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

doi:10.3969/j.issn.1674-8115.2025.10.008

Abstract

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

CLC Number:

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.

Figures/Tables 9

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

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

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

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

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)	‒	‒

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

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

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

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

TrendMD

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