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。

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.