Abstract
GPR99 holds promise as a potential therapeutic target for inflammatory diseases. GPR99 exhibits marked basal activity when coupled with the G(q) protein, its activation mechanism remains elusive. In this study, we determine the high-resolution structure of the human GPR99 in complex with the heterotrimeric miniG(q) in the ligand-free state using cryo-electron microscopy (cryo-EM). Our structural analysis and functional experiments reveal that the second extracellular loop (ECL2) of GPR99 occupies the orthosteric binding pocket, thereby promoting receptor self-activation. Moreover, we observe structural water molecules forming an extended polar network that connects ECL2 and the binding pocket, intricately linking these elements to the receptor's functional activity. Structure-based mutagenesis experiments further validate the critical role of ECL2 in intracellular signal transduction of GPR99, offering a structural basis for exploring its function under physiological or pathological conditions. Additionally, these findings also provide a crucial theoretical framework for the design of drugs targeting GPR99.