Significance The steroid hormone progesterone is highly involved in different physiological–pathophysiological processes, including bone formation and cancer progression. Understanding the working mechanisms, especially identifying the receptors of progesterone hormones, is of great value. In the present study, we identified GPR126 as a membrane receptor for both progesterone and 17-hydroxyprogesterone and triggered its downstream G protein signaling. We further characterized the residues of GPR126 that interact with these two ligands and found that progesterone promoted the progression of a triple-negative breast cancer model through GPR126-dependent Gi-SRC signaling. Therefore, developing antagonists targeting GPR126-Gi may provide an alternative therapeutic option for patients with triple-negative breast cancer.

The G protein–coupled bile acid receptor (GPBAR) is the membrane receptor for bile acids and a driving force of the liver–bile acid–microbiota–organ axis to regulate metabolism and other pathophysiological processes. Although GPBAR is an important therapeutic target for a spectrum of metabolic and neurodegenerative diseases, its activation has also been found to be linked to carcinogenesis, leading to potential side effects. Here, via functional screening, we found that two specific GPBAR agonists, R399 and INT-777, demonstrated strikingly different regulatory effects on the growth and apoptosis of non–small cell lung cancer (NSCLC) cells both in vitro and in vivo. Further mechanistic investigation showed that R399-induced GPBAR activation displayed an obvious bias for β-arrestin 1 signaling, thus promoting YAP signaling activation to stimulate cell proliferation. Conversely, INT-777 preferentially activated GPBAR-Gs signaling, thus inactivating YAP to inhibit cell proliferation and induce apoptosis. Phosphorylation of GPBAR by GRK2 at S310/S321/S323/S324 sites contributed to R399-induced GPBAR–β-arrestin 1 association. The cryoelectron microscopy (cryo-EM) structure of the R399-bound GPBAR-Gs complex enabled us to identify key interaction residues and pivotal conformational changes in GPBAR responsible for the arrestin signaling bias and cancer cell proliferation. In summary, we demonstrate that different agonists can regulate distinct functions of cell growth and apoptosis through biased GPBAR signaling and control of YAP activity in a NSCLC cell model. The delineated mechanism and structural basis may facilitate the rational design of GPBAR-targeting drugs with both metabolic and anticancer benefits.