GRK调控M1乙酰胆碱受体偏向性结合下游信号转导蛋白的机制研究

doi:10.3969/j.issn.1674-8115.2025.10.008

上海交通大学学报（医学版） ›› 2025, Vol. 45 ›› Issue (10): 1333-1341.doi: 10.3969/j.issn.1674-8115.2025.10.008

GRK调控M1乙酰胆碱受体偏向性结合下游信号转导蛋白的机制研究

魏嘉丽¹, 王冬雪², 王诗绮³, 徐见容³, 赵佩珅⁴, 赵兰雪¹()

^1.上海交通大学基础医学院药理学与化学生物学系，上海 ;201318
^2.中国科学院上海药物研究所，中科中山药物创新研究院，中山 528449
^3.上海中医药大学交叉科学研究院，上海 201203
^4.蒙纳士大学，蒙纳士制药科学研究院，墨尔本 3052

收稿日期:2025-01-14 接受日期:2025-03-13 出版日期:2025-10-28 发布日期:2025-10-28
通讯作者: 赵兰雪，副研究员，博士；电子信箱：annzoor@sjtu.edu.cn。
基金资助:
上海市高水平地方高校建设项目(ZXY24009);国家级中医西医会聚创新平台建设 [ZY(2025—2027)-2-2-1]

Mechanism of GRK subtypes modulating the unique binding properties of M1 acetylcholine receptor and transducers

WEI Jiali¹, WANG Dongxue², WANG Shiqi³, XU Jianrong³, ZHAO Peishen⁴, ZHAO Lanxue¹()

^1.Department of Pharmacology and Chemical Biology, Shanghai Jiao Tong University College of Basic Medical Sciences, Shanghai 201318, China
^2.Zhongshan Institute for Drug Discovery, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Zhongshan 528449, China
^3.Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
^4.Monash Institute of Pharmaceutical Sciences, Monash University, Melbourne 3052, Australia

Received:2025-01-14 Accepted:2025-03-13 Online:2025-10-28 Published:2025-10-28
Contact: ZHAO Lanxue, E-mail: annzoor@sjtu.edu.cn.
Supported by:
High-Level Local University Construction Project in Shanghai(ZXY24009);Three-year Action Plan for Shanghai TCM Development and Inheritance Program [ZY (2025?2027)-2-2-1]

摘要/Abstract

摘要：

目的·研究不同亚型G蛋白偶联受体激酶（G protein-coupled receptor kinase，GRK）在毒蕈碱型乙酰胆碱受体1（muscarinic acetylcholine receptor 1，M1受体）介导的偏向性信号转导过程中的作用机制，重点关注其调控M1受体与下游异源三聚体G蛋白（Gα_q-Gβ₁-Gγ₂）及β-抑制蛋白2（β-arrestin 2，βarr2）结合的分子效应。方法·构建基于生物发光能量共振转移（bioluminescence resonance energy transfer，BRET）的高灵敏度蛋白互作检测系统，选取6种结构及功能各异的M1受体激动剂/变构调节剂，系统测定在不同激动剂/变构调节剂刺激下M1受体与4种GRK亚型（GRK2/3/5/6）、βarr2以及G蛋白之间的动态相互作用。所有BRET实验数据均采用时间-效应曲线的曲线下面积（area under the curve，AUC）进行量化统计。首先，通过梯度浓度激动剂/变构调节剂处理及AUC拟合，建立浓度-效应曲线，综合分析各激动剂/变构调节剂相较于内源性激动剂氯化乙酰胆碱（acetylcholine chloride，ACh），在促进M1受体与GRK3/5、βarr2及G蛋白相互作用方面的效能差异；然后，将GRK按亚型类别分为GRK2/3和GRK5/6 2组，分别计算高浓度条件下M1受体与2类GRK互作的最大AUC值，进而评估不同类型GRK对M1受体与βarr2或G蛋白结合强度的调控倾向。结果·6种激动剂/变构调节剂均能有效诱导M1受体与GRK3的结合，但是它们也均能引起M1受体与GRK5发生解离；变构调节剂BQCA不仅能单独激活M1受体并引发其与下游信号转导蛋白的结合，还在与ACh联合处理时，使M1-G蛋白和M1-βarr2体系的浓度-效应曲线显著左移，提示其对ACh增幅作用主要是通过减小半数效应浓度；7组药物诱导的M1-βarr2与M1-G蛋白互作最大AUC之间存在中度正相关（r =0.722），但无统计学意义（P=0.067）；进一步分析表明，M1-GRK2/3与M1-GRK5/6互作最大AUC的比值，和M1-βarr2与M1-G蛋白互作最大AUC的比值同样呈正相关（r =0.760，P=0.047）。结论·M1受体可能在基础状态下即与GRK5/6预先结合，受体激动后两者解离，提示GRK5/6可能参与M1受体的失活或信号重编程；M1受体对不同GRK亚型的相对作用效率决定了其下游信号通路的偏好。

关键词: 毒蕈碱型乙酰胆碱受体1, G蛋白偶联受体激酶, G蛋白偶联受体, 信号偏向性

Abstract:

Objective ·To investigate the mechanisms by which different subtypes of G protein-coupled receptor kinases (GRKs) regulate the biased signaling transduction mediated by the muscarinic acetylcholine receptor 1 (M1 receptor), focusing on their molecular effects in modulating the binding of the M1 receptor to the downstream heterotrimeric G protein (Gα_q-Gβ₁-Gγ₂) and β-arrestin 2 (βarr2). Methods ·By establishing a highly sensitive protein interaction detection system based on bioluminescence resonance energy transfer (BRET), six M1 receptor agonists/allosteric modulators were selected to measure the dynamic interactions between the M1 receptor and four GRK subtypes (GRK2/3/5/6), βarr2, and the G protein under stimulation. All BRET data were statistically quantified using the area under the curve (AUC) of the time-response curves. First, concentration-effect curves were established by treatment with gradient concentrations of agonists/allosteric modulators and AUC fitting, to comprehensively analyze the differences in efficacy between each agonist/allosteric modulator and the endogenous agonist acetylcholine chloride (ACh) in promoting the interactions of M1 receptor with GRK3/5, βarr2, and the G protein; next, GRKs were divided into two groups based on subtypes: GRK2/3 and GRK5/6. The maximum AUC values for the interaction between the M1 receptor and the two GRK groups under high concentrations were calculated respectively, to further evaluate the regulatory propensity of different types of GRKs on the binding strength of the M1 receptor to βarr2 or the G protein. Results ·All six agonists/allosteric modulators effectively induced the association of the M1 receptor with GRK3, while simultaneousey inducing dissociation of the M1 receptor from GRK5. The allosteric modulator BQCA not only activated the M1 receptor alone and triggered its binding to downstream signaling proteins, but also, when co-treated with ACh, caused a significant leftward shift of the concentration-effect curves in the M1-G protein and M1-βarr2 systems, suggesting that its potentiation effect on ACh was mainly achieved by reducing the half-maximal effective concentration. A moderate positive correlation was observed between the maximum AUC values of M1-βarr2 and M1-G protein interactions induced by the seven groups of drug treatments (r =0.722, P=0.067). Further analysis showed that the ratio of the maximum AUC for M1-GRK2/3 interaction to that for M1-GRK5/6 interaction was also positively correlated with the ratio of the maximum AUC for M1-βarr2 interaction to that for M1-G protein interaction (r =0.760, P=0.047). Conclusion ·The M1 receptor may be pre-coupled with GRK5/6 under basal conditions, and they dissociate upon receptor activation, suggesting that GRK5/6 may be involved in M1 receptor inactivation or signal reprogramming. The relative efficiency of the M1 receptor's interaction with different GRK subtypes determines its preference for downstream signaling pathways.

Key words: muscarinic acetylcholine receptor 1 (M1 receptor), G protein-coupled receptor kinase (GRK), G protein-coupled receptor, signaling bias

中图分类号:

魏嘉丽, 王冬雪, 王诗绮, 徐见容, 赵佩珅, 赵兰雪. GRK调控M1乙酰胆碱受体偏向性结合下游信号转导蛋白的机制研究[J]. 上海交通大学学报（医学版）, 2025, 45(10): 1333-1341.

WEI Jiali, WANG Dongxue, WANG Shiqi, XU Jianrong, ZHAO Peishen, ZHAO Lanxue. Mechanism of GRK subtypes modulating the unique binding properties of M1 acetylcholine receptor and transducers[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2025, 45(10): 1333-1341.

图/表 9

图1 M1受体激动剂/变构调节剂诱导的M1受体与G蛋白结合浓度-效应曲线Note： Agonist responses were quantified by calculating the AUC of the kinetic traces for M1 receptor and G protein binding over a 20-minute period following stimulation with ACh, CCh (A), Pilo (B), Ipero (C), Xano (D), and BQCA (E), as well as after a 5-minute pre-treatment with 10 μmol/L BQCA prior to ACh application (F). The curves drawn through the data points represent the best fit of the operational model of agonism.

Fig 1 Concentration-response curves for M1-receptor/G-protein binding evoked by M1-receptor agonists/allosteric modulators

表1 M1受体激动剂/变构调节剂诱导的M1受体与G蛋白结合浓度-效应曲线的lgEC50 和 Emax

Tab 1 LgEC50 and Emax values of M1-receptor agonists/allosteric modulators obtained from concentration-response curves of M1 receptor and G-protein binding

Agonist/allosteric modulator	LgEC₅₀	E_max
ACh (n=4)	-5.903±0.099	385.883±21.039
CCh (n=3)	-5.301±0.141^①	271.541±48.441
Pilo (n=3)	-5.139±0.090^②	141.694±8.785^③
Ipero (n=3)	-7.540±0.093^③	381.134±10.272
Xano (n=3)	-5.094±0.079^④	276.680±15.139^⑤
BQCA (n=4)	‒	‒
ACh(B) (n=4)	-7.774±0.100^③	311.020±19.405

图2 M1受体激动剂/变构调节剂诱导的M1受体与βarr2结合浓度-效应曲线Note： Agonist responses were quantified by calculating the AUC of the kinetic traces for M1 receptor and βarr2 binding over a 20-minute period following stimulation with ACh, CCh (A), Pilo (B), Ipero (C), Xano (D), and BQCA (E), as well as after a 5-minute pre-treatment with 10 μmol/L BQCA prior to ACh application (F). The curves drawn through the data points represent the best fit of the operational model of agonism.

Fig 2 Concentration-response curves for M1-receptor/βarr2 binding evoked by M1-receptor agonists/allosteric modulators

表2 M1受体激动剂/变构调节剂诱导的M1受体与βarr2结合浓度-效应曲线的lgEC50 和 Emax

Tab 2 LgEC50 and Emax values of M1-receptor agonists/allosteric modulators obtained from concentration-response curves of M1 receptor and βarr2 binding

Agonist/allosteric modulator	LgEC₅₀	E_max
ACh (n=3)	-6.115±0.319	241.277±23.552
CCh (n=3)	‒	‒
Pilo (n=4)	‒	‒
Ipero (n=4)	-8.174±0.098^①	391.214±11.609^②
Xano (n=4)	‒	‒
BQCA (n=3)	‒	‒
ACh(B) (n=4)	-7.117±0.225	199.458±20.933

表3 M1受体激动剂/变构调节剂诱导的M1受体与GRK3结合浓度-效应曲线的lgEC50 和 Emax

Tab 3 LgEC50 and Emax values of M1-receptor agonists/allosteric modulators obtained from concentration-response curves of M1 receptor and GRK3 binding

Agonist/allosteric modulator	LgEC₅₀	E_max
ACh (n=3)	‒	‒
CCh (n=3)	-5.258±0.154	46.263±6.437
Pilo (n=3)	-7.248±0.268	18.780±1.114
Ipero (n=4)	-7.274±0.111^①	40.119±6.043
Xano (n=4)	‒	‒
BQCA (n=3)	-6.505±0.044	12.227±7.734
Ach(B) (n=4)	‒	‒

图3 M1受体激动剂/变构调节剂诱导的M1受体与GRK3结合浓度-效应曲线Note： Agonist responses were quantified by calculating the AUC of the kinetic traces for M1 receptor and GRK3 binding over a 20-minute period following stimulation with ACh, CCh (A), Pilo (B), Ipero (C), Xano (D), and BQCA (E), as well as after a 5-minute pre-treatment with 10 μmol/L BQCA prior to ACh application (F). The curves drawn through the data points represent the best fit of the operational model of agonism.

Fig 3 Concentration-response curves for M1-receptor/GRK3 binding evoked by M1-receptor agonists/allosteric modulators

图4 M1受体激动剂/变构调节剂诱导M1受体与GRK5解离浓度-效应曲线Note： Agonist responses were quantified by calculating the AUC of the kinetic traces for M1 receptor and GRK5 dissociation over a 20-minute period following stimulation with ACh, CCh (A), Pilo (B), Ipero (C), Xano (D), and BQCA (E), as well as after a 5-minute pre-treatment with 10 μmol/L BQCA prior to ACh application (F). The curves drawn through the data points represent the best fit of the operational model of agonism.

Fig 4 Concentration-response curves for M1-receptor/GRK5 disassociation evoked by M1-receptor agonists/allosteric modulators

表4 M1受体激动剂/变构调节剂诱导的M1受体与GRK5解离浓度-效应曲线的lgEC50 和 Emax

Tab 4 LgEC50 and Emax values of M1-receptor agonists/allosteric modulators obtained from concentration-response curves of M1 receptor and GRK5 disassociation

Agonist/allosteric modulator	LgEC₅₀	E_max
ACh (n=3)	-6.289±0.234	-24.886±0.972
CCh (n=3)	-6.004±0.193	-31.393±10.711
Pilo (n=4)	‒	‒
Ipero (n=4)	-8.655±0.253^①	-51.719±8.572^②
Xano (n=4)	-6.596±0.216	-21.004±9.831
BQCA (n=3)	‒	‒
Ach(B) (n=4)	-7.105±0.258	-37.911±3.360^③

图5 M1受体激动剂/变构调节剂诱导的M1受体与不同亚型GRK、βarr2、G蛋白互作最大AUC间的相关性分析Note： A. Correlation between the maximum AUCs of M1 receptor interactions with βarr2 and G protein. B. Correlation between the maximum AUCs of M1 receptor interactions with βarr2 and GRK2/3. C. Correlation between the maximum AUCs of M1 receptor interactions with βarr2 and GRK5/6. D. Correlation between the maximum AUCs of M1 receptor interactions with G protein and GRK2/3. E. Correlation between the maximum AUCs of M1 receptor interactions with G protein and GRK5/6. F. Correlation between the ratio of AUICM1-GRK2/3 and AUCM1-GRK5/6 and that of AUCM1-βarr2 and AUCM1-Gγ2.

Fig 5 Correlation analysis of the maximum AUCs for M1 receptor interactions with different GRKs subtypes, βarr2, and G protein induced by M1 receptor agonists/allosteric modulators

参考文献 27

[1]	GALVIN V C, YANG S T, PASPALAS C D, et al. Muscarinic M1 receptors modulate working memory performance and activity via KCNQ potassium channels in the primate prefrontal cortex[J]. Neuron, 2020, 106(4): 649-661.e4.
[2]	KAWAKAMI K, YANAGAWA M, HIRATSUKA S, et al. Heterotrimeric Gq proteins act as a switch for GRK5/6 selectivity underlying β-arrestin transducer bias[J]. Nat Commun, 2022, 13(1): 487.
[3]	DEAN B. Muscarinic M1 and M4 receptor agonists for schizophrenia: promising candidates for the therapeutic arsenal[J]. Expert Opin Investig Drugs, 2023, 32(12): 1113-1121.
[4]	KAOULLAS M G, THAL D M, CHRISTOPOULOS A, et al. Ligand bias at the muscarinic acetylcholine receptor family: opportunities and challenges[J]. Neuropharmacology, 2024, 258: 110092.
[5]	SCARPA M, MOLLOY C, JENKINS L, et al. Biased M1 muscarinic receptor mutant mice show accelerated progression of prion neurodegenerative disease[J]. Proc Natl Acad Sci USA, 2021, 118(50): e2107389118.
[6]	BRADLEY S J, MOLLOY C, VALUSKOVA P, et al. Biased M1-muscarinic-receptor-mutant mice inform the design of next-generation drugs[J]. Nat Chem Biol, 2020, 16(3): 240-249.
[7]	MATTHEES E S F, HAIDER R S, HOFFMANN C, et al. Differential regulation of GPCRs: are GRK expression levels the key?[J]. Front Cell Dev Biol, 2021, 9: 687489.
[8]	WANG D X, YAO Y J, WANG S Q, et al. Structural insights into M1 muscarinic acetylcholine receptor signaling bias between Gα_q and β-arrestin through BRET assays and molecular docking[J]. Int J Mol Sci, 2023, 24(8): 7356.
[9]	FOX R I, STERN M, MICHELSON P. Update in Sjögren syndrome[J]. Curr Opin Rheumatol, 2000, 12(5): 391-398.
[10]	SCHRAGE R, SEEMANN W K, KLÖCKNER J, et al. Agonists with supraphysiological efficacy at the muscarinic M2 ACh receptor[J]. Br J Pharmacol, 2013, 169(2): 357-370.
[11]	KAUL I, SAWCHAK S, CORRELL C U, et al. Efficacy and safety of the muscarinic receptor agonist KarXT (xanomeline-trospium) in schizophrenia (EMERGENT-2) in the USA: results from a randomised, double-blind, placebo-controlled, flexible-dose phase 3 trial[J]. Lancet, 2024, 403(10422): 160-170.
[12]	SHIREY J K, BRADY A E, JONES P J, et al. A selective allosteric potentiator of the M1 muscarinic acetylcholine receptor increases activity of medial prefrontal cortical neurons and restores impairments in reversal learning[J]. J Neurosci, 2009, 29(45): 14271-14286.
[13]	BERSTEIN G, BLANK J L, JHON D Y, et al. Phospholipase C-β1 is a GTPase-activating protein for Gq/11, its physiologic regulator[J]. Cell, 1992, 70(3): 411-418.
[14]	RAJAGOPAL S, SHENOY S K. GPCR desensitization: acute and prolonged phases[J]. Cell Signal, 2018, 41: 9-16.
[15]	KAHSAI A W, SHAH K S, SHIM P J, et al. Signal transduction at GPCRs: allosteric activation of the ERK MAPK by β-arrestin[J]. Proc Natl Acad Sci USA, 2023, 120(43): e2303794120.
[16]	LATORRACA N R, MASUREEL M, HOLLINGSWORTH S A, et al. How GPCR phosphorylation patterns orchestrate arrestin-mediated signaling[J]. Cell, 2020, 183(7): 1813-1825.e18.
[17]	GUREVICH E V, TESMER J J G, MUSHEGIAN A, et al. G protein-coupled receptor kinases: more than just kinases and not only for GPCRs[J]. Pharmacol Ther, 2012, 133(1): 40-69.
[18]	GIACOBINI E, CLAUDIO CUELLO A, FISHER A. Reimagining cholinergic therapy for Alzheimer's disease[J]. Brain, 2022, 145(7): 2250-2275.
[19]	SCARR E, SEO M S, AUMANN T D, et al. The distribution of muscarinic M1 receptors in the human hippocampus[J]. J Chem Neuroanat, 2016, 77: 187-192.
[20]	NGUYEN H T M, VAN DER WESTHUIZEN E T, LANGMEAD C J, et al. Opportunities and challenges for the development of M₁ muscarinic receptor positive allosteric modulators in the treatment for neurocognitive deficits[J]. Br J Pharmacol, 2024, 181(14): 2114-2142.
[21]	DRUBE J, HAIDER R S, MATTHEES E F, et al. GPCR kinase knockout cells reveal the impact of individual GRKs on arrestin binding and GPCR regulation[J]. Nat Commun, 2022, 13(1): 540.
[22]	DUAN J, LIU H, ZHAO F H, et al. GPCR activation and GRK2 assembly by a biased intracellular agonist[J]. Nature, 2023, 620(7974): 676-681.
[23]	CARMAN C V, PARENT J L, DAY P W, et al. Selective regulation of Gα_q/11 by an RGS domain in the G protein-coupled receptor kinase, GRK2[J]. J Biol Chem, 1999, 274(48): 34483-34492.
[24]	MATTHEES E S F, FILOR J C, JAISWAL N, et al. GRK specificity and Gβγ dependency determines the potential of a GPCR for arrestin-biased agonism[J]. Commun Biol, 2024, 7(1): 802.
[25]	EICHMANN T, LORENZ K, HOFFMANN M, et al. The amino-terminal domain of G-protein-coupled receptor kinase 2 is a regulatory Gβγ binding site[J]. J Biol Chem, 2003, 278(10): 8052-8057.
[26]	GARDNER J, EIGER D S, HICKS C, et al. GPCR kinases differentially modulate biased signaling downstream of CXCR3 depending on their subcellular localization[J]. Sci Signal, 2024, 17(823): eadd9139.
[27]	ROOK J M, ABE M, CHO H P, et al. Diverse effects on M₁ signaling and adverse effect liability within a series of M₁ ago-PAMs[J]. ACS Chem Neurosci, 2017, 8(4): 866-883.

GRK调控M1乙酰胆碱受体偏向性结合下游信号转导蛋白的机制研究

Mechanism of GRK subtypes modulating the unique binding properties of M1 acetylcholine receptor and transducers

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