G protein coupled receptors (GPCRs) are a superfamily of membrane receptors known for high signal transduction efficiencies. One of the key aspects of the GPCR signaling mechanism is the coupling interaction between the receptor and the G protein in response to external stimuli. We examined the pre-stimulus receptor-G protein coupling state by single-particle tracking (SPT) of M$_2$ muscarinic receptors and G$_i$ proteins in live cells.
M$_2$ receptors and G$_i$ proteins were genetically fused with fluorescent proteins (GFP and/or mCherry), expressed in CHO cells, and imaged on a Total Internal Reflection Fluorescence (TIRF) microscope. Single particles were identified in each frame of the TIRF movies and tracked using the TrackMate software. Mean-squared displacement (MSD) functions were computed for each single-particle trajectory. The diffusion parameters for receptors and G proteins were obtained by fitting their MSD functions to appropriate diffusion models.
Both the M$_2$ receptors and the G$_i$ proteins exhibited significant fractions of confined diffusion (compatible with the membrane compartment formed by actin microfilament-based meshwork) and active transportation (compatible with the rate of myosin trafficking along actin microfilaments). The motions of the M$_2$ receptors and of the G$_i$ proteins were distinctive from each other in the basal state of receptors, but they became similar when the receptors were activated by the agonist. Corroborated with dual-color fluorescence correlation spectroscopy measurements performed on the same samples, the SPT results supported a transient recruitment model without a stable pre-stimulus coupled complex.