G蛋白偶联受体（GPCR）家族是最大的一类细胞膜上受体，负责多种胞外信号的感知和传导。GPCR被胞外信号激活后引起细胞膜内侧的G蛋白解聚或者β-arrestin招募，进而通过一系列信号转导将胞外信号转换为胞内二级信使的积累、离子通道的开闭、激酶的级联反应或受体的内吞等生化事件，细胞因此对胞外信号作出响应。G蛋白偶联受体家族在细胞信号传递中的关键作用，使它成为众多已知和在研药物的靶点。

      GPCR领域的一个关键生物学问题是：各种受体如何识别各自的配体、激活并向下游传递信号。近年来，A家族的肽类受体成为GPCR靶向药物中最受关注的一类受体亚家族。然而，多数肽类受体的信号转导机制研究尚不充分。本课题的研究对象之一甘丙肽受体（GALRs）是一类特异性识别神经肽甘丙肽（GAL）的受体亚家族，包含GALR1-3。GAL和GALRs与进食，代谢，痛觉的生理过程以及抑郁和精神分裂的病理过程有关，尤其参与睡眠行为的调控。尽管早在上个世纪研究人员就发现了该类信号转导系统，其识别和结合配体的原理、高度特异地偶联特定类型G蛋白的机制还没有充分的研究。本论文通过单颗粒冷冻电镜（SPA）的结构生物学方法，首次解析了GALR1和GALR2在激活状态的信号转导复合物结构，观察到GAL折叠成为典型的helix-turn-helix的结构基序，以“平躺”的方式结合在GALRs的顶部。结合结构分析与大量的点突变验证，本文论证了GALRs亚型之间对GAL的识别存在明显不同，并进一步证明GALRs通过其ICL2（Intracellular loop 2）和ICL3的序列差异维持严格的G蛋白亚型特异性。另外，通过对GALR1富含组氨酸的配体结合口袋的分析和功能验证，本文提出Zn²⁺作为GALR1特异的负向别构调节剂参与对受体激活的调控。

      GPCR领域的另一个关键生物学问题是信号转导系统的脱敏（desensitization）。脱敏机制防止受体在配体持续暴露下被过度激活，从而对细胞造成损害。经典的脱敏系统是GRK/β-arrestin系统和RGS系统。近来，有报道发现当GPCR被激活后，游离的Gβγ亚基会被钾离子通道聚合结构域蛋白家族（Potassium channel tetramerization domain-containing protein, KCTD）的成员KCTD5识别并引导E3泛素连接酶Cullin-3对Gβγ进行泛素化降解。该系统代表第一个被报道的以泛素化降解的方式针对Gβγ的脱敏机制。相比对其他脱敏系统的认识，Cullin-3/KCTD5对Gβγ特异性的识别和降解的生化和结构生物学细节尚不清楚。因此，本研究的第二个部分利用单颗粒冷冻电镜方法，对该新型GPCR通路的脱敏机制进行了探索，首先确定了KCTD5和Gβγ以最大5:5的化学剂量比结合，进而解析了它们的高分辨率结构，详细论证了KCTD5招募Gβγ的结构基础。为了克服KCTD5与Cullin-3复合物不稳定的问题，本研究解析了KCTD5的同源蛋白KCTD7与Cullin-3的结构，并依据保守的互作模式成功搭建第一个Cullin-3/KCTD5/Gβγ的E3泛素连接酶和底物的复合物模型。

      总之，本研究运用单颗粒冷冻电镜方法深入探究了GPCR领域的两个关键生物学问题。 结合生化和分子生物学手段，一方面描述了GALRs激活、特异性偶联G蛋白的基础和众多细节，为进行潜在的激动剂、拮抗剂设计和深入理解各类GPCR的G蛋白选择性提供了详细的参考。另一方面，本研究描述了KCTD5作为接头蛋白招募Cullin-3和激活的Gβγ的结构生物学基础，第一次系统地报道了Cullin-3与KCTD家族较为完整的酶与底物的复合物模型并比较分析了其结构特点。另外，对遗传性渐进肌阵挛性抽动症（Progressive myoclonic epilepsy）的致病基因KCTD7的结构解析，拓展了对KCTD家族结构多样性的认识，为进一步理解和研究KCTD家族的生理和病理机制提供了很好的参考。

The G protein-coupled receptor (GPCR) family is the largest group of cell membrane receptors responsible for sensing and transmitting various extracellular signals. Upon activation by extracellular signals, GPCRs induce G protein dissociation or β-arrestin recruitment on the cytoplasmic side of the cell membrane, leading to a series of biochemical events such as the accumulation of intracellular second messengers, ion channel opening/closing, kinase cascade activation and receptor internalization, thereby allowing the cell to respond to extracellular signals. The crucial role of the GPCR family in cell signal transduction has made it a target for numerous known and ongoing drug developments.

A key question in GPCR biology is how various receptors recognize their respective ligands, activate, and transmit signals downstream. In recent years, the peptide receptor subfamily of the A family has become one of the most studied groups of GPCRs targeted by drugs. However, the signaling mechanisms of most peptide receptors is still not well understood. One of the research subjects in this study, the galanin receptors (GALRs), is a subfamily containing GALR1-3 that specifically recognizes the neuropeptide galanin (GAL). GAL and GALRs are associated with physiological processes such as feeding, metabolism, pain, sleep behavior as well as pathological processes such as depression and schizophrenia. Although researchers discovered this signaling transduction system decades ago, the principles of ligand recognition and binding, the mechanisms of highly specific coupling with certain types of G proteins by GALR subtypes remain unclear. In this research, through the single-particle cryo-electron microscopy analysis method (SPA), the respective structures of the GALR1 and GALR2 signaling complexes in the activated state were solved. It was observed that GAL folds into a typical helix-turn-helix structure and lays on the top of receptor plane. Through structural analysis and extensive point mutation studies, the current research demonstrated that there are significant differences in the recognition of GAL among GALR subtypes and further proved that GALRs maintain strict G protein subtype specificity through the sequence differences of their ICL2 (Intracellular loop 2) and ICL3. In addition, through analysis of GALR1’s histidine-rich ligand binding pocket, the current research proposed that Zn²⁺ serves as a GALR1-specific negative allosteric modulator (NAM) involved in the regulation of receptor activation.

Another key question in GPCR biology is desensitization of the signal transduction process. The desensitization mechanism prevents the receptor from being overactivated under constant exposure to ligands, which can cause damage to the cell. The classical desensitization systems are the GRK/β-arrestin system and the RGS system. Recently, it was reported that when GPCRs are activated, the free Gβγ subunit is recognized by a member of the potassium channel tetramerization domain-containing protein family (KCTD), KCTD5, which then recruits the E3 ubiquitin ligase Cullin-3 to ubiquitinate and degrade Gβγ. This is the first reported desensitization mechanism against Gβγ by ubiquitination and degradation. Compared to other desensitization systems, the biochemical and structural details of specific recognition and degradation of Gβγ by the Cullin-3/KCTD5 E3 ligase system are not yet clear. Therefore, in the second part of this research, the novel desensitization mechanism was explored using the SPA method. Firstly, it was determined that KCTD5 and Gβγ bind in a maximal stoichiometry of 5:5. The complex structure was subsequently resolved at high resolution, providing a detailed explanation of the structural basis of KCTD5 recruiting Gβγ. To overcome the instability issue of the KCTD5/Cullin-3 complex, the structure of the KCTD5 homolog, KCTD7, in complex with Cullin-3 was resolved. Based on the conserved interaction mode, the first Cullin-3/KCTD5/Gβγ E3 ubiquitin ligase/substrate complex model was successfully constructed.

In summary, this study used the SPA method to investigate two major questions in GPCR biology. Through a combination of biochemical and molecular biology methods, we described the basis and details of GALRs activation and specific coupling to G proteins, providing detailed references for potential agonist and antagonist design and a deeper understanding of the G protein selectivity of various GPCRs. Further, this study described the structural basis of KCTD5 as an adaptor protein that recruits Cullin-3 and activates Gβγ, and for the first time, systematically reported the complex structure of Cullin-3/KCTD family E3 ligase in complex with substrates. Additionally, solvation of the structure of KCTD7, the pathogenic gene for Progressive myoclonic epilepsy, helps us expand our understanding of the structural diversity of the KCTD family. These findings provide a detailed reference for further understanding and studying the physiological and pathological mechanisms of the KCTD family.