Temporal dependence of shifts in mu opioid receptor mobility at the cell surface after agonist binding observed by single-particle tracking

Abstract

Agonist binding to the mu opioid receptor (MOR) results in conformational changes that allow recruitment of G-proteins, activation of downstream effectors and eventual desensitization and internalization, all of which could affect receptor mobility. The present study employed single particle tracking (SPT) of quantum dot labeled FLAG-tagged MORs to examine shifts in MOR mobility after agonist binding. FLAG-MORs on the plasma membrane were in both mobile and immobile states under basal conditions. Activation of FLAG-MORs with DAMGO caused an acute increase in the fraction of mobile MORs, and free portions of mobile tracks were partially dependent on interactions with G-proteins. In contrast, 10-minute exposure to DAMGO or morphine increased the fraction of immobile FLAG-MORs. While the decrease in mobility with prolonged DAMGO exposure corresponded to an increase in colocalization with clathrin, the increase in colocalization was present in both mobile and immobile FLAG-MORs. Thus, no single mobility state of the receptor accounted for colocalization with clathrin. These findings demonstrate that SPT can be used to track agonist-dependent changes in MOR mobility over time, but that the mobility states observed likely arise from a diverse set of interactions and will be most informative when examined in concert with particular downstream effectors.

Principles and Applications of Single Particle Tracking in Cell Research

“…Diffusion on the membrane (e.g., lipid, [52] protein, [53] virus, [54] and particle [55] ); 2. Effects of agonists (DAMGO and morphine) on receptor mobility; [56] 3. Intracellular transport in different physiological states (e.g., in early apoptotic cells [57] ); 4.…”

“…The diffusion pattern of receptors is not only related to conformational changes of their own but also related to the drug treatment. By investigating the diffusion of FLAG-tagged mu opioid receptor (MOR) in the treatment of DAMGO, a high efficacy agonist, Metz et al [56] demonstrated that the activation of this agonist can lead to an acute increase in the percentage of mobile MORs by identifying their diffusion patterns and coefficients.…”

Modelling intermittent anomalous diffusion with switching fractional Brownian motion

“…The statistical analysis of particle trajectories recorded with single-particle tracking has revolutionised the field of cellular biophysics [1][2][3][4][5]. To name a few representative examples, exquisite information is found on lipid membranes [6][7][8], receptors [9][10][11], ion channels [12][13][14], nucleic acids [15,16], filaments [17], and organelles [18][19][20]. Further, synthetic particles can be used as probes to study cellular rheology [21][22][23].…”

Opioid-Modulated Receptor Localization and Erk1/2 Phosphorylation in Cells Coexpressing μ-Opioid and Nociceptin Receptors

“…Single-particle tracking (SPT) has been applied to show that MOPs can be in various mobility states under basal conditions [ 10 , 11 ]. It has also been used to track agonist-dependent changes in MOP mobility over time, but the mobility states were probably affected by a diverse set of downstream effectors [ 12 ]. The dynamic monomer-dimer equilibrium of MOP was described via SPT, demonstrating that the agonist DAMGO, but not morphine, induced dimer formation correlated with β-arrestin 2 binding to MOP [ 13 ].…”