Cholesterol content drives distinct pharmacological behaviours of µ-opioid receptor in different microdomains of the CHO plasma membrane

Abstract

Cholesterol in the plasma membrane of eukaryotic cells contributes to modulating the functions and signalling pathways of numerous transmembrane proteins, including G protein Coupled Receptors (GPCRs). We have previously shown that the function of the human micro-opioid receptor (hMOR) expressed in Saccharomyces cerevisiae is modulated by sterols including cholesterol. Here, we investigated the effects of cholesterol content on hMOR pharmacology and on hMOR partitioning in cholesterol-poor and -rich domains in eukaryotic mammalian cells (CHO). We show that cholesterol is required for the stabilization of a receptor conformation with high agonist affinity and for triggering G-protein activation after agonist binding to the receptor. Biochemical analysis of untreated and cholesterol-depleted membranes in cells expressing hMOR indicated that the receptor is only present in cholesterol poor domains, in the basal state. After agonist binding to untreated CHO membranes, two distinct populations of receptor were found in cholesterol-rich and -poor domains. Cholesterol depletion or treatment of CHO membranes with the G-protein-decoupling agent GppNHp prevented the redistribution, indicating that receptor activated states localized into cholesterol-rich domains. Pharmacological data and biochemical analysis indicate that distinct activated conformations of hMOR exist in CHO plasma membrane and correspond to microdomains differing by thickness and proportions of lipid components, including cholesterol.

Differential Effect of Membrane Cholesterol Removal on μ- and δ-Opioid Receptors

“…The results from the sucrose gradient membrane separation experiments are consistent with reports of the localization of -opioid receptors in caveolin-enriched membrane fractions as well as caveolin-poor fractions (9,23). In contrast, we found ␦-opioid receptors only in the same cellular fractions as transferrin receptor, a non-raft marker.…”

“…The receptor, however, is less tightly coupled to G proteins (47) and may depend on cholesterol to aid in this coupling even in non-raft regions of the plasma membrane. This is supported by evidence that cholesterol alone can stabilize receptors in a high affinity state (23), and increasing membrane viscosity with a cholesterol analog has been shown to improve the potency of the -opioid receptor agonist DAMGO to stimulate [ 35 S]GTP␥S binding (22). The heterogeneous distribution of -opioid receptors in caveolin-enriched and caveolin-poor fractions may explain the rather small effect of cholesterol depletion on acute -opioid signaling (either [ 35 S]GTP␥S binding or adenylyl cyclase inhibition).…”

“…For example, an increase in plasma membrane microviscosity by addition of cholesteryl hemisuccinate to SH-SY5Y cell membranes increased -opioid receptor coupling to G proteins (22). Conversely, removal of membrane cholesterol from Chinese hamster ovary cells has been shown to either decrease (23) or increase (24) the coupling of -opioid receptors to G proteins, as measured by [ 35 …”

Regulation ofµ-Opioid Receptors: Desensitization, Phosphorylation, Internalization, and Tolerance

“…Reports on the localization of agonist-bound MORs vary. In one study, morphine-bound receptors remained within the rafts but etorphine-bound receptors diffused out of raft domain (Zheng et al, 2008a), but another study found that DAMGO-bound MORs moved into rafts (Gaibelet et al, 2008). The lipid environment can have substantial effects on agonist binding (Lazar and Medzihradsky, 1992) and MOR activity.…”

Recent Developments in the Study of Opioid Receptors

“…Crystallization of the four OPrs also required the presence of cholesterol Manglik et al, 2012;Thompson et al, 2012;Wu et al, 2012), although the role of cholesterol as a factor determining the observed structures of these receptors is not discussed by the authors. It has long been known that modulating the cholesterol content of OPr-expressing cell membranes can alter the binding and signal transduction properties of the receptors (Lazar and Medzihradsky, 1992;Xu et al, 2006;Gaibelet et al, 2008;Zheng et al, 2012), although the authors differ in their proposed (non-mutually exclusive) mechanisms (e.g., altered membrane microviscosity, receptor partition into lipid rafts, facilitation of association with G proteins, facilitation of dimer formation, modulation of receptor palmitoylation).…”