Regulation of exocytosis by direct Gbg blockade of fusion

通过直接 Gbg 融合阻断来调节胞吐作用

• 批准号: 10542729
• 负责人: SIMON T ALFORD
• 金额: $ 40.23万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-03-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10542729
• 关键词: Acute; Address; Adrenergic Receptor; Affect; Affinity; Agonist; Atlases; Autoreceptors; Behavior; Binding; Biological Assay; Brain; Brain region; Code; Complex; Coupled; Data; Disease; Dose; Electrophysiology (science); Exocytosis; Exocytosis Inhibition; G-Protein-Coupled Receptors; GTP-Binding Proteins; Gait; Genetic; Hormonal; Hormone Receptor; Hormones; Impairment; Induced Hyperthermia; Insulin; Knowledge; Learning; Mediating; Membrane; Modification; Molecular; Monitor; Mus; Mutant Strains Mice; Neuromodulator; Neurotransmitters; Nociception; Peripheral; Pharmaceutical Preparations; Phase; Phenotype; Physiological; Physiology; Play; Protein Isoforms; Proteomics; RIPK1 gene; Regulation; Regulation of Exocytosis; Role; SNAP receptor; Secretory Cell; Signal Transduction; Site; Slice; Specificity; Stress; System; Testing; Therapeutic; Transgenic Organisms; Virus Diseases; Work; behavioral phenotyping; cultural competence; depressive symptoms; examination questions; genetic manipulation; hormone regulation; interest; inward rectifier potassium channel; knock-down; mouse model; nervous system disorder; neurotransmission; postsynaptic; preference; presynaptic; receptor; receptor binding; reconstitution; response; synaptic function; synergism; tool; voltage; Acute; Address; Adrenergic Receptor; Affect; Affinity; Agonist; Atlases; Autoreceptors; Behavior; Binding; Biological Assay; Brain; Brain region; Code; Complex; Coupled; Data; Disease; Dose; Electrophysiology (science); Exocytosis; Exocytosis Inhibition; G-Protein-Coupled Receptors; GTP-Binding Proteins; Gait; Genetic; Hormonal; Hormone Receptor; Hormones; Impairment; Induced Hyperthermia; Insulin; Knowledge; Learning; Mediating; Membrane; Modification; Molecular; Monitor; Mus; Mutant Strains Mice; Neuromodulator; Neurotransmitters; Nociception; Peripheral; Pharmaceutical Preparations; Phase; Phenotype; Physiological; Physiology; Play; Protein Isoforms; Proteomics; RIPK1 gene; Regulation; Regulation of Exocytosis; Role; SNAP receptor; Secretory Cell; Signal Transduction; Site; Slice; Specificity; Stress; System; Testing; Therapeutic; Transgenic Organisms; Virus Diseases; Work; behavioral phenotyping; cultural competence; depressive symptoms; examination questions; genetic manipulation; hormone regulation; interest; inward rectifier potassium channel; knock-down; mouse model; nervous system disorder; neurotransmission; postsynaptic; preference; presynaptic; receptor; receptor binding; reconstitution; response; synaptic function; synergism; tool; voltage

Summary Inhibition of secretion by Gi/o-coupled GPCRs is an important control mechanism used by many hormones and neuromodulators. It is well documented that activation of Gi/o-coupled GPCRs in secretory cells releases G subunits that inhibit Ca2+ entry through voltage-dependent Ca2+ channels (VDCCs), leading to reduced hormone release. However, a direct interaction between G and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins also inhibits transmitter hormone release in many systems. This mechanism is not only more acute and direct in controlling evoked release but also has the ability to modify spontaneous release. However, the mechanistic details of this SNARE-mediated modulation remain understudied, with many open questions. For example, which GPCRs work through this mechanism? We have also found that G inhibition of Ca2+ entry synergizes with G inhibition of SNARE-mediated exocytosis. We will address the mechanism of this synergism as well as it’s implications in physiology. Adding another layer of complexity is the diversity of G and G isoforms and the control of specificity of Gs. Using proteomic assays, we have found that specific G subunits (i.e. G12) bind to SNARE even without GPCR agonists whereas adding agonists enhances G12 binding but also brings new Gs (e.g. G23) to SNAREs. These data suggest that unique G subunits can differentially act on SNARE to achieve different degrees of modulation via GPCRs or even without GPCR activation. Therefore, we propose that SNARE-mediated G modulation of hormone release exerts its functional diversity by different combination of G subunits and different degree of SNARE binding. This leads us to focus on three specific aims that test (1) which GPCRs work through modulation of Ca2+ entry and which work through binding SNARE (2) what is the mechanism of synergism between the two G-mediated mechanisms and (3) what is the role of particular G and  subunits in GPCR regulation of hormone release. Given the huge diversity of GPCRs in CNS, given G’s close ties to modulation of exocytosis, and given their relevance to many hormonal and neurological disorders, this project will illuminate a more versatile modulation of secretion by different Gs, bridge the knowledge gap between tonic and phasic modulation of release via GPCRs in secretion, and unravel molecular mechanisms underpinning various hormonal and neurological disorders.

概括 GI/O耦合GPCR对分泌的抑制是许多激素和 神经调节剂。有充分的文献证明，秘密细胞中GI/O耦合GPCR的激活释放G 通过电压依赖性Ca2+通道（VDCC）抑制Ca2+进入的亚基，导致减少 释放马龙。但是，G与可溶性N-乙基米酰胺敏感因子之间的直接相互作用 附着蛋白受体（SNARE）蛋白也抑制许多系统中的发射机激素释放。这 机制不仅更加敏锐，而且在控制唤起的释放方面具有直接性，而且还具有修改的能力 自发释放。但是，该圈圈介导的调节的机械细节仍然存在 研究了，有许多开放的问题。例如，哪些GPCR通过这种机制起作用？我们 还发现G抑制Ca2+进入与G抑制军鼓介导的胞吐作用合成。 我们将解决这种协同作用的机制以及对生理学的影响。添加另一个 复杂性层是G和G同工型的多样性以及GS的特异性的控制。使用 蛋白质组学测定，我们发现特定的G亚基（即G12）即使没有GPCR 激动剂虽然增加激动剂增强了G12结合，但也将新的GS（例如G23）带到了鼻涕中。 这些数据表明，唯一的G亚基可以在SNARE上采取不同的作用，以实现不同的程度 通过GPCR甚至没有GPCR激活调节。因此，我们提出了圈套介导的g 马酮释放的调制通过不同组合的亚基和 不同程度的圈套结合。这使我们专注于三个测试的特定目标（1）GPCR 通过调制Ca2+进入以及通过绑定的圈套来工作（2） 两种G介导的机制之间的协同作用和（3）特定G和亚基的作用是什么 在GPCR释放的GPCR调节中。 鉴于G与胞吞作用的密切联系，鉴于中枢神经系统中GPCR的多样性，并鉴于它们 该项目与许多马匹和神经系统疾病有关，将阐明更通用的调节 通过不同的g的分泌，弥合了释放的补品和阶段调制之间的知识差距 分泌中的GPCR以及分子机制的分子机制，支撑着各种激素和神经系统 疾病。