Cholesterol Regulates ?-Opioid Receptor-Induced ?-Arrestin 2
Translocation to Membrane Lipid Rafts□
Yu Qiu, Yan Wang, Ping-Yee Law, Hong-Zhuan Chen, and Horace H. Loh
Department of Pharmacology, University of Minnesota, Minnesota (Y.Q., P.Y.L., H.H.L.); and Department of Pharmacology,
Institute of Medical Sciences, Shanghai JiaoTong University School of Medicine, Shanghai, China (Y.Q., Y.W., H.Z.C.)
Received December 30, 2010; accepted April 25, 2011
?-Opioid receptor (OPRM1) is mainly localized in lipid raft mi-
crodomains but internalizes through clathrin-dependent path-
ways. Our previous studies demonstrated that disruption of
lipid rafts by cholesterol-depletion reagent blocked the agonist-
induced internalization of OPRM1 and G protein-dependent
signaling. The present study demonstrated that reduction of
cholesterol level decreased and culturing cells in excess cho-
lesterol increased the agonist-induced internalization and de-
sensitization of OPRM1, respectively. Further analyses indi-
cated that modulation of cellular cholesterol level did not affect
agonist-induced receptor phosphorylation but did affect mem-
brane translocation of ?-arrestins. The translocation of ?-arres-
tins was blocked by cholesterol reduction, and the effect could
be reversed by incubating with cholesterol. OptiPrep gradient
separation of lipid rafts revealed that excess cholesterol re-
tained more receptors in lipid raft domains and facilitated the
recruitment of ?-arrestins to these microdomains upon agonist
activation. Moreover, excess cholesterol could evoke receptor
internalization and protein kinase C-independent extracellular
signal-regulated kinases activation upon morphine treatment.
Therefore, these results suggest that cholesterol not only can
influence OPRM1 localization in lipid rafts but also can effec-
tively enhance the recruitment of ?-arrestins and thereby affect
the agonist-induced trafficking and agonist-dependent signal-
ing of OPRM1.
Cholesterol, a major constituent of membrane lipids, plays
critical roles in structure and function of membrane proteins.
Cholesterol can directly interact with membrane proteins
and thus modulate protein functions. For example, choles-
terol is specifically required for the interaction between
large- and intermediate-conductance Ca2?-activated K chan-
nels (Romanenko et al., 2009). Cholesterol stabilizes the oxy-
tocin receptor to maintain the high-affinity state of the re-
ceptor for agonists (Gimpl et al., 2008). Moreover, cholesterol
can interact with sphingolipids and other lipids to segregate
into dynamic microdomains in the cell membranes (Lingwood
et al., 2009). Lipid rafts are such microdomains that can
cluster specific membrane proteins and thus regulate the
protein functions (Allen et al., 2007).
Internalization (endocytosis) is an important biological
process essential for many functions, including cell growth
and differentiation, pathogen entry, receptor signaling, and
down-regulation. The internalization of membrane proteins
can be mediated by clathrin-dependent and -independent,
lipid raft-dependent pathways (Le Roy and Wrana, 2005).
Cholesterol is shown to be essentially required in the forma-
tion of clathrin-coated endocytic vesicles (Rodal et al., 1999;
Subtil et al., 1999). However, several lines of evidence sug-
gest that the effect of cholesterol is far-reaching. Cholesterol
and lipid rafts are more profoundly involved in the clathrin-
dependent internalization pathway. Cholesterol depletion,
which releases epidermal growth factor receptor from lipid
rafts, inhibits agonist-induced receptor internalization with-
out impairing receptor function (Pike and Casey, 2002). Fur-
ther study indicates that the internalization of epidermal
growth factor receptor via clathrin-coated pits is started from
membrane rafts (Puri et al., 2005). Moreover, it has recently
been reported that lipid rafts and clathrin cooperate in the
internalization of the cellular prion protein (Sarnataro et al.,
This research was supported in parts by National Institutes of Health
National Institute on Drug Abuse [Grants DA007339, DA016674, DA000564,
DA011806, K05-DA00513] (the last to P.Y.L.); the National Great Basic Sci-
ence Project of China [Grant 2010CB529806]; and Shanghai Natural Science
foundation [Grant 10ZR1417000].
Article, publication date, and citation information can be found at
The online version of this article (available at http://molpharm.
aspetjournals.org) contains supplemental material.
ABBREVIATIONS: OPRM1, ?-opioid receptor; M?CD, methyl-?-cyclodextrin; ?Arr, ?-arrestin; HA, hemagglutinin; Ro-31-8425, bisindolylma-
leimide X; GFP, green fluorescent protein; DAMGO, [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin; N2A, neuro2A neuroblastoma cell; PIPES, piperazine-
N,N?-bis(2-ethanesulfonic acid); ERK, extracellular signal-regulated kinase; TR, transferrin receptor; PKC, protein kinase C.
Copyright © 2011 The American Society for Pharmacology and Experimental Therapeutics
Mol Pharmacol 80:210–218, 2011
Vol. 80, No. 1
Printed in U.S.A.
2009). The internalization of the integral membrane protein
CD317, which resides in the lipid rafts but internalizes
through clathrin-coated pits, is inhibited by dissociating it
with the rafts (Rollason et al., 2007).
?-Opioid receptor (OPRM1) belongs to the superfamily of G
protein-coupled receptors. As a large group of membrane
proteins, the function of G protein-coupled receptors has also
been broadly demonstrated to be regulated by cholesterol and
lipid rafts (Chini and Parenti, 2004; Barnett-Norris et al.,
2005). OPRM1 is shown to reside mainly in lipid raft mi-
crodomains, and its signaling can be either impaired or en-
hanced upon lipid raft disruption by cholesterol removal with
methyl-?-cyclodextrin (M?CD) in different cell types (Huang
et al., 2007; Zheng et al., 2008a; Levitt et al., 2009). More-
over, the agonist-induced internalization of OPRM1 is shown
to be blocked by M?CD treatment (Zhao et al., 2006). Because
OPRM1 internalizes through clathrin-coated pits (Minnis et
al., 2003; von Zastrow, 2003), how cholesterol depletion
blocks the clathrin-dependent internalization is unclear. The
internalization of GPCRs is initiated by receptor phosphory-
lation and subsequent recruitment of ?-arrestins (?Arr),
which couple receptors to the clathrin-coated pits (Goodman
et al., 1996; Ferguson, 2001; von Zastrow et al., 2003) and
uncouple receptors from G proteins to terminate receptor
signaling (desensitization) (Lefkowitz and Shenoy, 2005), in-
dicating that receptor internalization and desensitization share
common pathways. Thus, whether cholesterol manipulation
can affect OPRM1 desensitization needs to be clarified.
Therefore, we carried out the current study to investigate
the role of cholesterol in OPRM1 internalization and desen-
sitization. Our results showed that cholesterol manipulation
by incubating with cholesterol-depletion reagents or with ex-
cess cholesterol could decrease or increase the agonist-induced
internalization and desensitization of OPRM1. These effects
could be attributed to the compartmentation of the receptor and
recruitment of ?Arr to lipid raft microdomains.
Materials and Methods
Cells and Materials. Murine neuroblastoma Neuro2A (N2A)
cells stably expressing hemagglutinin-tagged ?-opioid receptor (HA-
OPRM1) (the Bmaxand Kdvalues for [3H]diprenorphine were deter-
mined to be 1.9 pmol/mg protein and 0.30 ? 0.04 nM, respectively)
were maintained in Dulbecco’s modified Eagle’s medium supple-
mented with 10% fetal bovine serum, 100 unit/ml penicillin, 100
?g/ml streptomycin, and 250 ?g/ml G418 in a 10% CO2incubator at
37°C. M?CD and cholesterol were purchased from Sigma-Aldrich
(St. Louis, MO). Simvastatin and bisindolylmaleimide X hydrochlo-
ride (Ro-31-8425) were purchased from EMD Biosciences. ?Arr2-
GFP (in pEGFP-N1) was kindly provided by Dr. Mario Ascoli (Uni-
versity of Iowa, Iowa City, IA). Anti-?Arr1 and ?Arr2 antibodies
were kindly provided by Dr. Martin Oppermann (University of Go ¨t-
tingen, Go ¨ttingen, Germany).
Determination of Receptor Internalization by Fluores-
cence-Activated Cell Sorting Analysis. Receptor internalization
was quantified by fluorescence-activated cell sorting analysis as
described previously (Qiu et al., 2003). In brief, after incubation with
1 ?M agonist for the indicated time intervals, cells were chilled on ice
to terminate receptor trafficking, and cell surface receptors were
visualized by incubating the cells with anti-HA antibody (1:1000),
followed by incubation with the Alexa Fluor 488 (Invitrogen, Carls-
bad, CA)-conjugated anti-mouse IgG antibody (1:1000). Surface re-
ceptor staining intensity of the antibody-labeled cells was analyzed
using fluorescence flow cytometry (FACScan; BD Biosciences, San
Jose, CA). To exclude the possible effects of cholesterol manipulation
on cell-surface receptor level or antibody immunoreactivities, control
cells without agonist treatment were treated with the same tested
concentrations of M?CD or cholesterol. Receptor internalization was
quantified as the percentage loss of cell surface fluorescence in
agonist-treated cells. For cells transfected with ?Arr2-GFP or
pEGFP-N1 vector, the cell surface receptors were labeled with Alexa
Fluor 633-conjugated anti-mouse IgG antibody and cells expressing
GFP were gated to determine agonist-induced receptor internalization.
Determination of Receptor Desensitization by Intracellu-
lar cAMP Assay. The intracellular cAMP level was measured as
described previously (Zhao et al., 2006). Cells in 96-well plates were
exposed to agonist for the indicated time intervals. The medium was
then removed and replaced with 100 ?l of Krebs-Ringer-HEPES
buffer (110 mM NaCl, 25 mM glucose, 55 mM sucrose, 10 mM
HEPES, 5 mM KCl, 1 mM MgCl2, and 1.8 mM CaCl2, pH 7.4) with
0.5 mM 3-isobutyl-1-methylxanthine, 10 ?M forskolin, and with or
without agonist. Then the cells were incubated for 15 min at 37°C
and terminated by heating at 90°C for 8 min. The measurement of
cAMP level in supernatant was performed by using AlphaScreen
cAMP detection kit (PerkinElmer Life and Analytical Sciences,
Waltham, MA). Receptor desensitization was calculated as the per-
centage loss of the ability of agonist to inhibit forskolin-stimulated
intracellular cAMP production in agonist-treated cells.
Receptor Phosphorylation Assay. Cells cultured in 100-mm
dishes were incubated with 1 ?M DAMGO for 30 min at 37°C. The
reactions were terminated on ice. Cells were washed with phosphate-
buffered saline at 4°C and subsequently lysed in 0.5 ml of lysis buffer
[0.5% Triton X-100, 10 mM Tris, pH 7.4, 150 mM NaCl, and 25 mM
KCl, with 0.1 mM phenylmethylsulfonyl fluoride, 40 ?g/ml Complete
protease inhibitor mixture (Roche Applied Science, Indianapolis,
IN), 50 mM sodium fluoride, 10 mM sodium pyrophosphate, and 0.1
mM sodium vanadate as phosphatase inhibitors]. After centrifuga-
tion at 12,000g for 5 min, the supernatant was immunoprecipitated
with 1 ?l of mouse anti-HA (Covance Research Product, Princeton,
NJ) and rProtein G agarose beads (Invitrogen) at 4°C overnight.
Then the beads were washed six times with cell lysis buffer and were
extracted with SDS-PAGE sample buffer. Approximately equal
amount of proteins was resolved by SDS-PAGE and transferred to
polyvinylidene difluoride membranes. The phosphorylated OPRM1
receptors were detected by anti-phospho-Ser375of OPRM1 antibody
(OPRM1phosphoSer375; Cell Signaling Technology, Danvers, MA)
and were normalized to the total immunoprecipitated receptors.
?-Arrestin Translocation Assay. The agonist-induced translo-
cation of endogenous ?Arr to the cell membrane was analyzed as
described previously (Huttenrauch et al., 2002). Cells cultured in
150-mm dishes were incubated with 1 ?M [D-Ala2, N-Me-Phe4,
Gly5-ol]-enkephalin (DAMGO) for 10 min at 37°C. The cells were
then placed on ice and scraped into 3 ml of buffer A (10 mM PIPES,
100 mM KCl, 3 mM NaCl, and 3.5 mM MgCl2, pH 7.0) containing 0.1
mM phenylmethylsulfonyl fluoride, 40 ?g/ml Complete protease in-
hibitor mixture. The cells were homogenized and sonicated and sub-
jected to centrifugation at 1000g for 20 min. The supernatant was
loaded on a discontinuous gradient of 50, 35, and 20% sucrose in
buffer A and centrifuged at 160,000g and 4°C for 2 h. The superna-
tant (cytosol) was removed. The 35/50% sucrose interface (mem-
brane) was collected and diluted in 3 ml of buffer A and centrifuged
at 160,000g and 4°C for 15 min again. The pellet was resuspended in
40 ?l of detergent buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5
mM EDTA, 1% Triton X-100, and 0.05% SDS with protease inhibi-
tors). Approximately equal amount of proteins was resolved by SDS-
polyacrylamide gel electrophoresis and transferred to polyvinylidene
difluoride membranes. ?Arr1 and ?Arr2 were detected by monoclo-
nal anti-?Arr1 and ?Arr2 antibodies (1:500) and determined with
the analysis software ImageQuant (GE Healthcare, Chalfont St.
Giles, Buckinghamshire, UK).
Lipid Raft Separation. Separation of the lipid rafts from other
membrane domains by OptiPrep density gradient was carried out as
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Address correspondence to: Ping-Yee Law, Department of Pharmacology,
University of Minnesota, 6-120 Jackson Hall, 321 Church St. S.E., Minneap-
olis, MN, 55455-0217. E-mail: firstname.lastname@example.org
Qiu et al.