Identification And Functional Study Of Steroid Hormone Ligands For Two AGPCRs And Their Molecular Mechanism Of Ligand Recognition

Adhesion G protein-coupled receptor(aGPCRs)is the second largest family in the G protein-coupled receptor(GPCRs)superfamily,with 33 members.For 1-9 subfamilies.aGPCRs function as key molecular switches in many important physiological processes in organisms,including immune responses,brain development,angiogenesis,bone growth,reproduction,and cell fate determination.Although aGPCRs regulate a variety of physiological functions,the known ligand information for most aGPCRs is very limited,which greatly limits the study of aGPCRs-mediated biological effects.Therefore,the discovery of endogenous ligands for adhesion receptors It will become a key part of studying the function of aGPCRs.Recent studies in our laboratory have found that glucocorticoids are the endogenous ligands of the adhesion receptor ADGRG3,and play a regulatory role by activating the downstream Gi pathway of the receptor.Other groups have found that the sterol analog 3-αacetoxydihydrodeoxygedunin(3-α-DOG)is a partial agonist of ADGRG1 and ADGRG5.Notably,the ligand-binding pockets of these adhesion-like receptors are relatively conserved among adhesion-like receptors belonging to the same subfamily.Therefore,whether steroid hormones are universal as the ligands of adhesion receptors,and whether different subfamily receptors have different steroid hormone ligands,are scientific issues worthy of further study.We systematically screened the members of the ADGRG subfamily to which ADGRG1,ADGRG3,and ADGRG5 belong,and found that ADGRG2 and ADGRG6 can also be activated by steroid hormones,and identified their ligands as dehydroepiandrosterone(DHEA),respectively.)/dehydroepiandrosterone sulfate(DHEAS)and progesterone(Progesterone,P4)/17α-Hydroxyprogesterone(170HP).Interestingly,we found that DHEA,DHEAS,progesterone,and 170HP are all sex hormones that are closely related to the functioning of the reproductive system.For example,DHEA and DHEAS can increase the levels of androgens in the testis and epididymis,and can also improve the ovarian environment in patients with decreased ovarian reserve and increase the probability of pregnancy;progesterone and 170HP can regulate ovulation,initiate and maintain the pregnancy process,and promote breast development.Play an important role.Therefore,whether ADGRG2 and ADGRG6,as membrane receptors of these hormones,are involved in regulating the corresponding biological functions,it is worthy of further investigation.In addition,we found that there is an"orthogonal" phenomenon in the pairing between steroid hormones and adhesion receptors,steroid hormones such as glucocorticoids,progesterone,etc.only act on specific adhesion receptors,so these adhesion receptors The molecular mechanism by which the bodyspecifically recognizes different steroid hormones also remains to be elucidated.ADGRG2 is a receptor closely related to male reproduction.Previous studies have found that ADGRG2 is coupled to cystic fibrosis transmembrane conductance regulator(CFTR)to regulate chloride current and water in male efferent tubules.Salt metabolism balance.Here,we first found that DHEA and DHEAS activated ADGRG2 downstream Gs signaling,but not Gq and β-arrestin-1/2 signaling,suggesting that DHEA and DHEAS may have regulatory effects on the coupling and function of ADGRG2 and CFTR.Second,we isolated male mouse efferent tubule non-ciliated cells and overexpressed ADGRG2 and CFTR in HEK293 cells.Electrophysiological experiments demonstrated that DHEA activates ADGRG2,promotes receptor coupling to CFTR,and rapidly modulates CFTR currents.In addition,we also found that Deoxycorticosterone(DOC),a neutral antagonist of ADGRG2,inhibited DHEA-induced cAMP accumulation,and electrophysiological experiments demonstrated that DOC antagonized DHEA-regulated DHEA-mediated cAMP accumulation in male mouse efferent tubule non-ciliated cells.CFTR current.Finally,we solved three high-resolution structures of Apo-ADGRG2-Gs,DHEA-ADGRG2-βT-Gs,DHEA-ADGRG2-FL-Gs,and predicted the interaction between the antagonist DOC and ADGRG2 by molecular dynamics simulation.patterns,revealing the molecular mechanism by which ADGRG2 recognizes different steroid ligands.The above findings will provide a powerful tool for exploring the function of ADGRG2 in the reproductive system,and also open up new ideas for the treatment of male infertility.As a member of the same ADGRG subfamily,ADGRG6 is activated by progesterone,which in malignancies promotes the development of uterine and breast cancers.Whether ADGRG6,as a membrane receptor for progesterone and 170HP,is involved in breast cancer progression is unclear.Therefore,we established a triple-negative breast cancer model to study the signal transduction mechanism and function of progesterone and 170HP acting on membrane receptors.First,we explored the ability of progesterone and 170HP to activate different G protein signaling pathways downstream of ADGRG6,and found that progesterone and 170HP activated the Gi pathway at low concentrations,while 170HP activated the Gs pathway at high concentrations.Further functional studies found that knockdown of ADGRG6 in triple-negative breast cancer cell lines blocked the pro-proliferative effect of progesterone and slowed the growth of xenograft tumors in nude mice.Furthermore,we found that progesterone plays a role in promoting cancer mainly by activating the ADGRG6-Gi-SRC pathway.Finally,we also found that progesterone and 170HP bind to the seven-transmembrane(7TM)of ADGRG6,in which two amino acids K1001 and F1012 of the second extracellular loop specifically recognize 170HP but not progesterone The key factor.In summary,our study identified the steroid hormone ligands of ADGRG2:DHEA,DHEAS and DOC,and also found that DHEA,DHEAS and DOC acting on ADGRG2 can regulate the chloride ion homeostasis of the export tubule,revealing that ADGRG2 specifically recognizes the molecular mechanism of DHEA and DOC,which provides a theoretical basis for the development of drugs targeting ADGRG2,and also puts forward new ideas for the treatment of male infertility.In addition,our study also identified the steroid hormone ligands of ADGRG6:progesterone and 17-hydroxyprogesterone,and found that the action of progesterone and 17-hydroxyprogesterone on ADGRG6 can promote the development of triplenegative breast cancer,clarifying that ADGRG6 specifically recognizes Molecular mechanism of progesterone and 17-hydroxyprogesterone.This provides a theoretical reference for the design of drugs targeting ADGRG6 and new insights for the treatment of triple-negative breast cancer.