Dynamic Internal Motion of GPCR on Live Cells

Abstract

G protein-coupled receptors (GPCRs) are seven-transmembrane receptors, which transmit extracellular signals inside cells via activation of G proteins. GPCRs are involved in a wide variety of physiological functions such as signal sensing, immune system, and neurotransmission. Indeed, half of the drugs directly or indirectly target GPCRs. Although structures and function of GPCRs have been well studied, little has been known about the real-time dynamics in signal transduction. In this study, we used Diffracted X-ray Tracking (DXT) and Diffracted X-ray Blinking (DXB) techniques for the analysis. They elucidate protein dynamics through the analysis of diffraction spots from the gold nanocrystals labeled on the protein surface, and are very precise single-molecular analytical techniques. Serotonin receptor subtype 2A (5-HT2A) was transiently expressed on HEK 293 cells, and the gold nanocrystals were attached to the N-terminally introduced FLAG-tag. Short range measurement was performed by DXT (100/frame), which tracks the movement of diffraction spots. At microsecond time scale, internal motion of 5-HT2A was much higher in the ligand-free condition (2.70 Å/ms) than that of the ligand-binding condition (0.87 Å/ms). In contrast, long range measurement by DXB (100 ms/frame) presented opposite results. DXB analyses the continuation of signals by calculating the auto-correlation function of each pixel from recorded data. At millisecond time scale, internal motion of the ligand binding 5-HT2A was faster than that of ligand-free condition. These dual-natured data may suggest that we succeeded in extracting two different modes of 5-HT2A motion on live cells; fluctuation of 5-HT2A on the cell membrane at micro-second range, and ligand-induced dynamic motion of 5-HT2A at milli-second range. This is probably a first report to demonstrate the ligand induced intramolecular motion of GPCRs on live cells.