Kinetics of the initial steps of G protein-coupled receptor-mediated cellular signaling revealed by single-molecule imaging.
We report on an in vivo single-molecule study of the signaling kinetics of G protein-coupled receptors (GPCR) performed using the neurokinin 1 receptor (NK1R) as a representative member. The NK1R signaling cascade is triggered by the specific binding of a fluorescently labeled agonist, substance P (SP). The diffusion of single receptor-ligand complexes in plasma membrane of living HEK 293 cells is imaged using fast single-molecule wide-field fluorescence microscopy at 100 ms time resolution. Diffusion trajectories are obtained which show intra- and intertrace heterogeneity in the diffusion mode. To investigate universal patterns in the diffusion trajectories we take the ligand-binding event as the common starting point. This synchronization allows us to observe changes in the character of the ligand-receptor-complex diffusion. Specifically, we find that the diffusion of ligand-receptor complexes is slowed down significantly and becomes more constrained as a function of time during the first 1000 ms. The decelerated and more constrained diffusion is attributed to an increasing interaction of the GPCR with cellular structures after the ligand-receptor complex is formed.
Lill
Yoriko
Y
Nano-Optics group, National Competence Center for Research in Nanoscale Science, Institute of Physics, University of Basel, 4056 Basel, Switzerland.
Martinez
Karen L
KL
Lill
Markus A
MA
Meyer
Bruno H
BH
Vogel
Horst
H
Hecht
Bert
B
D002460:Cell Line; D006801:Humans; D007700:Kinetics; D008856:Microscopy, Fluorescence / Q000379:methods; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry* / Q000502:physiology; D018040:Receptors, Neurokinin-1 / Q000737:chemistry* / Q000502:physiology; D012680:Sensitivity and Specificity; D015398:Signal Transduction / Q000502:physiology*; D013997:Time Factors
D043562:Receptors, G-Protein-Coupled; D018040:Receptors, Neurokinin-1