Abstract
Differential regulation of the μ-opioid receptor (MOP) has been linked to the development of opioid tolerance and dependence which both limit the clinical use of opioid analgesics. At a cellular level, MOP regulation occurs via receptor phosphorylation, desensitization, plasma membrane redistribution, and internalization. Here, we used fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP) to detect and quantify ligand-dependent changes in the plasma membrane organization of MOP expressed in human embryonic kidney (HEK293) cells. The low internalizing agonist morphine and the antagonist naloxone did not alter constitutive MOP plasma membrane organization. In contrast, the internalizing agonist DAMGO changed MOP plasma membrane organization in a pertussis toxin-insensitive manner and by two mechanisms. Firstly, it slowed MOP diffusion in a manner that was independent of internalization but dependent on GRK2/3. Secondly, DAMGO reduced the surface receptor number and the proportion of mobile receptors, and increased receptor clustering in a manner that was dependent on clathrin-mediated endocytosis. Overall, these results suggest the existence of distinct sequential MOP reorganization events at the plasma membrane and provide insights into the specific protein interactions that control MOP plasma membrane organization.
Highlights
The μ-opioid receptor (MOP) is the G protein-coupled receptor (GPCR) that mediates the analgesic effects of opioids such as morphine, fentanyl, and codeine
MOP has been demonstrated to partition into lipid rafts and the dynamics of MOP diffusion at the plasma membrane contribute to the specific signaling responses elicited by different opioid ligands (Huang et al, 2007; Gaibelet et al, 2008; Zheng et al, 2008)
The distribution of opioid receptors, including the MOP, into different nanoscale plasma membrane domains has been shown to be influenced by cholesterol (Rogacki et al, 2018), and MOP mobility, surface density, and the dynamics of plasma membrane lipids is affected by ethanol (Vukojevic et al, 2008b)
Summary
The μ-opioid receptor (MOP) is the GPCR that mediates the analgesic effects of opioids such as morphine, fentanyl, and codeine. Lateral mobility of MOP and MOP-G protein coupling is changed differentially by the activating agonist (Sauliere-Nzeh Ndong et al, 2010) in a manner depending on the membrane cholesterol content (Melkes et al, 2016) Together, these studies suggest a link between distinct functional states of MOP and the dynamic organization of receptors within the plasma membrane. We have previously reported that DAMGO and morphine elicit different spatiotemporal signaling profiles, and that these are dictated by the lateral redistribution of MOP within the plasma membrane, rather than internalization (Halls et al, 2016) These previous studies lacked the temporal resolution required to investigate rapid diffusion changes of the receptor at the cell surface
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