GPR20是一个A类孤儿G蛋白偶联受体（GPCR），由于其不同的高表达而成为胃肠间质瘤（GIST）的潜在治疗靶点。最近，在GIST治疗的临床试验中，开发了一种包含GPR20结合抗体（Ab046）的抗体药物结合物（ADC）。在没有任何已知配体的情况下，GPR20在基础活性高的情况下会不断激活Gi蛋白，但它如何实现这种高基础活性仍不清楚。在这里，我们报道了人类GPR20复合物的三个冷冻电镜结构，包括在无Ab046 Fab和有Ab046 Fab存在的情况下与Gi耦合的GPR20和无Gi的GPR20。值得注意的是，这些结构显示一个独特折叠的N末端螺旋上覆盖着跨膜区域，我们的突变研究表明该盖区在刺激GPR20的基础活性中发挥了关键作用。我们还揭示了GPR20和Ab046之间的分子相互作用，这可能有助于设计具有增强亲和力或新功能的工具抗体以用于GPR20。此外，我们报道了一个未指定密度占据的正位点，其中可能对于探索去孤儿化机会是至关重要的。©2023作者。

GPR20 is a class-A orphan G protein-coupled receptor (GPCR) and a potential therapeutic target for gastrointestinal stromal tumors (GIST) owing to its differentially high expression. An antibody-drug conjugate (ADC) containing a GPR20-binding antibody (Ab046) was recently developed in clinical trials for GIST treatment. GPR20 constitutively activates Gi proteins in the absence of any known ligand, but it remains obscure how this high basal activity is achieved. Here we report three cryo-EM structures of human GPR20 complexes including Gi-coupled GPR20 in the absence or presence of the Fab fragment of Ab046 and Gi-free GPR20. Remarkably, the structures demonstrate a uniquely folded N-terminal helix capping onto the transmembrane domain and our mutagenesis study suggests a key role of this cap region in stimulating the basal activity of GPR20. We also uncover the molecular interactions between GPR20 and Ab046, which may enable the design of tool antibodies with enhanced affinity or new functionality for GPR20. Furthermore, we report the orthosteric pocket occupied by an unassigned density which might be essential for exploring opportunities for deorphanization.© 2023. The Author(s).