Multimodal intrinsic activation of GPCRs in ultrastable plasma membrane nanodomains

Abstract G protein-coupled receptors (GPCRs) mediate many physiological functions and are key targets in drug development 1-3 . A long-held tenet of molecular pharmacology is that GPCRs can spontaneously sample preexisting active conformations. This concept is pivotal to our understanding of ligand pharmacology 4 , however, direct evidence supporting it has only been obtained with reconstituted receptors 5-12 . Here, we introduce a method for quantitatively imaging the intrinsic activation probability of GPCRs directly at the plasma membrane of live cells, utilizing fluorescent conformational biosensors 13,14 . Our findings unveil a remarkable spatial multimodality in intrinsic activation probability, with a significant majority (up to 99%) of plasma membrane-expressed receptors showing negligible spontaneous activation. In contrast, the remaining minority of receptors exhibits spontaneous activation up to 22-fold higher than previously estimated. Experiments and theoretical calculations revealed that receptors diffuse into and out of ultralong-lived (∼5 minutes) nanodomains where the local membrane curvature allosterically enhances activation in the absence and presence of ligands. Extensive testing across five prototypic GPCRs indicates spatial nanoscale multimodality is ubiquitous, but varying in magnitude depending on the receptor and cell type. Upending conventional wisdom, this study reveals that drug efficacy is not a constant number but a spatiotemporal function ε (x, y, z, t) whose properties define and multiplex the signaling potency and efficacy of ternary complexes of GPCRs and likely other plasma membrane-receptors. Graphical Abstract GPCR spontaneous activation and intrinsic efficacy are not uniform across the plasma membrane but exhibit ultralong-lived spatial multimodality . Spatial variations in the curvature and composition of the plasma membrane, lead to the emergence of ultralong-lived nanodomains with contrasting physicochemical properties that allosterically regulate GPCR conformations. This results in a multimodal landscape of intrinsic efficacy ϵ (x, y, z, t) that ultimately governs cell signaling. XY scalebar: 500 nm. Z-range: 100 nm.