Protease-activated receptors (PARs) are a unique group within the G protein-coupled receptor superfamily, orchestrating cellular responses to extracellular proteases via enzymatic cleavage, which triggers intracellular signaling pathways. Protease-activated receptor 1 (PAR1) is a key member of this family and is recognized as a critical pharmacological target for managing thrombotic disorders. In this study, we present cryo-electron microscopy structures of PAR1 in its activated state, induced by its natural tethered agonist (TA), in complex with two distinct downstream proteins, the G_q and G_i heterotrimers, respectively. The TA peptide is positioned within a surface pocket, prompting PAR1 activation through notable conformational shifts. Contrary to the typical receptor activation that involves the outward movement of transmembrane helix 6 (TM6), PAR1 activation is characterized by the simultaneous downward shift of TM6 and TM7, coupled with the rotation of a group of aromatic residues. This results in the displacement of an intracellular anion, creating space for downstream G protein binding. Our findings delineate the TA recognition pattern and highlight a distinct role of the second extracellular loop in forming β-sheets with TA within the PAR family, a feature not observed in other TA-activated receptors. Moreover, the nuanced differences in the interactions between intracellular loops 2/3 and the Gα subunit of different G proteins are crucial for determining the specificity of G protein coupling. These insights contribute to our understanding of the ligand binding and activation mechanisms of PARs, illuminating the basis for PAR1’s versatility in G protein coupling.

蛋白酶激活受体 （PAR） 是 G 蛋白偶联受体超家族中的一个独特组，通过酶切割协调细胞对细胞外蛋白酶的反应，从而触发细胞内信号通路。蛋白酶激活受体 1 （PAR1） 是该家族的关键成员，被认为是管理血栓性疾病的关键药理学靶标。在这项研究中，我们展示了 PAR1 在其激活状态下的冷冻电子显微镜结构，由其天然栓系激动剂 （TA） 诱导，分别与两种不同的下游蛋白 G_q 和 G 异源三聚体形成复合物。TA 肽位于表面口袋内，通过显着的构象变化促进 PAR1 激活。与涉及跨膜螺旋 6 （TM6） 向外移动的典型受体激活相反，PAR1 激活的特征是 TM6 和 TM7 同时向下移动，再加上一组芳香族残基的旋转。这导致细胞内阴离子的置换，为下游 G 蛋白结合创造空间。我们的研究结果描绘了 TA 识别模式，并强调了第二个细胞外环在 PAR 家族内与 TA 形成β片中的独特作用，这是在其他 TA 激活受体中未观察到的特征。此外，细胞内环 2/3 和不同 G 蛋白的 Gα 亚基之间相互作用的细微差异对于确定 G 蛋白偶联的特异性至关重要。这些见解有助于我们了解 PAR 的配体结合和激活机制，阐明了 PAR1 在 G 蛋白偶联中多功能性的基础。