Elucidating The Structural Organization Of G-protein Coupled Signaling Systems

阐明 G 蛋白偶联信号系统的结构组织

基本信息

批准号：
8565505
负责人：
John K Northup
金额：
$ 20.4万
依托单位：
NATIONAL INSTITUTE ON DEAFNESS AND OTHER COMMUNICATION DISORDERS
依托单位国家：
美国
项目类别：
财政年份：
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/8565505
关键词：
Adenylate Cyclase Adrenergic Agents Adrenergic Antagonists Affect Agonist Alprenolol Attenuated Binding Bioluminescence Bipolar Disorder Carbamazepine Cell surface Cells Complex Coupled Cyclic AMP Data Disease Dissociation Dopamine Dopamine Antagonists Dopamine Receptor Drug usage Energy Transfer GTP-Binding Proteins Goals Heterotrimeric GTP-Binding Proteins Hormones Human G(i) Alpha Proteins Isoproterenol Ligands Light Lithium Mediating Membrane Molecular Moods Neurotransmitters Organism Pathway interactions Patients Pharmaceutical Preparations Pharmacologic Substance Population Propranolol Proteins Regulation Sepharose Signal Pathway Signal Transduction Solutions Spiperone Stimulus System Therapeutic Work adrenergic base beta-adrenergic receptor brain tissue human DRD4 protein human disease in vivo novel therapeutics organizational structure propylsulfonic acid protein complex receptor response treatment effect valproate

项目摘要

Beta-adrenergic receptors (bAR) activate the stimulatory heterotrimeric G protein (Gs) while D2-like dopamine receptors (D2DR) activate the inhibitory heterotrimeric G protein (Gi). Activation of Gs and Gi cause the stimulation and inhibition of adenylyl cyclase (AC), respectively. Beta2-adrenergic (b2AR) and D4 dopamine (D4DR) receptors are prototypical bAR and D2DR that co-immunoprecipitate when they are exogenously co-expressed in HEK 293 cells suggesting the existence of a signaling complex containing both receptors. Bioluminescence resonance energy transfer (BRET) was used to demonstrate that these receptors as well as G proteins and AC were closely associated as a signaling complex in HEK 293 cells, and to show that activation of Gi results in a conformational change of the heterotrimeric Gi that does not involve the dissociation of the G-alpha-i-subunit from the Gbeta-gamma-subunit complex. To determine if a signaling complex containing both bAR and D2DR exists in vivo, brain tissue was dissolved in a solution of 3-3-cholamidopropyl)dimethylammonio-2-hydroxy-1-propanesulfonate (CHAPSO), and the soluble bAR was precipitated with alprenolol-Sepharose. The precipitate was able to bind the D2DR ligand 125Iiodosulpride, and its binding was blocked by the D2DR antagonist spiperone, but not by the bAR antagonist propranolol, indicating that the precipitate also contained D2DR. If propranolol was present during incubation of the CHAPSO extract with alprenolol-Sepharose, no specific 125Iiodosulpride binding could be detected in the precipitate indicating that the D2DR was associated directly with the bAR or was part of a complex that included the bAR, and not non-specifically bound by the alprenolol-Sepharose. The molecular basis for the therapeutic action of mood stabilizing drugs including carbamazepine, lithium, and valproate is being investigated. These drugs are used to treat bipolar disorder, a serious disease that affects 1% of the population. The most popular hypothesis is that these mood stabilizing drugs reduce the level of cyclic AMP produced in response to neurotransmitter-mediated activation of AC by an as yet ill defined mechanism. HEK 293 cells co-expressing b2AR and D4DR were treated with therapeutic concentrations of these drugs. Both carbamazepine and lithium reduced the isoproterenol-mediated stimulation of AC and attenuated the ability of dopamine to inhibit AC. As with patient treatment the effects of the drugs were not immediate, but required prolonged exposure. The data suggest that the drug effects are the result of a reduction in cell surface b2AR and D4DR (Beta-adrenergic receptors (bAR) activate the stimulatory heterotrimeric G protein (Gs) while D2-like dopamine receptors (D2DR) activate the inhibitory heterotrimeric G protein (Gi). Activation of Gs and Gi cause the stimulation and inhibition of adenylyl cyclase (AC), respectively. Beta2-adrenergic (b2AR) and D4 dopamine (D4DR) receptors are prototypical bAR and D2DR that co-immunoprecipitate when they are exogenously co-expressed in HEK 293 cells suggesting the existence of a signaling complex containing both receptors. Bioluminescence resonance energy transfer (BRET) was used to demonstrate that these receptors as well as G proteins and AC were closely associated as a signaling complex in HEK 293 cells, and to show that activation of Gi results in a conformational change of the heterotrimeric Gi that does not involve the dissociation of the G-alpha-i-subunit from the Gbeta-gamma-subunit complex. To determine if a signaling complex containing both bAR and D2DR exists in vivo, brain tissue was dissolved in a solution of 3-3-cholamidopropyl)dimethylammonio-2-hydroxy-1-propanesulfonate (CHAPSO), and the soluble bAR was precipitated with alprenolol-Sepharose. The precipitate was able to bind the D2DR ligand 125Iiodosulpride, and its binding was blocked by the D2DR antagonist spiperone, but not by thebAR antagonist propranolol, indicating that the precipitate also contained D2DR. If propranolol was present during incubation of the CHAPSO extract with alprenolol-Sepharose, no specific 125Iiodosulpride binding could be detected in the precipitate indicating that the D2DR was associated directly with the bAR or was part of a complex that included the bAR, and not non-specifically bound by the alprenolol-Sepharose. The molecular basis for the therapeutic action of mood stabilizing drugs including carbamazepine, lithium, and valproate is being investigated. These drugs are used to treat bipolar disorder, a serious disease that affects 1% of the population. The most popular hypothesis is that these mood stabilizing drugs reduce the level of cyclic AMP produced in response to neurotransmitter-mediated activation of AC by an as yet ill defined mechanism. HEK 293 cells co-expressing b2AR and D4DR were treated with therapeutic concentrations of these drugs. Both carbamazepine and lithium reduced the isoproterenol-mediated stimulation of AC and attenuated the ability of dopamine to inhibit AC. As with patient treatment the effects of the drugs were not immediate, but required prolonged exposure. The data suggest that the drug effects are the result of a reduction in cell surface b2AR and D4DR (Rebois, R. V., Maki, K, Meeks, J. A., Fishman, P. H., Hebert, T. E. and Northup, J. K. D2-like dopamine and beta-adrenergic receptors form a signaling complex for bimodal regulation of adenylyl cyclase Cell Signaling, in press).

β-肾上腺素能受体（BAR）激活刺激异三聚体G蛋白（GS），而D2样多巴胺受体（D2DR）激活抑制性异三聚体G蛋白（GI）。 GS和GI的激活分别导致刺激和抑制腺苷酸环化酶（AC）。 β2-肾上腺素能（B2AR）和D4多巴胺（D4DR）受体是典型的bar和d2dr，当它们在HEK 293细胞中外源共表达时，它们表明存在两个受体的信号复合物。 Bioluminescence resonance energy transfer (BRET) was used to demonstrate that these receptors as well as G proteins and AC were closely associated as a signaling complex in HEK 293 cells, and to show that activation of Gi results in a conformational change of the heterotrimeric Gi that does not involve the dissociation of the G-alpha-i-subunit from the Gbeta-gamma-subunit complex.为了确定在体内含有BAR和D2DR的信号传导复合物是否存在于体内，将脑组织溶解在3-3-胆碱丙基）二甲基氨基-2-羟基-1-羟基-1-丙酸酯（Chapso）的溶液中，并将可溶剂棒与丙二醇 - 乙糖一起沉淀。沉淀物能够结合D2DR配体125iodosulpride，其结合被D2DR拮抗剂尖龙阻塞，但没有由杆拮抗剂普萘洛尔（Propranolol）阻塞，表明沉淀物还含有D2DR。如果在Chapso提取物与藻烯醇 - 丝氨酸孵育过程中存在普萘洛尔，则在沉淀物中无法检测到未检测到的125iododospride结合，表明D2DR直接与条形杆直接相关，或者是棒的一部分，或不是包括棒的一部分，而不是非特异性地由丙二醇 - sepharose结合。正在研究情绪稳定药物的治疗作用的分子基础，包括卡马西平，锂和丙戊酸。这些药物用于治疗躁郁症，这是一种严重的疾病，影响了1％的人群。最流行的假设是，这些情绪稳定药物降低了响应神经递质介导的AC激活AC的循环AMP水平，但尚未定义的机制。用这些药物的治疗浓度处理共表达B2AR和D4DR的HEK 293细胞。卡马西平和锂都降低了异丙肾上腺素介导的AC刺激，并减弱了多巴胺抑制AC的能力。与患者治疗一样，药物的影响不是立即的，而是需要长时间的暴露。数据表明，该药物的作用是细胞表面B2AR和D4DR（β-肾上腺素能受体（BAR）降低的结果，激活了刺激性异三聚体G蛋白（GS），而D2样多巴胺受体（D2DR）激活了抑制性异三元素G蛋白（GI）的抑制作用。（AC），分别是β2-肾上腺素（B2AR）和D4多巴胺（D4DR）受体是原型的bar和D2DR，当它们在HEK 293细胞中共同表达，它们在HEK 293细胞中表达了它们，这表明存在于两个受体中的信号传递（均具有bret）。 AC在HEK 293细胞中作为信号传导复合物密切相关，并表明GI的激活导致异三聚体GI的构象变化，这不涉及Gbeta-Gamma-subunit与Gbeta-Gbeta-Gamma-Gamma-subunit的解离。为了确定在体内含有BAR和D2DR的信号传导复合物是否存在于体内，将脑组织溶解在3-3-胆碱丙基）二甲基氨基-2-羟基-1-羟基-1-丙酸酯（Chapso）的溶液中，并将可溶剂棒与丙二醇 - 乙糖一起沉淀。沉淀物能够结合D2DR配体125iodosulpride，其结合被D2DR拮抗剂尖龙阻塞，但没有由BAR拮抗剂丙二醇固醇阻塞，表明该沉淀物还含有D2DR。如果在Chapso提取物与藻烯醇 - 丝氨酸孵育过程中存在普萘洛尔，则在沉淀物中无法检测到未检测到的125iododospride结合，表明D2DR直接与条形杆直接相关，或者是棒的一部分，或不是包括棒的一部分，而不是非特异性地由丙二醇 - sepharose结合。正在研究情绪稳定药物的治疗作用的分子基础，包括卡马西平，锂和丙戊酸。这些药物用于治疗躁郁症，这是一种严重的疾病，影响了1％的人群。最流行的假设是，这些情绪稳定药物降低了响应神经递质介导的AC激活AC的循环AMP水平，但尚未定义的机制。用这些药物的治疗浓度处理共表达B2AR和D4DR的HEK 293细胞。卡马西平和锂都降低了异丙肾上腺素介导的AC刺激，并减弱了多巴胺抑制AC的能力。与患者治疗一样，药物的影响不是立即的，而是需要长时间的暴露。数据表明，该药物的作用是细胞表面B2AR和D4DR降低的结果（Rebois，R。V.，Maki，K，Meeks，J.A.