High-Affinity Functional Fluorescent Ligands for Human β-Adrenoceptors.

Area of Science:

• Biochemistry
• Cell Biology
• Microscopy

Background:

• G-protein coupled receptors (GPCRs) are crucial cell surface proteins involved in numerous physiological processes.
• Visualizing GPCRs in their native cellular environment is essential for understanding their dynamic functions.
• Existing imaging techniques often face limitations in resolution, specificity, or require complex experimental setups.

Purpose of the Study:

• To develop novel red-emitting fluorescent probes for visualizing G-protein coupled receptors (GPCRs) in living cells.
• To create probes compatible with advanced microscopy techniques like Stimulated Emission Depletion (STED) and Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET).
• To assess the probes' ability to maintain receptor functionality and enable simultaneous imaging and functional studies.

Main Methods:

• Synthesis of red-emitting fluorescent probes targeting β-adrenergic receptors (βARs).
• Evaluation of probe binding affinity and selectivity using TR-FRET assays.
• High-resolution imaging of β₂ARs in pancreatic CAPAN cells using confocal and STED microscopy.
• Assessment of probe compatibility with functional assays measuring cyclic adenosine monophosphate (cAMP) levels and receptor internalization.

Main Results:

• Developed probes exhibit nanomolar binding affinity and high selectivity for β₂AR over β₁AR.
• Carazolol-derived probes are fluorogenic, enabling no-wash imaging experiments.
• STED microscopy achieved a two-fold improvement in lateral optical resolution compared to confocal microscopy.
• Revealed the formation of β₂AR microdomains in native pancreatic cells.
• Probes successfully maintained functional properties, allowing simultaneous imaging and modulation of cellular responses.

Conclusions:

• The novel fluorescent probes provide a powerful tool for high-resolution imaging of GPCRs in living cells.
• These probes facilitate the study of receptor microdomain formation and dynamics.
• The probes' ability to retain functional properties opens avenues for simultaneous imaging and functional analysis of GPCRs.
• This work advances the understanding of GPCR behavior and signaling in native cellular contexts.