Abstract
G protein-coupled receptors (GPCRs) are fundamental mediators of cellular signaling, yet the influence of mechanical forces, particularly membrane tension, on their activity remains poorly understood. Here, we investigate the role of membrane tension in modulating GPCR signaling dynamics using a combination of optogenetic tools and live-cell imaging. We employed a light-sensitive GPCR chimera (opto-β2AR) to activate Gαs signaling in HEK293T cells while simultaneously monitoring membrane tension via a FRET-based tension sensor (Flipper-TR). Our results demonstrate that acute increases in membrane tension, induced by hypo-osmotic shock or mechanical stretch, significantly potentiate GPCR-mediated cAMP production. Conversely, membrane tension reduction via hyper-osmotic shock attenuates signaling. Optogenetic activation of the GPCR revealed that tension-dependent modulation occurs within seconds, suggesting a rapid, membrane-localized mechanism. Using pharmacological inhibitors and siRNA knockdown, we identified that the mechanosensitive ion channel Piezo1 and the actin cytoskeleton are critical mediators of this tension-GPCR crosstalk. Live-cell imaging further showed that GPCR activation itself induces local membrane tension changes, establishing a bidirectional feedback loop. These findings reveal membrane tension as a dynamic regulator of GPCR signaling, with implications for understanding mechanotransduction in physiological and pathological contexts.