Regulation of exocytosis by direct Gbg blockade of fusion

摘要 抑制Gi/o偶联GPCRs的分泌是许多激素和 神经调节剂。已有文献证明，分泌细胞中Gi/o偶联GPCRs的激活会释放G 抑制钙离子通过电压依赖性钙通道(VDCC)内流的亚基，导致 荷尔蒙释放。然而，G-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)蛋白在许多系统中也抑制递质激素的释放。这 机制不仅在控制诱发释放方面更加尖锐和直接，而且还具有修改的能力 自发释放。然而，这种陷阱介导的调制的机械细节仍然存在 未得到充分研究，有许多悬而未决的问题。例如，哪些GPCR通过这一机制发挥作用？我们 还发现G-抑制钙内流与G-抑制SNARE介导的胞吐作用具有协同作用。 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。添加另一个 复杂性层面是G和G异构体的多样性以及GS的特异性控制。 蛋白质组学分析发现，即使在没有GPCRs的情况下，特定的G亚基(即G12)也能与SNARE结合 激动剂在增加激动剂增强G12结合的同时，也带来了新的GS(如G23)。 这些数据表明，独特的G亚基可以不同地作用于SNARE，以实现不同程度的 通过GPCRs或甚至在没有GPCRs激活的情况下调节。因此，我们提出了SNARE介导的G 激素释放的调节通过G-亚基和G-GFP亚基的不同组合发挥其功能多样性 不同程度的圈套束缚。这导致我们将重点放在测试(1)哪些GPCRs 通过调节Ca~(2+)进入和通过结合SNARE起作用(2)什么是机制 G介导的两种机制之间的协同作用以及(3)特定G和亚基的作用是什么 在GPCRs中调节激素释放。 鉴于中枢神经系统中GPCRs的巨大多样性，鉴于G的S与胞吐作用的调节密切相关，并给出了他们的 与许多荷尔蒙和神经紊乱相关，这个项目将阐明一种更多功能的调节 不同的GS的分泌，弥合了强直和相位调节释放之间的知识差距 分泌物中的GPCRs，并解开支持各种激素和神经的分子机制 精神错乱。

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.