GPR64 Signal Transduction And Molecular Mechanism Of Its Coupling To G-proteins

Background:G Protein Coupled Receptors (GPCRs) are one class of membrane protein which encompass more than 800 members in huan genome, mutations of these receptors are associated with many diseases and 40% of precriptional drugs targets to GPCRs. All GPCRs have an extracellular N-terminus, an intracellular C tail,7 transmembrane helices, three extracellular loops and three intracellular loops. GPCRs respond to extracellular signal and transduce the signal into cells and then cause a series of biochemical reaction. GPCRs need to interact with G-protein or arrestin to active them and produce second messengers to active downstream signal pathways. The heterotrimeric G protein is composed of three subunits, ?, ?, and ?. To date,20 types of Ga subunits,5 types of G? subunits, and 10 types of Gy subuits have been identified. Different Ga subunits can initiate the production of different second messengers, leading to divergent downstream pathways. Different GPCRs can interact with different G-proteins to regulate different physiological functions. GPR64 belongs to adhesion GPCR subfamilies, containing a long N-terminus and a GPCR proteolytic site, expressed in human and mouse epididymis, human prostate cancer, parathyroid gland and central nervous system. Recently, studies have shown that GPR64 plays an important role in liquid reabsorption to adjust sperm concentration in the male reproductive system. However, the signal transduction downstream of GPR64 and molecular mechanism underlying its coupling to G proteins are unknown.Objects:We use second messengers measurement to detect the signal pathway of GPR64. Subsequently, we look for the key residues of GPR64 which are responsible for its G protein coupling activity.Methods:We use the GloSensor cAMP assay to quantify second messenger cAMP, the NFAT-luciferase measurement to measure its Gq coupling,the quickchange to make GPR64 mutations for further investigation of the signaling properties of these mutants.Results:The result of GloSensor cAMP assay and NFAT-DLR show that GPR64 couples to Gs and Gq signal pathway to increase the production of cAMP and Ca2+,respectively. GPR64FL's ability of increasng cAMP and NEAT is lower than that of GPR64?. Different GRP64 mutants display different effects in cAMP or NFAT regulation.Conclusions:GPR64 can couple to Gs and Gq to activate downstream signal pathways. GPR64 couples to Gs through H696/M697, V704, F705, whereas GPR64 couples to Gq through H696/M697, Y708, R803/K804.