Proteomics in HT1080 WT and GPR56 KO cells

G-protein-coupled receptors (GPCRs) mediate most cellular responses to hormones, neurotransmitters as well as environmental stimulants. However, whether GPCRs participate in modulation of tissue homeostasis through ferroptosis remains unclear. By GPCR cDNA library screening, here we identify that GPR56/ADGRG1 remodels ferroptosis plasticity. Loss of GPR56 sensitizes cells to ferroptosis and deficiency of GPR56 deteriorates ferroptosis-mediated liver injury induced by Doxorubicin (DOX) or ischemia-reperfusion (IR). Mechanistically, GPR56 decreases the abundance of phospholipids containing free polyunsaturated fatty acids (PUFAs) by promoting endocytosis-lysosomal degradation of CD36. By screening a panel of steroid hormones, we identified that 17α-hydroxypregnenolone (17-OH PREG) acts as an agonist of GPR56 to antagonize ferroptosis and efficiently attenuates liver injury before or after insult. Moreover, disease associated GPR56 mutants were unresponsive to 17-OH PREG activation and insufficient to defend against ferroptosis. Together, our findings uncover that 17-OH PREG-GPR56 axis-mediated signal transduction works as a new anti-ferroptotic pathway to maintain liver homeostasis, providing novel insights into the potential therapy for liver injury.