The M2 Muscarinic Receptor Signaling Complex Resolved by Single Molecule Tracking in Live Cells

Many aspects of the cellular signaling pathways via G protein-coupled receptors (GPCRs) are not completely understood. In particular, three questions have been the focus of much attention and debate: the oligomeric status of the receptor, the coupling strength between the receptor and the G protein, and the regulation of the receptors in response to external stimuli. Here we examine those questions by 2D tracking the M2 muscarinic receptors with Total Internal Reflection Fluorescence Microscopy (TIRFM) in the membrane of live cells. M2 Receptors and G proteins were genetically fused with fluorescent proteins (GFP and mCherry) and expressed in Chinese Hamster Ovary (CHO) cells. The distributions of oligomeric sizes and diffusion coefficients for both the receptors and the G proteins were extracted from single-particle tracking data obtained with TIRFM. Both of the receptors and G proteins were found to be localized in membrane micro-domains, showing multi-step photobleaching and exhibited a wide range of diffusion behaviors on a slower time scale than single protein controls. Corroborated with dual-color fluorescence correlation spectroscopy on the same samples, we propose that multiple oligomeric receptors and oligomeric G proteins sizes co-exist in close proximity inside a spatially confined signaling scaffold in the membrane of living cells. In addition, two-color simultaneous tracking of co-expressed receptors and G proteins shows preliminary evidence for receptor-G protein decoupling and receptor internalization upon induction with excessive agonist.