Membrane disruption attenuates agonist potency in prostanoid receptors.

G protein-coupled receptors (GPCRs) are key signal transducers and the target of about one-third of all FDA-approved drugs. Many structural and pharmacological studies rely on disrupted membrane conditions, such as purified receptors in artificial systems or radioligand binding assays using membrane fragments, even though it had not been systematically studied whether membrane integrity affects GPCR function. To address this, we developed Förster resonance energy transfer (FRET)-based GPCR conformation sensors to directly measure receptor activation in both intact and disrupted membranes. Our results show that while some GPCRs remain unaffected, prostanoid receptor conformation sensors exhibit a strong dependence on membrane integrity: their agonist and antagonist potencies decrease up to 30-fold upon membrane disruption, revealing a crucial role of the membrane integrity in ligand-receptor affinity. Validation with wildtype receptors in functional signaling assays confirmed that these effects reflect genuine receptor characteristics rather than unspecific signals from the sensor design. We ruled out several factors that could explain the loss of affinity but were unable to fully elucidate the mechanism behind this phenomenon. Nevertheless, this effect may introduce bias into structural and pharmacological studies. It is therefore essential to account for membrane integrity and to employ optimized experimental strategies to ensure robust and reliable data interpretation.