Selective activation of intracellular β1AR using a spatially restricted antagonist.

G-protein-coupled receptors (GPCRs) regulate several physiological and pathological processes and represent the target of approximately 30% of FDA-approved drugs. GPCR-mediated signaling was thought to occur exclusively at the plasma membrane. However, recent studies have unveiled their presence and function at subcellular membrane compartments. There is a growing interest in studying compartmentalized signaling of GPCRs. This requires development of novel tools to separate GPCRs signaling at the plasma membrane from the ones initiated at intracellular compartments. We took advantage of the structural and pharmacological information available for β1-adrenergic receptor (β1AR), an exemplary GPCR that functions at subcellular compartments, and rationally designed spatially restricted antagonists. We generated a cell impermeable β1AR antagonist by conjugating a suitable pharmacophore to a sulfonate-containing fluorophore. This cell-impermeable antagonist only inhibited β1AR on the plasma membrane. In contrast, a cell permeable β1AR agonist containing a non-sulfonated fluorophore, efficiently inhibited both the plasma membrane and Golgi pools of β1ARs. Furthermore, the cell impermeable antagonist selectively inhibited the phosphorylation of downstream effectors of PKA proximal to the plasma membrane in adult cardiomyocytes while β1AR intracellular pool remained active. Our tools offer promising avenues for investigating compartmentalized β1AR signaling in various context, potentially advancing our understanding of β1AR-mediated cellular responses in health and disease. They also offer a general strategy to study compartmentalized signaling for other GPCRs in various biological systems.