Harwood Clare R, Sykes David A, Redfern-Nichols Theo, Underwood Owen, Nicholson Colin, Khoshgrudi Armin N, Koers Eline J, Ladds Graham, Briddon Stephen J, Veprintsev Dmitry B
Division of Physiology, Pharmacology and Neuroscience, School of Life Sciences, University of Nottingham, Nottingham, United Kingdom.
Centre of Membrane Proteins and Receptors (COMPARE), University of Birmingham and University of Nottingham, Nottingham, United Kingdom.
Front Pharmacol. 2025 Mar 27;16:1367991. doi: 10.3389/fphar.2025.1367991. eCollection 2025.
The β-adrenoceptor (βAR) is a class A G protein-coupled receptor (GPCR). It is therapeutically relevant in asthma and chronic obstructive pulmonary disease (COPD), where βAR agonists relieve bronchoconstriction. The βAR is a prototypical GPCR for structural and biophysical studies. However, the molecular basis of agonist efficacy at the βAR is not understood. We hypothesised that the kinetics of GPCR-G protein interactions could play a role in determining ligand efficacy. By studying a range of agonists with varying efficacy, we examined the relationship between ligand-induced mini-G binding to the βAR and ligand efficacy, along with the ability of individual ligands to activate the G protein in cells.
We used NanoBRET technology to measure ligand-induced binding of purified Venus-mini-G to βAR-nLuc in membrane preparations under both equilibrium and kinetic conditions. In addition, we examined the ability of these βAR agonists to activate the heterotrimeric G protein, measured using the G-CASE protein biosensor in living cells. This assay detects a reduction in NanoBRET between the nano-luciferase (nLuc) donor on the Gα subunit and Venus acceptor on the Gγ upon G protein activation.
The 12 βAR agonists under study revealed a broad range of ligand potency and efficacy values in the cellular G-CASE assays. Kinetic characterisation of mini-G binding to the agonist βAR complex revealed a strong correlation between ligand efficacy values (E) and mini-G affinity ( ) and its association rate ( ). In contrast, there was no correlation between ligand efficacy and reported ligand dissociation rates (or residence times).
The association rate ( ) of the G protein to the agonist βAR complex is directly correlated with ligand efficacy. These data support a model in which higher-efficacy agonists induce the βAR to adopt a conformation that is more likely to recruit G protein. Conversely, these data did not support the role of agonist binding kinetics in determining the molecular basis of efficacy.
β-肾上腺素能受体（βAR）是A类G蛋白偶联受体（GPCR）。它在哮喘和慢性阻塞性肺疾病（COPD）的治疗中具有重要意义，βAR激动剂可缓解支气管收缩。βAR是用于结构和生物物理研究的典型GPCR。然而，βAR激动剂效力的分子基础尚不清楚。我们推测GPCR-G蛋白相互作用的动力学可能在决定配体效力方面发挥作用。通过研究一系列效力不同的激动剂，我们研究了配体诱导的微型G与βAR的结合与配体效力之间的关系，以及单个配体在细胞中激活G蛋白的能力。
我们使用纳米BRET技术在平衡和动力学条件下测量纯化的维纳斯-微型G与膜制剂中βAR-nLuc的配体诱导结合。此外，我们研究了这些βAR激动剂激活异源三聚体G蛋白的能力，使用G-CASE蛋白生物传感器在活细胞中进行测量。该测定法检测G蛋白激活后Gα亚基上的纳米荧光素酶（nLuc）供体与Gγ上的维纳斯受体之间纳米BRET的降低。
所研究的12种βAR激动剂在细胞G-CASE测定中显示出广泛的配体效价和效力值。微型G与激动剂βAR复合物结合的动力学表征显示配体效力值（E）与微型G亲和力（ ）及其缔合速率（ ）之间存在强相关性。相比之下，配体效力与报道的配体解离速率（或停留时间）之间没有相关性。
G蛋白与激动剂βAR复合物的缔合速率（ ）与配体效力直接相关。这些数据支持一种模型，即效力更高的激动剂诱导βAR采用更可能募集G蛋白的构象。相反，这些数据不支持激动剂结合动力学在决定效力分子基础中的作用。
