| G protein-coupled receptor(GPCR)is the largest membrane protein superfamily in the human genome.There have been found more than 800 GPCRs,accounting for almost 3%of the genome code proteins.GPCRs are widely distributed and distr-ibuted throughout all tissues and organs in the human body,which are involved in regulating almost all life activities such as perception,reproduction,growth,development and neuronal activity of the body.For example,photoreceptors can regulate visual,olfactory receptors can determine odor recognition,adrenergic receptors are involved in the regulation of heart rate and blood pressure,chemokine receptors are involved in the regulation of the development of the immune system and inflammation,fatty acid receptors are involved in energy metabolism,and adenosine receptors are involved in neurodevelopmental and tissue damage protection,opioid receptors regulate pain and drug addiction,and so on.GPCR plays a key role in maintaining the normal physiological state of the body,and its dysfunction can lead to many diseases such as Alzheimer's disease,Parkinson's disease,dwarfism,color blindness and asthma.According to statistics,about one-third of the clinical drugs currently on the market have direct function on GPCRs,and above 1/10 of the top 200 best-selling drugs in the world made GPCRs as drug targets.Some very famous drugs including losartan,propranolol,ranitidine and tegaserod.Therefore,the scientific research of GPCR shows extremely important clinical significance and application value,and the related basic research has got 10 Nobel Prizes.The acute secretion of hormones triggered by the activation of GPCR plays an important role in many physiological processes in the body.It has been widely accepted that GPCR which is activated by ligands,plays signal transduction function mainly through two types of downstream effector molecules:G protein and ?-arrestin.Nowadays,it is a hot spot for drug research to develop a ligand that only activates G protein or ?-arrestin signaling pathway.Traditionally,the G-protein-mediated receptor signaling is very fast,occurring primarily on the cell membrane,typically from a few seconds to a few minutes,known as the first-wave signaling pathway;whereas?-arrestin-mediated receptors signaling is slower than the G protein signaling,which begins after the receptor complex enters the endoplasmic reticulum.It usually occurs after 5 minutes and is called the second wave signal pathway.For example,G protein subunit G?y can act directly on GIRK,leading to the opening of the channel,but the?-arrestin signaling pathway can not rapidly activate ion channels.The angiotensin receptor AT1R is an important drug target for clinical treatment of hypertension and heart failure.In our study of AT1R;we found that when AT1R is activated by the ?-arrestin-biased ligand TRV120027,the downstream PLC? and TRPC3 can be recruited by the ATIR-?-arrestinl signaling complex,thereby activating the calcium channel TRPC3 on the cell membrane,leading to acute secretion of catecholamine hormones such as adrenaline(within 1 minute).In addition,when we replaced the C-terminus of ?-arrestin-1 with the C-terminus of ?-arrestin-2,or blocked the interaction of ?-arrestinl and PLCy with a specific poly-Pro peptide,it would block the TRV120027-induced activation of TRPC3.Our further studies indicate that GPCR-?-arrestin-1-TRPC3,a rapid signal transduction pathway,plays an essential role in the function of angiotensin receptors and M-type acetylcholine receptors under physiological and pathological context.Our discovery breaks the traditional concept that ?-arrestin can only mediate the second-wave signaling pathway of GPCR.It has been first demonstrated that GPCR can exert the first wave signal transduction effect through ?-arrestin1,which will be very usedul for the research of GPCR in the future.It also revealed the potential side effect of the clinical drug TRV120027.For example,TRV120027 may promote the acute secretion of adrenaline through the ?-arrestin-1 pathway,which is unfavorable for the treatment of hypertension and heart failure.However,if we can combine TRV120027 with the poly-Pro peptide that inhibits the activity of ?-arrestin-1,it can not only ensure the positive therapeutic effect of TRV 1 20027,but also eliminates its potential side effects.Research purposesThe biased ligand of AT1R can lead to different physiological effects by selectively activating the G protein pathway or the ?-arrestin pathway.It has been reported that the ?-arrestin biased ligand of AT1R shows the better therapeutic effect on the treatment of hypertension and heart failure than the traditional antagonist losartan.However,the adrenaline and norepinephrine produced by ATI R when activated by traditional ligands such as Ang? are detrimental to the treatment of cardiovascular diseases.At present,the ?-arrestin biased ligand TRV120027 has entered the phase III clinical stage.The main purpose of this study is to study the downstream signal transduction pathway and the secretion of hormones after AT1R is activated by ?-arrestin biased ligand.Research methods1.The carbon fiber electrode was used to detect the secretion of catecholamines in mouse primary chromaffin cells.2.By using Fura-2,a calcium ion probe,to measure the concentration of intracellular calcium.3.Detect the co-localization between AT1R,?-arrestin-1,TRPC3 by using immunofluorescence.4.Detect the interaction between AT1R,?-arrestin.TRPC3 and PLCyl by co-immunoprecipitation(Co-IP)assay.5.Bioresonance energy transfer experiments(BRET)assay were used to detect the recruitment of downstream ?-arrestin by ATIR and the interaction between?-arrestin-1 and TRPC3.6.Detection of intracellular IP1 levels and the secretion of epinephrine and norepinephrine by using the related ELISA kit.7.Detect the interaction between ?-arrestin-1 and PLC?1-SH3 by GST-pull down assay.8.Statistical analysis:All data were presented as mean± SEM.Statistical comparisons were performed by using GraphPad Prism5 software with ANOVA,P<0.05 was considered statisticaly significant.Research results1.ATIR beta-arrestin biased ligand can lead to hormone secretion.We test with primary chromaffin cells isolated from the adrenal medulla of mice and found that not only Gq-biased ligands can cause chromophoric cells to secrete catecholamines when stimulated with different biased ligands for ATIR,?-arrestin-biased ligands such as TRV120026,TRV120027 can also lead to hormone secretion.2.After activated by the biased ligand,ATIR lead to hormone secretion by the?-arrestin-1 pathway rather than the ?-arrestin-2 pathway.We used Gq knockout mice,?-arrestin-1 knockout mice,and ?-arrestin-2 knockout mice for such experiments.It has been found that Gq or ?-arrestin-2 knockout did not affect the secretion caused by the biased ligand.However,after knocking out?-arrestin-1,ATIR was stimulated with a ?-arrestin-biased ligand such as TRV120027,but no secretion was detected.It revealed that during this process,ATIR lead to-hormone secretion completely by the ?-arrestin-1 pathway.3.The secretion caused by the ?-arrestin-1 pathway is achieved by the influx of extracellular calcium ions.In order to detect the source of intracellular calcium ions during the secretion process caused by the ?-arrestin-1 pathway,we replaced the extracellular environment with a 0 Ca2+,solution.In the absence of extracellular calcium ions,the hormone secretioncan not be detected even if it is stimulated with ?-arrestin-biased ligand.It indicated that the extracellular calcium ions is important for the ?-arrestin-1 pathway-mediated secretion process.4.The ?-arrestin-1 pathway activates the calcium channel TRPC3 on the cell membrane.Extracellular calcium ions flow into the cells usually through the calcium channel on the cell membrane.In order to study the calcium influx caused by the activation of calcium channels on the cell membrane in this process,we used a series of blockers of calcium channel and found that when we specifically blocking TRPC3 on the cell membrane,the intracellular calcium signal could not be detected.It is suggested that the ?-arrestin-1 pathway may lead to the activation of TRPC3 on the cell membrane.which causes extracellular calcium ions to flow into the cell.5.?-arrestin-1 can interact directly with TRPC3.To investigate how ?-arrestin-1 activates TRPC3 on cell membranes,we first examined whether there is direct interactions by immunofluorescence assay.The results showed that ?-arrestin-1 and TRPC3 on the cell membrane were co-localized after stimulated with the biased ligand TRV120027.Moreover,we demonstrated that TRV 120027 can induce a direct interaction between ?-arrestin-1 and TRPC3 by co-immunoprecipitation(Co-IP)assay and bioluminescence resonance energy transfer(BRET)assay.6.AT]R/?-arrestin-1/TRPC3 can form a complex that regulates intracellular calcium.Additionaly,we find there is a direct interaction between ATIR and TRPC3 through Co-IP.Therefore,it may take function by forming a tertiary complex AT1R/?-arrestin-1/TRPC3.We co-expressed these three proteins in HEK293 cells and stimulated with TRV120027 to detect changes in intracellular calcium signaling.And the increase in calcium signal can be blocked by the inhibitor of TRPC3.It indicates that ATI R/?-arrestin-1/TRPC3 act together to regulate intracellular calcium.7.PLCyl can participate in the formation of signaling complex thus can affect secretion.Previous reports indicate that PLC?1 is critical for the activation of TRPC3.In order to investigate whether PLC?1 is involved in this process,we detect whether?-arrestin-1 can directly interact with PLCyl by CoIP experiments,and we find that the specific Poly-Pro peptide can block the interaction between ?-arrestin-1 and PLC?1,consequently abolishing the activation of TRPC3,which can affect the hormone secretion of chromaffin cells.8.The ?-arrestin-1/TRPC3 pathway is a general mechanism for GPCR.In order to explore whether the ?-arrestin-1/TRPC3 mechanism is specifically act on AT1R,or it is a general mechanism.We used a range of GPCR agonists,including acetylcholine,cholecystokinin,and oxytocin to detect whether the hormone secretion induced by ligand can be inhibited by TRPC3 inhibitor.It was found that acetylcholine receptor-mediated hormone secretion can be inhibited by PYR3.Furthermore,in experiments with ?-arrestin-1 knockout mice,hormonr secretion was significantly reduced.This phenomenon indicating that the ?-arrestin-1/TRPC3 pathway is also applicable to the acetylcholine receptor.Therefore,the mechanism we have discovered may be general mechanism. |
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