Quantification of Molecular Interactions in Living Cells in Real Time using a Membrane Protein Nanopattern

Reichmuth, A. M., Zimmermann, M., Wilhelm, F., Frutiger, A., Blickenstorfer, Y., Fattinger, C., Waldhoer, M., Voros, J. (2020). Analytical Chemistry, 92(13), 8983–8991.
https://doi.org/10.1021/acs.analchem.0c00987

Cell-based focal molography enables real-time quantification of specific intracellular protein interactions at membrane receptors in living cells, free from interference by off-target signaling.

The Challenge

Understanding G protein-coupled receptor (GPCR) signaling requires monitoring specific molecular interactions at receptors in living cells. Traditional label-free biosensors cannot distinguish receptor-specific events from nonspecific cellular changes such as morphological shifts or off-target receptor activation. Fluorescence-based assays offer specificity but require molecular modifications that can alter the biology being studied.

The Approach

Using focal molography, the authors created a transmembrane mologram by spatially arranging SNAP-tagged beta-2 adrenergic receptors (β2ARs) in the plasma membrane of HEK293 cells. The receptors self-assembled from the sensor chip surface into the cell membrane, forming a coherent nanopattern. This diffractive arrangement renders only target-receptor interactions visible while nonspecific cellular changes remain undetectable.

Key Results

• Real-time intracellular binding kinetics: Agonist-induced mass recruitment at the receptor was resolved in real time, with signal increasing over 20 minutes post-stimulation and partially reversing upon addition of competitive antagonist ICI 118,551
• Identification of recruited intracellular partner: Combined molographic and fluorescence readout on the same chip identified beta-arrestin-2 as the protein recruited to the receptor; cells lacking beta-arrestin-2 showed no molographic response to agonist stimulation
• Quantitative agreement with established assays: Dose-response curves from cell-based molography matched classical BRET arrestin recruitment assays for both isoproterenol and formoterol, validating the method as quantitatively reliable
• Insensitive to off-target signaling: Stimulation of off-target adenosine A2A/A2B receptors with NECA produced no measurable molographic signal, while the same cells responded to beta-2AR agonists, confirming that focal molography reports exclusively on the coherently arranged receptor

Why It Matters

This work extends focal molography from purified protein assays to living cells, enabling label-free, real-time monitoring of intracellular signaling events at specific membrane receptors. The method provides a new tool for GPCR pharmacology, receptor deorphanization, and drug discovery, with potential extension to other membrane protein classes.

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