GRK Mediates μ-Opioid Receptor Plasma Membrane Reorganization

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.

Homologous Regulation of Mu Opioid Receptor Recycling by Gβγ, Protein Kinase C, and Receptor Phosphorylation

“…In SpH-MOR cells treated with PTX 14-16 hours before imaging, DAMGO-induced MOR clustering into endocytic domains was not inhibited (Fig. 2, A and B), consistent with previous studies (Halls et al, 2016;(Gondin et al, 2019)). Fluorescence values normalized to the first frames after DAMGO addition were quantified for control and PTX-treated cells as an index of receptor internalization.…”

The use of fluorescence correlation spectroscopy to monitor cell surface β2‐adrenoceptors at low expression levels in human embryonic stem cell‐derived cardiomyocytes and fibroblasts

“…The faster-diffusing component (dwell time τ D1 ; 20-80 µs) was consistent with free unbound SNAP-488 dye diffusing within the detection volume. 43 The slower diffusing component (τ D2 ) represented the SNAPβ2AR expressed on the cell membrane. The diffusion coefficient (D) of this second component, however, was very similar between cell lines ( Figure 6F, CRISPR: 0.12 ± 0.01, n = 24; OE: 0.10 ± 0.01 µm 2 s −1 , n = 30, respectively).…”

“…37,38 One approach with the sensitivity to monitor receptors in membrane microdomains at very low receptor expression levels is fluorescence correlation spectroscopy (FCS). [39][40][41][42][43][44] Here, we have generated haplotype-specific SNAP-tagged β2AR human ES cell lines and applied FCS to monitor cell surface receptors in differentiated fibroblasts and cardiomyocytes.…”

Ligand-dependent spatiotemporal signaling profiles of the μ-opioid receptor are controlled by distinct protein-interaction networks

“… DAMGO and morphine differentially assemble protein-interaction networks to control MOR spatiotemporal signaling. A, DAMGO activation of the MOR leads to rapid recruitment of GRK2 and β-arrestins and facilitates a redistribution of the MOR at the plasma membrane (66). The DAMGO-stimulated receptor is in close proximity to IQGAP1, a large scaffolding protein that is required for the activation of Rac1 and nuclear ERK.…”