Abstract
Signal transduction mechanisms and successive regulatory processes alter the lateral mobility of β2-adrenergic receptor (β2-AR). In this work we combined modern single particle tracking methods in order to analyze the diffusion dynamics of SNAP-tagged β2AR in HEK 293 wild-type cells and HEK 293 β-arrestin knockout cells before and after agonist stimulation. For analysis of trajectories we first used mean squared displacement (MSD) analysis. Secondly, we applied an advanced variational Bayesian treatment of hidden Markov models (vbSPT) in combination with the recently introduced packing coefficient (Pc), which together provided a detailed model of three discrete diffusive states, state transitioning and spatial confinement. Interesting to note, state switching between S3 (fast-diffusing) and S1 (slow-diffusing) occurred sequentially over an intermediate state S2. After ligand stimulation more SNAP-tagged β2AR in HEK 293 wild-type cells switched occupancy into the slow-diffusing state, whereas less receptors were found in the fast diffusive state. Unexpectedly, all three states showed a fraction of confined receptor mobility that increased under stimulation, but confinement sizes were unaffected. Receptor diffusion characteristics were comparable in HEK 293 β-arrestin knockout cells under basal conditions and only minor but non-significant changes occurred upon stimulation, as expected from the depletion of β-arrestin, an important regulatory protein. The data presented here on the occurrence of different diffusion states, their transitioning and variable spatial confinements clearly indicate that lateral mobility of β2AR is much more complex than previously thought.
Significance Statement G protein-coupled receptors (GPCRs) constitute the largest protein family targeted by approved drugs. In the course of signal transduction, GPCRs undergo various biological states, which correlate to their lateral diffusion behavior and are influenced by functional interactions and the local environment at the plasma membrane. Investigation by single particle tracking has the potential to give insight into open questions about lateral diffusion dynamics. By combination of recent methods, we developed a sophisticated approach that tracks dynamic changes in receptor diffusion and takes their spatial confinement into account. Using these methods, we demonstrate that agonist stimulation of β2-adrenergic receptor evoked strong alterations to diffusion characteristics, such as increased fractions of slow-diffusing and confined receptors.
Footnotes
N.S. and H.B. performed research; S.F and H.H. designed research; N.S., H.B., S.F. and H.H. wrote the paper
The authors do not declare any conflict of interest.