Subcellular activation of β- adrenergic receptors using a spatially restricted antagonist

• 成果类型: Article
• 署名作者: Liccardo, Federica; Morstein, Johannes; Lin, Ting-Yu; Pampel, Julius; Lang, Di; Shokat, Kevan M.; Irannejad, Roshanak
• 署名单位: University of California System; University of California San Francisco; University of California System; University of California San Francisco; University of California System; University of California San Francisco; Howard Hughes Medical Institute; University of California System; University of California San Francisco
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-14631
• DOI: 10.1073/pnas.2404243121
• 发表日期: 2024-10-01
• 关键词: phosphorylation; TRAFFICKING; myocytes; reveals

摘要

Gprotein- coupled receptors (GPCRs) regulate several physiological and pathological processes and represent the target of approximately 30% of Food and Drug Administration- 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 tools to separate GPCR signaling at the plasma membrane from the ones initiated at intracellular compartments. We leveraged the structural and pharmacological information available for f3- adrenergic receptors (f3ARs) and focused on f3 1AR as exemplary GPCR that functions at subcellular compartments, and rationally designed spatially restricted antagonists. We generated a cell- impermeable f3 AR antagonist by conjugating a suitable pharmacophore to a sulfonate- containing fluorophore. This cell- impermeable antagonist only inhibited f3 1AR on the plasma membrane. In contrast, a cell- permeable f3 AR antagonist containing a nonsulfonated fluorophore efficiently inhibited both the plasma membrane and Golgi pools of f3 1ARs. Furthermore, the cell- impermeable antagonist selectively inhibited the phosphorylation of PKA downstream effectors near the plasma membrane, which regulate sarcoplasmic reticulum (SR) Ca2+ release in adult cardiomyocytes, while the f3 1AR Golgi pool remained active. Our tools offer promising avenues for investigating compartmentalized f3 AR signaling in various contexts, potentially advancing our understanding of f3 AR- mediated cellular responses in health and disease. They also offer a general strategy to study compartmentalized signaling for other GPCRs in various biological systems.