Intracellular Signaling In Endocrine Cells

内分泌细胞的细胞内信号传导

• 批准号: 7594119
• 负责人: STANKO S. STOJILKOVIC
• 金额: $ 125.69万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7594119
• 关键词: Accounting; Action Potentials; Adenylate Cyclase; Agonist; Alternative Splicing; Androgens; Anterior Pituitary Gland; Arginine; Attenuated; Binding; C-terminal; Calcium; Calcium Channel; Calcium Oscillations; Calcium Signaling; Calcium ion; Calcium-Activated Potassium Channel; Cardiac; Cations; Cell membrane; Cell model; Cells; Characteristics; Compatible; Complex; Confocal Microscopy; Coupled; Coupling; Cyclic AMP; Cyclic Nucleotides; Data; Distal; Dose; Down-Regulation; Endocrine; Endothelin; Endothelin A Receptor; Endothelin Receptor; Event; Excision; Exhibits; Exons; Family; Fire - disasters; GTP-Binding Proteins; Generations; Gonadotropins; Heterogeneity; Hormonal; Hormones; Introns; Ion Channel; Ions; Ivermectin; Kinetics; Ligand Binding Domain; Ligands; Lysine; Mediating; Membrane; Membrane Potentials; Messenger RNA; Metabolism; Modeling; Molecular; Molecular Biology; Mutate; Mutation; Neurons; Operative Surgical Procedures; P2X-receptor; Pathway interactions; Pattern; Pharmacology; Phosphorylation Site; Pituitary Gland; Population; Potassium; Property; Protein Kinase C; Proteins; Purinoceptor; RNA Splicing; Rate; Rattus; Recombinants; Recycling; Regulation; Reporting; Role; Secretory Cell; Signal Pathway; Signal Transduction; Site; Sodium; Sodium Channel; Somatostatin; Somatotropin-Releasing Hormone; Tetrodotoxin; Time; Tissues; Transcript; Transmembrane Domain; Variant; Work; base; casein kinase II; cell type; desensitization; enhanced green fluorescent protein; extracellular; inorganic phosphate; lactotroph; large-conductance calcium-activated potassium channels; mathematical model; mutant; novel; receptor; receptor function; research study; response; theories; three dimensional structure; tool; voltage

Numerous plasma membrane channels have been characterized in pituitary cells, but the mechanism underlying their electrical activity is still not known. Our current efforts focus on the role of sodium-conducting channels in the control of electrical activity in pituitary cells. In these cells, removal of sodium reversibly hyperpolarized the membrane potential and suppressed calcium oscillations, followed by a reduction in the level of intracellular calcium concentration to near steady-state levels. In contrast, the blockade by tetrodotoxin of voltage-dependent sodium channels, which are expressed in these cells, was ineffective. One group of the channels that could account for such effects of removal of extracellular sodium are hyperpolarization-activated channels, which have a distinct role in the control of membrane excitability in spontaneously active cardiac and neuronal cells. Using molecular biology and electrophysiological tools, we identified these channels in pituitary cells. Our results further indicated that these channels were under tonic activation by the basal level of cAMP. We also combined experiments and theory to clarify the mechanisms underlying spontaneous and receptor-controlled electrical activity using pituitary somatotrophs as a cell model. Experiments support the role of a sodium-conducting and tetrodotoxin-insensitive channel in controlling spontaneous and growth hormone-releasing hormone-stimulated pacemaking, the latter in a cAMP-dependent manner; an opposing role of spontaneously active inwardly rectifying potassium (Kir) channels and G-protein-regulated Kir channels in somatostatin-mediated inhibition of pacemaking; as well as a role of voltage-gated calcium channels in spiking and large conductance (BK-type) calcium-activated potassium channels in plateau bursting. The mathematical model is compatible with a wide variety of experimental data involving pharmacology and extracellular ion substitution and supports the importance of constitutively active tetrodotoxin-insensitive sodium and Kir channels in maintaining spontaneous pacemaking in pituitary somatotrophs. The model also suggests that these channels are involved in the up- and down-regulation of electrical activity by growth hormone-releasing hormone and somatostatin. In the model, the plateau bursting is controlled by two functional populations of BK channels, characterized by distance from the voltage-gated calcium channels. The rapid activation of the proximal BK channels is critical for the establishment of the plateau, whereas slow recruitment of the distal BK channels terminates the plateau. Spontaneous electrical activity of pituitary cells is stimulated and/or inhibited by numerous hormones. Our ongoing work is focused on the role of endothelins, ATP, and androgens in control of voltage-gated calcium influx and hormone secretion. Endothelins are produced by pituitary cells and functional endothelin-A receptors are expressed in all five major secretory cell types. In gonadotrophs, stimulation of these receptors leads to activation of the Gq/11 signaling pathway, accompanied by the oscillatory calcium release from intracellular pools and gonadotropin secretion and facilitation of voltage-gated calcium influx. However, in somatotrophs and lactotrophs endothelins inhibit spontaneous voltage-gated calcium influx through the Gi/o signaling pathway. These observations raised the possibility that multiple receptor subtypes are generated by alternative RNA splicing of the pituitary endothelin-A receptor and exhibit comparable binding characteristics but are coupled to different G proteins and intracellular signaling. Consistent with this hypothesis, we reported recently on the isolation of cDNAs of endothelin-A transcripts from rat anterior pituitary, which are generated by alternative RNA splicing. Deletion of exon 2 and insertion of fragments from intron 1 and 2 accounted for formation of three misplaced proteins, whereas the insertion of a fragment from intron 6 resulted in generation of a functional plasma membrane receptor, termed the endothelin-A-C13 receptor. In this splice variant, the C-terminal 382S-426N sequence of the wild type receptor was substituted with a shorter 382A-399L sequence, resulting in alteration of the putative domains responsible for coupling to Gq/11 and Gs proteins and the endocytotic recycling, as well as in deletion of the predicted protein kinase C/casein kinase 2 phosphorylation sites. The mRNA transcripts for the splice receptor were identified in normal and immortalized pituitary cells and several other tissues. The pharmacological profiles of recombinant wild type and spliced receptors were highly comparable, but the coupling of the splice receptor to the calcium-mobilizing signaling pathway was attenuated, causing a rightward shift in the agonist potency. Furthermore, the efficacy of the spliced receptor to stimulate adenylyl cyclase signaling pathway and to internalize was significantly reduced. These results indicate for the first time the presence of a novel endothelin-A splice receptor, which could contribute to the functional heterogeneity among secretory pituitary cell types. P2X receptors are a family of ligand-gated cation channels composed of two transmembrane domains, with N- and C-termini located intracellularly and a large extracellular loop containing the ATP binding domain. We progressed in identifying the residues important for agonist binding and gating, using ivermectin (IVM), an alosteric modulator. All experiments were done with enhanced green fluorescent protein-tagged receptors to identify cells expressing receptors for electrophysiological recordings and to visualize the subcellular distribution of receptors by confocal microscopy. In the presence of IVM, all low or non-responsive mutants responded to agonists in a dose-dependent manner. The results further indicated that lysines 67 and 313 and arginine 295 have a critical role in forming the proper three-dimensional structure of P2X4 receptor for agonist binding and/or channel gating. To study the roles of the sequence downstream of ATP binding domain, we mutated the numerous conserved residues in the Lys313-Ile333 ectodomain sequence. The rates of wild type channel opening and closing in the presence of ATP, but not the rate of washout-induced closing, were dependent on agonist concentration. All mutants other than I317A were expressed in the plasma membrane at comparable levels. The majority of mutants showed significant changes in the peak amplitude of responses and the EC50 values for ATP. When stimulated with the supramaximal ATP concentration, mutants also differed in the kinetics of their activation, deactivation, and/or desensitization. The results suggest a critical role of the Lys313 residue in receptor function other than coordination of the phosphate group of ATP and a possible contribution of the Tyr315 residue to the agonist-binding module. The pattern of changes of receptor function by mutation of other residues was consistent with the operation of the Gly316-Ile333 sequence as a signal transduction module between the ligand binding domain and the channel gate in the second transmembrane domain. Finally, to characterize IVM binging site at P2X4 receptors we generated several chimeric and single-point mutants. Experiments with chimeric receptors revealed that the Val49-Val61 but not the Val64-Tyr315 ectodomain sequence is important for the effects of IVM on channel deactivation. Receptor-specific mutations placed in the Gly29-Val61 and Asp338-Leu358 regions showed the importance of Trp50, Val60, and Val357 residues in IVM regulation of the rate of channel deactivation, but not on the maximum current amplitude. These results suggest that the transmembrane domains and the nearby ectodomain region contribute to the eff

在垂体细胞中已经表征了许多质膜通道，但其电活动的基础机制尚不清楚。我们目前的努力集中在钠导通道在控制垂体细胞中电活动中的作用。在这些细胞中，去除钠可逆地超极化膜电位并抑制钙振荡，然后将细胞内钙浓度的水平降低至接近稳态的水平。相反，在这些细胞中表达的电压依赖性钠通道的四毒素的阻断是无效的。一组可以解释去除细胞外钠的影响的通道是超极化激活的通道，在自发活性心脏和神经元细胞中膜兴奋性的控制中具有明显的作用。使用分子生物学和电生理工具，我们在垂体细胞中确定了这些通道。我们的结果进一步表明，这些通道在CAMP的基础水平下受到滋补。我们还将实验和理论结合在一起，以阐明使用垂体植物营养物作为细胞模型的自发和受体控制的电活动的机制。实验支持钠导电和四毒素不敏感的通道在控制自发和生长激素释放激素刺激的起搏器中的作用，后者以cAMP依赖性方式；自发活跃的在天生抑制剂介导的起搏器抑制中，自发活跃的内向钾（KIR）通道（KIR）通道和G蛋白调节的KIR通道的相对作用；以及电压门控钙通道在高原爆发中钙激活的钾通道中的峰值和大电导（BK型）的作用。该数学模型与涉及药理学和细胞外离子取代的多种实验数据兼容，并支持组成型活性的四毒素不敏感的钠和KIR通道在维持垂体孕育体中自发起搏方面的重要性。该模型还表明，这些通道通过生长激素释放激素和生长抑素参与电活动的上调和下调。在模型中，高原爆发由两个BK通道的功能群控制，其特征在于距电压门控钙通道的距离。近端BK通道的快速激活对于建立高原至关重要，而远端BK通道的缓慢募集终止了高原。 垂体细胞的自发电活动被许多激素刺激和/或抑制。我们正在进行的工作集中于内皮蛋白，ATP和雄激素在控制电压门控钙涌入和激素分泌方面的作用。内皮蛋白是由垂体细胞产生的，功能性内皮素A受体均在所有五种主要分泌细胞类型中表达。在促性腺营养中，对这些受体的刺激导致GQ/11信号通路的激活，并伴随着细胞内池的振荡性钙释放，促性腺激素分泌以及电压门控钙的促进。然而，在母营养和乳突中，内皮蛋白会通过GI/O信号传导途径抑制自发性电压门控钙的涌入。这些观察结果提高了多种受体亚型是通过垂体内皮素A受体的替代RNA剪接而产生的，并具有可比的结合特性，但与不同的G蛋白和细胞内信号传导偶联。与这一假设一致，我们最近报道了从大鼠前垂体中的内皮素A转录物的cDNA分离，这些转录本是由替代RNA剪接产生的。外显子2的缺失以及内含子1和2的片段的插入占了三种放错蛋白质的形成，而内含子6中片段的插入导致产生功能性质膜受体，称为内皮素-A-C13受体。 In this splice variant, the C-terminal 382S-426N sequence of the wild type receptor was substituted with a shorter 382A-399L sequence, resulting in alteration of the putative domains responsible for coupling to Gq/11 and Gs proteins and the endocytotic recycling, as well as in deletion of the predicted protein kinase C/casein kinase 2磷酸化位点。在正常和永生的垂体细胞和其他几个组织中鉴定出剪接受体的mRNA转录物。重组野生型和剪接受体的药理谱是高度可比性的，但是剪接受体与钙振动信号通路的偶联被减弱，从而导致激动剂效力的向右移动。此外，剪接受体刺激腺苷酸环化酶信号通路和内在化的功效显着降低。这些结果首次表明存在新型内皮素A剪接受体的存在，这可能有助于分泌性垂体细胞类型之间的功能异质性。 P2X受体是由两个跨膜结构域组成的配体门控阳离子通道的家族，其n-和c-termini位于细胞内，一个含有ATP结合结构域的大细胞外环。 我们进步了，使用酸酯（IVM）（IVM），鉴定对激动剂结合和门控重要的残基。所有实验均使用增强的绿色荧光蛋白标记的受体进行，以鉴定表达电生理记录的受体的细胞，并通过共焦显微镜可视化受体的亚细胞分布。在IVM存在的情况下，所有低或非反应性突变体都以剂量依赖性方式对激动剂做出反应。结果进一步表明，赖氨酸67和313和精氨酸295在形成适当的P2X4受体的适当三维结构中，用于激动剂结合和/或通道门控。为了研究ATP结合结构域下游序列的作用，我们突变了LYS313-ELE333胞外域序列中的众多保守残基。在ATP存在的情况下，野生型通道开口和关闭的速率，但不取决于引起的闭合率，取决于激动剂的浓度。除I317a以外的所有突变体在质膜中均以可比的水平表达。大多数突变体在响应的峰值幅度和ATP的EC50值上显示出显着变化。当受到超大性ATP浓度刺激时，突变体的激活，失活和/或脱敏的动力学也有所不同。结果表明，除了ATP磷酸盐组的配位以外，LYS313残基在受体功能中的关键作用以及Tyr315残基对激动剂结合模块的可能贡献。通过其他残基突变通过突变的受体功能变化模式与Gly316-Ele333序列的运行一致，作为在第二跨膜结构域中配体结合域和通道栅极之间的信号转导模块。 最后，为了表征P2X4受体的IVM弯曲位点，我们产生了几个嵌合和单点突变体。 嵌合受体的实验表明，VAL49-VAL61而不是Val64-Tyr315胞外域序列对IVM对通道失活的影响很重要。放置在GLY29-VAL61和ASP338-LEU358区域中的受体特异性突变显示了TRP50，Val60和Val357残基在通道失活速率的IVM调节中的重要性，但不在最大电流幅度上。这些结果表明，跨膜结构域和附近的外生域区域有助于EFF