G protein-coupled receptors, that transduce their effects by coupling to specific G proteins. Within the dopamine receptor family, the adenylyl cyclase stimulatory receptors include the D₁ and D₅ subtypes. The D₁ and D₅ dopamine receptors are genetically distinct, sharing >80% sequence homology within the highly conserved seven transmembrane spanning domains, but displaying only 50% overall homology at the amino acid level. When expressed in transfected GH₄C₁ rat pituitary cells, both D₁ and D₅ receptors stimulate adenylyl cyclase and have identical affinities toward dopaminergic agonists and antagonists. In order to analyze specific signaling pathways mediated by activation of either D₁ or D₅ receptors, we have identified the G proteins that are coupled to these receptors. Through functional analyses and competition binding studies, and from immunoprecipitation techniques, using antisera against the various α subunits of G proteins, we have established that both D₁ and D₅ receptors couple to G_sα. In addition, D₁ receptors are also coupled to G_oα. Since G_oα has been implicated in the regulation of Ca²⁺, K⁺, and Na⁺ channels, this finding would suggest that D₁ receptors can mediate the functional activity of these ion channels. There is also evidence to indicate that D₅ receptors couple to G_zα, a novel G protein abundantly expressed in neurons. Thus, despite similar pharmacological properties, such differential coupling of D₁ and D₅ receptors to G proteins other than G_sα, indicates that dopamine can transduce varied signaling responses upon the simultaneous stimulation of both these receptors.     

Dopamine-induced functional activation of Gαq mediated by dopamine D1-like receptor in rat cerebral cortical membranes

Although multiple roles of dopamine through D₁-like (D₁ and D₅) and D₂-like (D₂, D₃, and D₄) receptors are initiated primarily through stimulation or inhibition of adenylyl cyclase via G_s/olf or G_i/o, respectively, there have been many reports indicating diverse signaling mechanisms that involve alternative G protein coupling. In this study, dopamine-induced Gα_q activation in rat brain membranes was investigated. Agonist-induced Gα_q activation was assessed by increase in guanosine-5′-O-(3-[³⁵S]thio)triphosphate ([³⁵S]GTPγS) binding to Gα_q determined by [³⁵S]GTPγS binding/immunoprecipitation assay in rat brain membranes. Dopamine-stimulated Gα_q functionality was highest in cortex as compared to hippocampus or striatum. In cerebral cortical membranes, this effect was mimicked by benzazepine derivatives with agonist properties at dopamine D₁-like receptors, that is, SKF83959, SKF83822, R(+)-SKF81297, R(+)-SKF38393, and SKF82958, but not by the compounds with dopamine D₂-like receptor agonist properties except for aripiprazole. Against expectation, stimulatory effects were also induced by SKF83566, R(+)-SCH23390, and pergolide. The pharmacological profiling by using a series of antagonists indicated that dopamine-induced response was mediated through dopamine D₁-like receptor, which was distinct from the receptor involved in 5-HT-induced response (5-HT_2A receptor). Conversely, the responses induced by SKF83566, R(+)-SCH23390, and pergolide were most likely mediated by 5-HT_2A receptor, but not by dopamine D₁-like receptor. Caution should be paid when interpreting the experimental data, especially in behavioral pharmacological research, in which SKF83566 or R(+)-SCH23390 is used as a standard selective dopamine D₁-like receptor antagonist. Also, possible clinical implications of the agonistic effects of pergolide on 5-HT_2A receptor has been mentioned.     

Differential Regulation of D1 Dopamine Receptor- and of A2a Adenosine Receptor-Stimulated Adenylyl Cyclase by μ-, δ1-, and δ2-Opioid Agonists in Rat Caudate Putamen

Abstract: Inhibition and stimulation of adenylyl cyclase by opioid and D₁ dopamine or A_2a adenosine agonists, respectively, were characterized in the caudate putamen of rats. D₁ dopamine receptors have been reported to be localized preferentially on striatonigral neurons and A_2a adenosine receptors on striatopallidal neurons. The aim of the present study was to evaluate the effects of μ-[Tyr-d -Ala-Gly-(N-Me)Phe-Gly-ol (DAMGO)], δ₁-[Tyr-d -Pen-Gly-Phe-d -Pen (DPDPE)], and δ₂- ([d -Ala²]deltorphin-II [DT-II]) opioid agonists on the D₁ dopamine receptor- and A_2a adenosine receptor-stimulated adenylyl cyclase in membranes from rat caudate putamen. The results show that DAMGO, DPDPE, and DT-II inhibit forskolin-stimulated adenylyl cyclase [selectively antagonized by d -Phe-Cys-Tyr-d -Trp-Orn-Thr-Pen-Thr-NH₂ (CTOP; μ antagonist), 7-benzylidenenaltrexone (BNTX; δ₁ antagonist), and naltriben (NTB; δ₂ antagonist), respectively], but only μ- and δ₂-opioid agonists inhibit D₁ dopamine-stimulated adenylyl cyclase (antagonized by CTOP and NTB, respectively). Furthermore, DT-II and DPDPE inhibit A_2a adenosine-stimulated adenylyl cyclase (antagonized by NTB and BNTX, respectively), whereas DAMGO did not inhibit A_2a adenosine-stimulated adenylyl cyclase activity. These results suggest that μ-, δ₁-, and δ₂-opioid receptors display differential localization and provide neurochemical evidence suggesting the differential location of the δ₁ and δ₂ subtypes. μ-Opioid receptors may be preferentially expressed by striatonigral neurons, δ₁- by striatopallidal neurons, and δ₂- by these two striatal efferent neuron populations.     

Differential Requirements of Sodium for Coupling of Cannabinoid Receptors to Adenylyl Cyclase in Rat Brain Membranes

Abstract: Sodium is generally required for optimal inhibition of adenylyl cyclase by G_l/o-coupled receptors. Canna-binoids bind to specific receptors that act like other members of the G_l/o-coupled receptor superfamily to inhibit adenylyl cyclase. However, assay of cannabinoid inhibition of adenylyl cyclase in rat cerebellar membranes revealed that concentrations of NaCI ranging from 0 to 150 mM had no effect on agonist inhibition. This lack of effect of sodium was not unique to cannabinoid receptors, because the same results were observed using baclofen as an agonist for GABA_B receptors in cerebellar membranes. The lack of sodium dependence was region-specific, because assay of cannabinoid and opioid inhibition of adenylyl cyclase in striatum revealed an expected sodium dependence, with 50 mM NaCI providing maximal inhibition levels by both sets of agonists. This difference in sodium requirements between these two regions was maintained at the G protein level, because agonist-stimulated low K_m GTPase activity was maximal at 50 mM NaCI in striatal membranes, but was maximal in the absence of NaCI in cerebellar membranes. Assay of [³H]WIN 55212–2 binding in cerebellar membranes revealed that the binding of this labeled agonist was sensitive to sodium and guanine nucleotides like other G_l/o-coupled receptors, because both NaCI and the nonhydrolyzable GTP analogue Gpp(NH)p significantly inhibited binding. These results suggest that differences in receptor-G protein coupling exist for cannabinoid receptors between these two brain regions.     

Essential roles of dopamine D4 receptors and the type 1 adenylyl cyclase in photic control of cyclic AMP in photoreceptor cells

Light and dopamine regulate many physiological functions in the vertebrate retina. Light exposure decreases cyclic AMP formation in photoreceptor cells. Dopamine D₄ receptor (D₄R) activation promotes light adaptation and suppresses the light‐sensitive pool of cyclic AMP in photoreceptor cells. The key signaling pathways involved in regulating cyclic AMP in photoreceptor cells have not been identified. In the present study, we show that the light‐ and D₄R‐signaling pathways converge on the type 1 Ca²⁺/calmodulin‐stimulated adenylyl cyclase (AC1) to regulate cyclic AMP synthesis in photoreceptor cells. In addition, we present evidence that D₄R activation tonically regulates the expression of AC1 in photoreceptors. In retinas of mice with targeted deletion of the gene (Adcy1) encoding AC1, cyclic AMP levels and Ca²⁺/calmodulin‐stimulated adenylyl cyclase activity are markedly reduced, and cyclic AMP accumulation is unaffected by either light or D₄R activation. Similarly, in mice with disruption of the gene (Drd4) encoding D₄R, cyclic AMP levels in the dark‐adapted retina are significantly lower compared to wild‐type retina and are unresponsive to light. These changes in Drd4?/? mice were accompanied by significantly lower Adcy1 mRNA levels in photoreceptor cells and lower Ca²⁺/calmodulin‐stimulated adenylyl cyclase activity in retinal membranes compared with wild‐type controls. Reduced levels of Adcy1 mRNA were also observed in retinas of wild‐type mice treated chronically with a D₄R antagonist, L‐745870. Thus, activation of D₄R is required for normal expression of AC1 and for the regulation of its catalytic activity by light. These observations illustrate a novel mechanism for cross‐talk between dopamine and photic signaling pathways regulating cyclic AMP in photoreceptor cells.     

Evidence for a Distinct D1Like Dopamine Receptor that Couples to Activation of Phosphoinositide Metabolism in Brain

Abstract: Dopamine and the D₁, receptor agonist SKF 38393 activate the phospholipase C-rnediated hydrolysis of phosphoinositides in brain slices. This action is selectively inhibited by SCH-23390, thus suggesting its mediation through the dopamine D₁ receptor. To determine if the dopamine receptor that mediates Phosphoinositide hydrolysis is the adenylyl, cyclase-linked D₁ receptor or a different subtype of the dopamine D₁ receptor, 20 benzazepine compounds that were previously characterized as selective dopamine D₁ receptor agonists were tested for stimulation of Phosphoinositide hydrolysis in rat striatal slices and for activation of adenylyl cyclase in rat striatal membranes. The compounds displayed a range of potencies and efficacies in stimulating adenylyl cyclase or Phosphoinositide hydrolysis. Compounds such as SKF 81427 and SKF 38393 were as efficacious as dopamine in stimulating Phosphoinositide hydrolysis, whereas other compounds, including SKF 85174 and SKF 86284, although showing high efficacy in stimulating cyclic AMP, failed to stimulate inositol phosphate formation. There was no correlation between the potencies (r= 0.016; p < 0.95) or efficacies (r=?0.294; p < 0.24) of the tested compounds in stimulating cyclic AMP formation and phosphoinositide hydrolysis. These observations indicate that the D₁-like dopamine receptor that mediates phosphoinositide hydrolysis is pharmacologically distinct from the classic D₁ receptor that is coupled to stimulation of cyclic AMP formation.     

Dopamine receptor localization in the mammalian retina

After a short history of dopamine receptor discovery in the retina and a survey on dopamine receptor types and subtypes, the distribution of dopamine receptors in the retinal cells is described and correlated with their possible role in cell and retinal physiology. All the retinal cells probably bear dopamine receptors. For example, the recently discovered D_1B receptor has a possible role in modulating phagocytosis by the pigment epithelium and a D₄ receptor is likely to be involved in the inhibition of melatonin synthesis in photoreceptors. Dopamine uncouples horizontal and amacrine cell-gap junctions through D₁-like receptors. Dopamine modulates the release of other transmitters by subpopulations of amacrine cells, including that of dopamine through a D₂ autoreceptor. Ganglion cells express dopamine receptors, the role of which is still uncertain. Müller cells also are affected by dopamine. A puzzling action of dopamine is observed in the ciliary retina, in which D₁- and D₂-like receptors are likely to be involved in the cyclic regulation of intraocular pressure. Most of the dopaminergic actions appears to be extrasynaptic and the signaling pathways remain uncertain. Further studies are needed to better understand the multiple actions of dopamine in the retina, especially those that implicate rhythmic regulations.     

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.     

Coupling of dopamine receptors to G proteins: studies with chimeric D2/D3 dopamine receptors

D₂ and D₃ dopamine receptors belong to the superfamily of G protein-coupled receptors; they share a high degree of homology and are structurally similar. However, they differ from each other in their second messenger coupling properties. Previously, we have studied the differential coupling of these receptors to G proteins and found that while D₂ receptor couples only to inhibitory G proteins, D₃ receptor couples also to a stimulatory G protein, Gs. We aimed to investigate the molecular basis of these differences and to determine which domains in the receptor control its coupling to G proteins. For this purpose four chimeras were constructed, each composed of different segments of the original D₂ and D₃ receptors. We have demonstrated that chimeras with a third cytoplasmic loop of D₂ receptor couple to Gi protein in a pattern characteristic of D₂ receptor. On the other hand chimeras containing a third cytoplasmic loop of D₃ receptor have coupling characteristics like those of D₃ receptor, and they couple also to Gs protein. These findings demonstrate that the third cytoplasmic loop determines and accounts for the coupling of dopamine receptors D₂ and D₃ to G proteins.     

Roles of G protein and beta-arrestin in dopamine D2 receptor-mediated ERK activation

ERK activation by dopamine D₂ receptor (D₂R) has been extensively characterized in various cell types including brain tissues. However, the involvement of β-arrestin in the D₂R-mediated ERK activation is not clear yet. Three different strategies were employed in this study to determine the roles of G protein or β-arrestin in D₂R-mediated ERK activation. The cellular level of β-arrestins was reduced by RNA interference and pertussis toxin-insensitive Gi proteins were used to identify the G protein involved. Finally point mutations of D₂R in which coupling with G protein was abolished but the interaction with β-arrestin was increased, were employed to determine whether the affinity between D₂R and β-arrestin is a critical factor for β-arrestin-mediated ERK activation. Our results show that G_i2 protein is involved in D₂R-mediated ERK activation but β-arrestins are either not involved or play minor role.