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.
Highlights
The mu opioid receptor (MOR) is a G-protein coupled receptor (GPCR) responsible for many of the physiological effects of endogenous opioids, as well as clinically important exogenously administered opioids
Clear differences were observed between the mean square displacement (MSD) under basal conditions and those in cells treated with DAMGO or morphine (Fig. 2A)
The present single-molecule study was undertaken to characterize time-dependent changes in FLAG-MOR diffusion induced by acute and extended applications of the MOR agonists morphine and DAMGO and to understand if specific mobility states of the MOR could clearly reflect interactions with specific effectors. Both mobile and immobile populations of FLAG-MORs were detected, evident in the bimodal distribution of the anomalous exponent α and consistent with recent work showing that MORs exist both in lipid-enriched nanodomains and more freely-distributed in the plasma membrane prior to agonist binding[13]
Summary
The mu opioid receptor (MOR) is a G-protein coupled receptor (GPCR) responsible for many of the physiological effects of endogenous opioids, as well as clinically important exogenously administered opioids (e.g. morphine, codeine, fentanyl). We set out to use single-particle tracking (SPT) to assess how the mobility of the MOR changes over time during agonist application reasoning that we should be able to detect signaling and desensitized receptors based on distinct mobilities as the receptor interacts with differing membrane and intracellular molecules (perhaps discrete nanodomains) in these states. Specific mobility states of individual receptors detected with SPT may correlate with distinct interactions with select effectors and other binding partners. Another study reported short-term confinement within specific membrane compartments followed by diffusion of the receptor between these compartments[7] Neither of these studies examined the effect of agonist binding on mobility. AtT20 cells stably transfected with Flag-MOR provide a good system for examining real-time dynamics of the MOR in the plasma membrane before and during agonist exposure
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
