Agonist‐specific conformational dynamics at the β 2 ‐adrenoceptor dictate allosteric modulation of Gαs signalling and bronchodilation

Abstract Background and Purpose β 2 ‐adrenoceptor (β 2 AR) agonists are the cornerstone of asthma therapy and promote bronchodilation through Gαs signalling in airway smooth muscle (ASM), but their efficacy is limited by β‐arrestin‐mediated β 2 AR desensitization. We recently described allosteric modulators (AMs) of β 2 AR that preferentially enhance isoproterenol‐mediated β 2 AR‐Gαs signalling in ASM. Orthosteric β‐agonists differ in efficacy, kinetics and signalling bias, and how this diversity shapes allosteric modulation of β 2 ARs remains unclear. We investigated whether AMs could selectively modulate β 2 AR‐Gαs signalling and bronchodilation in response to endogenous and clinically relevant β‐agonists. Experimental Approach The effects of a positive AM (PAM37) and two negative AMs (NAM36 and NAM42) were examined on β‐agonist‐induced Gαs and β‐arrestin recruitment and downstream signalling (cAMP, PKA and ERK) in HEK293 cells expressing β 2 ARs and primary human ASM cells. Bronchodilation was assessed using murine precision‐cut lung slices and in vivo lung mechanics. Molecular dynamics (MD) simulations were performed to delineate structural determinants of agonist‐dependent AM activity. Key Results PAM37 enhanced, whereas NAMs attenuated β 2 AR‐Gαs coupling, signalling and bronchodilation with no effect on β‐arrestin‐ERK signalling, establishing Gαs signalling and functional bias. AM effects differed in magnitude and kinetic profiles across β‐agonists, with fenoterol uniquely resistant to modulation. MD simulations revealed that fenoterol stabilizes a quasi‐inactive β 2 AR conformation characterized by an TM6 shift that occludes the allosteric pocket, providing a structural basis for its resistance. Conclusions and Implications These findings establish orthosteric ligand‐dependent allostery as a fundamental feature of β 2 AR pharmacology. PAM37 represents a tool to selectively enhance bronchodilation by β‐agonists in asthma.