Submembrane geography dictates GPCR signalling compartmentation and physiological function

G-protein-coupled receptors (GPCRs) are key mediators of intercellular signalling and targets for the vast majority of current therapeutics. Numerous biochemical and biophysical studies have uncovered that individual GPCRs activated at different subcellular locations can mediate distinct functional effects due to compartmentalization of the downstream second messengers in subcellular nanodomains. However, the reasons why a single receptor subtype activated at the same subcellular compartment such as plasma membrane leads to various functional responses have remained poorly understood. Here, we used beta1-adrenergic receptor and cardiomyocytes as classical examples of a therapeutically relevant GPCR and highly compartmentalized cell, to study communication between membrane receptors and subcellular cAMP nanodomains based on a combination of nanoscale scanning ion conductance microscopy assisted local receptor activation with Foerster resonance energy transfer based live cell imaging of the cAMP pathway. Unexpectedly, this approach revealed that beta1-adrenoceptors located in cardiomyocyte T-tubular membrane are strongly coupled to the regulation of cardiac contractility, whereas receptors residing in the outer non-tubular membrane preferentially engage nuclear nanodomains linked to hypertrophy. This unique mechanism of spatially segregated signalling was maintained in failing rodent and human hearts, indicating that it is conserved across species and preserved in disease. Finally, we explored potential therapeutic targeting of this mechanism by generating submembrane-specific beta-blockers acting exclusively at the outer non-tubular membrane. These improved compounds could ameliorate hypertrophy and cardiac remodeling without reducing contractility in vitro and in vivo. Together, these findings uncover an unexpected level of spatial organization of GPCR signaling within the same subcellular compartment and show that not only its identity but also its submembrane location determines cellular signaling and functional response engaged by the same GPCR.