Regulation of exocytosis by direct Gbg blockade of fusion

总结 通过Gi/o偶联的GPCR抑制分泌是许多激素使用的重要控制机制， 神经调质有充分的文献证明，分泌细胞中Gi/o偶联的GPCR的激活释放G蛋白， 抑制Ca 2+通过电压依赖性Ca 2+通道（VDCCs）进入的亚基，导致降低 荷尔蒙释放然而，G蛋白与可溶性N-乙基马来酰亚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递质激素释放。这 机制不仅在控制诱发释放方面更为敏锐和直接，而且具有调节 自发释放然而，这种SNARE介导的调节机制的细节仍然存在， 未被充分研究，有许多开放的问题。例如，哪些GPCR通过该机制发挥作用？我们 还发现，G蛋白对Ca 2+进入的抑制与G蛋白对SNARE介导的胞吐作用的抑制协同作用。 我们将讨论这种协同作用的机制以及它在生理学上的意义。添加另一 复杂层是G β和G β亚型的多样性以及G β特异性的控制。使用 通过蛋白质组学分析，我们发现即使没有GPCR，特异性G蛋白亚基（即G蛋白1亚基2）也能与SNARE结合 激动剂，而添加激动剂增强G β 1 β 2结合，但也为SNARE带来新的G β 2（例如G β 2 β 3）。 这些数据表明，独特的G蛋白亚基可以不同程度地作用于SNARE，以实现不同程度的抑制。 通过GPCR或甚至在没有GPCR激活的情况下调节。因此，我们认为SNARE介导的G蛋白酶 激素释放的调节通过G β亚基的不同组合发挥其功能多样性， 不同程度的束缚。这使我们关注三个具体的目标，测试（1）哪些GPCR 通过调节Ca 2+进入起作用，哪些通过结合SNARE起作用（2） 两种G β介导机制之间的协同作用以及（3）特定G β和G β亚基的作用 在激素释放的GPCR调节中。 考虑到CNS中GPCR的巨大多样性，考虑到GPCR与胞吐调节的密切联系，以及考虑到它们的生物学特性， 与许多激素和神经系统疾病相关，该项目将阐明一个更通用的调制 的分泌，通过以下途径弥合紧张性和阶段性释放调节之间的知识差距： GPCR在分泌中的作用，并阐明了支持各种激素和神经系统的分子机制 紊乱

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.