Function And Signal Transduction Of Adhesion G Protein-Coupled Receptor GPR64

Objective:Adhesion G protein-coupled receptors(Adhesion GPCRs)are a type of GPCR structure that has only recently been studied with much attention.Because the adhesion-like G protein-coupled receptors have complex structures,their research is difficult,only have little research on the function in vivo.In addition,most of the adhesion-like G protein-coupled receptors are still orphan receptors,and their ligands are unknown,so the function of G-protein-coupled receptors,signal transduction studies and ligand discovery of adhesion-like G-protein coupled receptors are of great significance.At present,according to relevant statistics,about 15%of couples worldwide suffer from infertility,and male factors account for more than 50%.Male infertility has become a concern.GPR64(also known as Adgrg2 or HE6)is mainly expressed in the epididymis and plays an important role in male fertility.This important function has caused us great interest in this receptor because it is an increase in male fertility or potential targets for male contraception.In the mouse study,GPR64 can cause leading to accumulation of sperm in the efferent ductules,but the specific cause is not clear.Recently,the endogenous GPR64 mutation known to humans is one of the causes of obstructive azoospermia in humans,but its signal transduction pathway is not clear.In addition,GPR64 is currently an orphan receptor and has no ligand.Currently,only the stimulating peptide derived from the Stachel sequence is known,and it cannot provide effective work for its functional studies.Therefore,this article mainly explores the function of GPR64 in the reproductive system,signal transduction,agonist peptide ligand optimization and its binding mode to GPR64 receptor.Research purposes1:Whether GPR64 plays an important role in the reproductive system.2:which downstream target protein regulates GPR64 in the efferent ductules reabsorption function?3:How the downstream G proteins regulation of endogenous mutations C570Y,K990E of GPR64 known to human disease regulation mechism?4:GPR64-known Stachel sequence-derived agonist peptide ligand optimization and binding of this ligand to the GPR64 receptor model.MethodsIn this paper,real-time quantitative PCR(Real-time PCR)was used to study the expression of 31 adhesion GPCRs in the epididymis;real-time quantitative PCR(Real-time PCR)was used to detect the efferent ductules and GPR64-promoter adenovirus-labeled cells the expression of important ion channels related to water regulation and Gs and Gq proteins;whether the construction of GPR64 frameshift mutant knockout mice was successful at the genetic level by common PCR and cDNA sequencing techniques;Western blot experimental methods were used to verify the protein expression of GPR64 in epididymis and HEK-293 cell lines.HE staining was used to detect WT mice and GPR64-/Y mice sperm accumulation in the epididymis and export tubules;GPR64-promoter adenovirus-labeled efferent ductules primary cells for later experiments;Using various channel inhibitors to study the important ion channels in the efferent ductules for water reabsorption;The Immunofluorescence staining technique was used to detected the expression of GPR64,CFTR,Gs,Gq in the epididymis or the efferent ductules;The concentration of Cl-was detected by the chloride ion MQAE probe in the cells after ligation of the the WT GPR64-/Y mice.The intracellular pH value of the WT mouse and the GPR64-/Y mice ligated efferent ductules was detected by hydrogen ion BCECF-AM probe.The whole cell current of the primary GPR64-promoter adenovirus-labeled export tube single cell and its HEK-293 cell recombination system was recorded by patch clamp method.The human endogenous GPR64 mutant plasmid known as C570Y,K990E were constructed by Quick change.GloSensor cAMP technology was used to detect GPR64 activation in Gs signaling pathway in HEK-293 cells;NFAT-Luciferase dual luciferase reporter detection system was used to detect GPR64 activation in Gq signaling pathway in HEK-293 cells;Membrane receptor ELISA was used to detect the expression of different mutants of GPR64.Alanine scanning was used to detect the key amino acids of GPR64 agonist peptide.The modified Bret method was used to detect the optimized agonist peptide V13F3Me derived from Stachel sequences binding activity to different GPR64 mutants.Results1.GPR64 expression was higher under normal conditions and lipopolysaccharide(LPS)-induced epididymal inflammation conditionsThe expression of 31 adhesion GPCRs in the epididymis was analyzed by Real-time PCR.It was found that the most of adhesion GPCRs were expressed in the epididymis.Among them,we found that the three highly expressed adhesion GPCRs include CD87,CELSR1 and GPR64.At the same time,in the case of LPS model-induced epididymal inflammation,only GPR64 was abundantly expressed in the above three highly expressed adhesion GPCRs.According to the results of this study and the known GPR64 function related literature summary,the GPR64 reproductive system has important functions.2.The construction of GPR64 frameshift mutant knockout mouse was successful.Using the CRISPR-cas9 technique,a 2 bp nucleotide was deleted and add of 10 bp nucleotides in the first exon region of GPR64,resulting in a frameshift mutation.Translation of GPR64 is stopped in the signal peptide region and used for all splice variants.The construction of GPR64 frameshift mutant knockout mice was successfully verified at the gene level by common PCR and genomic DNA sequencing,but the gene level sometimes does not represent changes in protein levels.We later examined the expression of GPR64 knockout mouse protein,GPR64 was expressed in the cell membrane,and the expression of GPR64 in knockout mice and WT mice was detected by isolating epididymal head membrane protein,and finally the construction of GPR64 knockout mice was verified to be successful.3.GPR64 knockout mice have reduced fertility and efferent ductules sperm accumulationBy counting the number of WT and GPR64-/Y knockout mouse embryos,the results showed that the fertility decreased after GPR64 knockout;GPR64-/Y mice were found the number of spermatozoa was reduced by detecting the sperm count in the epididymis tail and HE staining also showed the GPR64-/Y mice epididymis caput and coupus sperm area revealed statistical decrease;HE staining of the efferent ductules and statistical sperm area revealed that GPR64-/Y knockout mouse sperm showed massive accumulation in the efferent ductules.4.GPR64 knockout affects testicular water reabsorptionAfter GPR64 protein knockout,we tested the weight of testis under normal conditions and found that the weight of GPR64-/Y testis was significantly heavier than that of WT,indicating that GPR64 may play an important role in water reabsorption.In addition,we done the epididymal head ligation experiment.It ws also confirmed that GPR64 plays an important role in testicular water reabsorption.5.GPR64 knockout mice did not affect the development of epididymis,but affected the water reabsorption of the efferent ductules.Since GPR64 is expressed in the epididymal head,we hypothesize that GPR64 knockout mice affect the development of the epididymis to affect water reabsorption.The results indicate that GPR64-Y does not affect the development of the epididymis;in addition,80%of the water in the testis is water reabsorption via the output tubule Absorption,we separated the output tubules and measured the width of the efferent ductules under normal conditions.As a result,the width did not change,indicating that GPR64 did not affect the development of the epididymis and the efferent ductules width under normal conditions.We further refer to the literature,using the method of ligation at both ends of the efferent ductules,observe the change of the output tube width at different times.The results show that the GPR64 knockout mouse efferent ductules bulge obviously with the extension of the ligation time,indicating that the GPR64 plays an important role in the efferent ductules water reabsorption6.Efferent ductules water reabsorption function is related to CFTR ion channel regulationWe selected important ion regulatory channels for the efferent dutules such as CFTR(cystic fibrosis transferase regulatory protein),Anoctamin-1(ANO1),sodium-Hydrogen transporter 1(NHE1),sodium-hydrogen back transporter 3(NHE3),adenoma down-regulated protein(DRA),solute carrier family 26 member 9(SLC26A9),carbonic anhydrase II(CAII),chloride Channel fitting 1(CLCA1),We screened by real-time PCR for the high expression ion channels of the efferent ductules and efferent ductules GPR64 promoter-labeled primary cells.We selected CFTR,ANO1,NHE1,DRA,NKCC,SLC26a9,CAII with high expression of GPR64-promoter-labeled cells were selected.After the normal efferent ductules was ligated,the inhibitor of the above channel was used,The time swelling of efferent ductules showed that the CFTR inhibitor CFTRinh-172 could mimic the phenotype of GPR64 knockout mice,indicating that the GPR64 knockout mouse efferent ductules water reabsorption dysfunction may be related to CFTR ion channel regulation.7.GPR64 knockout mice has impact on the function of CFTR ion channelsIn order to investigate whether the water reabsorption dysfunction of GPR64 knockout mouse is related to the regulation of CFTR ion channel.First,we used immunofluorescence to confirm that GPR64 and CFTR have the same expression position,and they are expressed in non-ciliated cells of efferent ductules.We determined WT mice and GPR64-/Y mice chloride ion concentration and pH value after 48 hours of the efferent ductules ligation.We found that the GPR64-/Y knockout mice has a high chloride ion concentration and a high pH value,and the chloride ion and hydrogen ion homeostasis are unbalanced;It was further confirmed that GPR64-/Y knockout mice affect CFTR-mediated chloride current.However,Real-time PCR results showed that GPR64 knockdown did not affect CFTR expression.In summary,GPR64 knockdown affects CFTR function,but does not affect its expression.8.The function of GPR64 regulates CFTR function is related to the downstream protein Gq,?-arrestinl plays an important role in the formation of GPR64 and CFTR complexes.We used immunofluorescence staining and real-time PCR to detect the expression of downstream G protein in the output tubules,and found that Gq and GPR64 are expressed in the same cells,in non-ciiiated cells;and We further verified Gq+/-knockout mice the concentration of chloride and hydrogen ions in the efferent ductules after 48 hours of ligation.the results revealed that the Gq protein had a high ion concentration and a high pH after knockout,which indirectly reflected the CFTR function affected by Gq protein(we verified Gq+/-in the elife article Gq+/-knockout the mouse cell current is lower and the inhibitors of Gs and Gq protein in normal primary GPR64-promoter-labeled cells all confirmed that Gq protein plays an important role in mediating the function of GPR64 to regulate CFTR,and this part of the work I am involved.But these parts of the datas have been used in the graduation thesis of the former collaborator DaoLai-Zhang);the late experiment found that the concentration of chloride and hydrogen ions in the tissue cells of the ?-arrestin1-/-and?-arrestin2-/-knockout mice of the efferent ductules after 48 hours of ligation.The?-arrestinl knockout mouse has a high chloride ion concentration and pH value,and the ?-arrestinl protein affects the function of CFTR.(At the same time,we used the CO-IP experment to verify that GPR64 and CFTR proteins can form complexes in the elife article,and that ?-arrestinl plays a role in the formation of complexes,immunofluorescence.The results confirmed that p-arrestin1 knockdown affects the colocalization of CFTR with GPR64 and this part of the work I am involved.But these parta of the datas have been used in the graduation thesis of the former collaborator Daolai Zhang).9.HEK-293 cell line confirmed that GPR64 can constitutively activate Gs and Gq protein viabilityUsing GloSensor cAMP assay in HEK-293 cells,GPR64FL and GPR64beta can be used to constitutively activate endogenous cAMP levels,which can constitutively activate Gs signaling pathway;NFAT-Luciferase dual luciferase reporter assay in HEK-293 cells systematic detection of GPR64FL and GPR64beta activates the transcriptional level of NFAT and constitutively activates the Gq signaling pathway.10.GPR64 mutation K990E is known to be associated with human reproductive diseases that can reduce the Gs and Gq proteins activityThe GPR64FL-K990E,GPR64Beta-K990E mutant plasmids were constructed by Quick change.GloSensor cAMP technology was used to detect the effect of GPR64FL-K990E and mutation on Gs signaling pathway in HEK-293 cells.The results showed that K990E mutation can reduce the constitutive activation of endogenous cAMP and decrease the activity of Gs protein in GPR64;In HEK-293 cells the NFAT-Luciferase dual luciferase reporter assay system was used to detect NFAT transcript levels,indicating that GPR64FL-K990E can reduce the constitutive activation of NFAT transcription of GPR64 and reduce Gq protein activity.11.GPR64 mutation K990E is associated with human reproductive disease that can reduce CFTR currentThe HEK-293 cell recombination system was used to detect the ability of GPR64FL-K990E mutation.To detect whether GPR64FL-K990E mutation can regulate the chloride current of CFTR by whole-cell patch clamp technique.The results showed that the GPR64FL-K990E mutation resulted in decreasing the CFTR ability and decreased chloride current.12.The first and third positions of the agonist peptide P15 derived from the known Stachel sequence of GPR64 are critical for its activity.The first to last amino acids are sequentially mutated to alanine by alanine scanning technology.The key amino acid of peptide p15 is red-labeled T S F G I L L D L S R T S L P.The mutation of these amino acids can completely destroy the peptide in activating of cAMP.And by truncating the amino acids at both ends of the peptide p15,it was found that the amino acids at both ends were essential for the activity of p15,and the first,third,fifth,sixth,and seventh were the more conserved amino acids in the Adhesion GPCR.The amino acid mutation and the non-natural amino acid modification were used,and the results showed that the first T became the transgenic amino acid V and the third F became the unnatural amino acid Phe(4Me).13.GPR64-known Stachel sequence-derived agonist-optimized short peptide V13FMe enhances signal transduction for GS,Gq,?-arrestin1,and ?-arrestin2 compared to p15GPR64-known Stachel sequence-derived agonist-optimized short peptide V13FMe has a better activation effect on GPR64 than p15,and activates Gs,Gq,?-arrestinl and ?-arrestin2 signaling pathways,and activates Gs.The semi-activated concentration of the signal pathway was 471.5nmol/L,the semi-activated concentration of P15 was 81.4?mol/L,and the semi-activated concentration of V13FMe was 172 times that of P15.At the same time,compared with P15,the Gq,?-arrestin1 and ?-arrestin2 signaling pathways can be activated.At the same time,the semi-activated concentration EC50 of activated Gq is 214.5 ?mol/L,and the semi-activated concentration EC50 of activated P-arrestinl signaling pathway is 20?mol./L,the semi-activated concentration EC50 of the activated ?-arrestin2 signaling pathway was 30?mol/L.14.GPR64 mutants W757A,I758A,V768A,F824A,F826A disappeared affinity for GPR64 after the activating polypeptide[FITC]V13FMeFITC-labeled GPR64 activating polypeptide[FITC]V13FMe at different concentrations(10-10,10-9,10-8.10-7,10-6,10-5,1×10-5,2×10-5,104 mol/L),which studies its binding ability to different GPR64 mutation maps,showed that GPR64-?GPS-beta has a good binding ability to activated short peptides.but its mutants W757A,I758A,V768A,F824A,F826A mutations,and the short peptide binding capacity was completely lost,indicating that these four amino acid positions are closely related to GPR64 binding to V13FMe short peptide.15.GPR64 mutant affects the binding ability of the polypeptide V13FMe affects the downstream cAMP level.For the GPR64?-GPS-beta different mutants,the principle of Gs-mediated second messenger cAMP can be detected by GloSensor method.The results showed that compared with WT(GPR64?-GPS-beta),the binding ability of different GPR64 mutants to V13FMe decreased,resulting in decreased downstream cAMP activation levels,such as W757A,I758A,N759A,V762A,W824A,F826A.However,V13FMe did not change the binding ability of S760A,F772A,F828A,V836A,I844A and F845A,but its cAMP level decreased,which may be caused by the change of the ability of binding downstream Gs protein.Conclusions1.GPR64 is expressed at a higher level in the epididymis and the induction of epididymal inflammation.2.The GPR64 frameshift mutant knockout mouse construction was successful.3.After GPR64 knockout,with the decrease number of epididymal sperm and the phenomenon of sperm accumulation in the efferent ductules is accompanied.4.GPR64 knockout affects testicular and efferent ductules water reabsorption.5.GPR64 mainly affects the water reabsorption function of the efferent ductules by regulating the CFTR ion channel.6.GPR64 mainly affects the water reabsorption function of the efferent ductules CFTR channels by regulating Gq protein.7.The p-arrestinl protein plays an important role in the formation of GPR64 and CFTR complexes in the efferent ductules.8.GPR64 mutation K990E is known to be involved in human reproductive-related disease that can affecting Gs and Gq signaling pathways.9.GPR64 mutation K990E is known to be involved in human reproduction-related disease that can affect CFTR current.10.The first and third positions of the agonist peptide P15 derived from the known Stachel sequence of GPR64 are critical for providing its activity.11.GPR64-known Stachel sequence-derived agonist peptide-optimized short peptide V13FMe is enhanced for GS,Gq,?-arrestin1,and ?-arrestin2 signal transduction compared to p15.12.GPR64 mutants W757A,I758A,V768A,F824A,F826A disappeared affinity for GPR64 after the activating polypeptide[FITC]V13FMe.13.GPR64 mutant affects the binding ability of the polypeptide V13FMe and affects the downstream cAMP level.