The team of Xu Huaqiang, a researcher at the Shanghai Institute of Materia Medica of the Chinese Academy of Sciences, the team of Jinpeng Sun, a professor of Shandong University, and the team of Professor Zhang Yan of Zhejiang University, have analyzed the cryo-EM structure of glucocorticoid and its membrane receptor GPR97 and Go protein complex for the first time. The first resolved high-resolution structure of adhesion-like GPCRs with ligands and G protein complexes, related research results are published on Nature.
Adhesion G protein-coupled receptors (aGPCRs) are members of the GPCR superfamily. They play key molecular switches in some important physiological processes of the organism, such as brain development and water and salt regulation, inflammation and cell fate determination. Compared with other members of the GPCR superfamily, in addition to the classic 7-pass transmembrane core (7TM), aGPCRs also have a longer extracellular region, which constitutes a domain with different functions. It is generally believed that aGPCRs can be activated by extracellular matrix proteins or soluble small molecules. However, it is still unclear whether small molecule ligands can directly bind to 7TM and activate receptors.
Glucocorticoids play an important role in regulating the body`s development, growth, metabolism and immunity. It is the most important regulator of the body`s stress response and one of the most widely used anti-inflammatory and immunosuppressive agents in the clinic. Classical theory believes that glucocorticoids bind to glucocorticoid nuclear receptors and pass through nuclear pores to regulate the expression of related genes in the nucleus. This mode of action usually requires a longer reaction time and is called a genomic mechanism. Xu Huaqiang`s research group analyzed the glucocorticoid nuclear receptor and dexamethasone (Cell, 110: 93-105) and the endogenous glucocorticoid—hydrocortisone (Cell Research, 24) in 2002 and 2014, respectively. : 713–726), revealing the mechanism of glucocorticoid recognition and functional regulation of its nuclear receptors, which promoted the development of glucocorticoid receptor-targeted drugs.
In addition, glucocorticoids have been found to quickly cause changes in cells and the body, which suggests that there may be membrane receptors for glucocorticoids in the organism, which can mediate the rapid response of glucocorticoids. Studies have found that the rapid response of glucocorticoids is closely related to G protein. Gi inhibitor PTX can inhibit the rapid action of glucocorticoids, and based on this, it is speculated that GPCR is a potential membrane receptor for glucocorticoids.
Jinpeng Sun and the team of Professor Yi Fan of Shandong University conducted receptor physiology and endogenous ligand discovery on GPR97, and found that hydrocortisone, cortisone, and 11-deoxycortisol, including glucocorticoids, are in The internal endogenous steroid hormones can activate GPR97. Among them, dexamethasone has a stronger GPR97 activation ability, and it is finally confirmed that Go is the G protein pathway coupled after GPR97 activation.
Based on the previous work, the cooperative team used single-particle cryo-electron microscopy technology to analyze the structure of the complex formed after the activation of GPR97 by the exogenous ligand beclomethasone (BCM) and the endogenous ligand hydrocortisone (cortisol). Finally, the complex structures of the two ligand-activated GPR97 receptors and Go protein were obtained, with a resolution of 3.1 angstroms and 2.9 angstroms respectively.
Compared with other members of the GPCR subfamily, the 7TM of GPR97 presents a unique spatial distribution, and its helix exhibits a different length from other receptors. According to traditional theory, aGPCR`s unique extracellular GAIN domain and 7TM play its core function as a whole in the process of activating GPCR. However, the researchers found for the first time in the structure that glucocorticoids bind to an oval orthographic binding pocket in the GPR97 7TM core. In addition, GPR97 also exhibits a unique activation mechanism that is different from other Class A GPCR members. The GPR97 sequence does not contain conservative motifs such as PIF, DRY and NPxxY, which first recognize the ligand through toggle switch W6.53 and are activated. The activated receptor binds the receptor TM3-TM5-TM6 together with the first discovered upper Quaternary core (UQC), and then binds to the Go protein through HLY motif.
Receptor 7TM forms a large intracellular G protein binding pocket. All three intracellular loops are involved in the interaction between the receptor and G protein. The intracellular loop is closely related to the constitutive activation of the receptor. In this study, the researchers also explained for the first time the key role of palmitoylation of G protein in its coupling to GPCR. The study found for the first time that the C351 position of the α5 helix of Gαo was modified by palmitoylation, and further verified the unique role of this modification in the coupling of Go and GPR97.
In summary, the cooperative team discovered the high-affinity membrane receptor for glucocorticoids for the first time, and analyzed the structure of the GPR97 complex with the Go protein in the adhesion-like GPCR family under the activation of glucocorticoids through single-particle cryo-electron microscopy. This reveals the molecular mechanism by which glucocorticoids recognize and activate membrane GPR97 and the receptor coupling Go protein at near-atomic resolution. This achievement will play an important role in demonstrating and promoting the study of glucocorticoid membrane receptor function and understanding of the activation mechanism of adhesion-like GPCRs.
