The Other Side of Opioid Receptor Signalling: Regulation by Protein-Protein Interaction

Abstract

Opiate drugs mediate their analgesic, euphoriant, and rewarding effects by activating opioid receptors. Pharmacological and molecular studies have demonstrated the existence of three opioid receptor subtypes, μ, δ, and κ- that couple predominantly to Gi/Go types of G proteins to regulate the activity of a diverse array of effector systems. Ample experimental evidence has demonstrated that these receptors can physically interact with a variety of accessory proteins, confirming that signal transduction of the opioid receptors is not restricted to heterotrimeric G protein activation. Such interactions can alter the effectiveness of agonist-driven cell signalling, determine the signals generated and alter the trafficking, targeting, fine tuning and cellular localization of these receptors by providing a scaffold that links the receptors to the cytoskeletal network. The current review will summarize opioid receptor interacting partners and their role as currently understood. Increasing knowledge of the mechanisms by which these interactions are regulated is expected to address problems related to phenomena such as pain perception, tolerance and dependence that occur upon chronic opiate administration and define whether disruption of such interactions may contribute to the development of novel therapeutic strategies.

Full text

80 Current Drug Targets, 2012, 13, 80-102
1389-4501/12 $58.00+.00 © 2012 Bentham Science Publishers
The Other Side of Opioid Receptor Signalling: Regulation by Protein-
Protein Interaction
Zafiroula Georgoussi*,1, Eirini-Maria Georganta1 and Graeme Milligan2
1Laboratory of Cellular Signalling and Molecular Pharmacology, Institute of Biology, National Centre for Scientific
Research “Demokritos”, Aghia Paraskevi 15310, Athens, Greece
2Molecular Pharmacology Group, Institute of Neuroscience and Psychology, College of Medical, Veterinary and Life
Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, UK
Abstract: Opiate drugs mediate their analgesic, euphoriant, and rewarding effects by activating opioid receptors.
Pharmacological and molecular studies have demonstrated the existence of three opioid receptor subtypes, µ, δ, and κ-
that couple predominantly to Gi/Go types of G proteins to regulate the activity of a diverse array of effector systems.
Ample experimental evidence has demonstrated that these receptors can physically interact with a variety of accessory
proteins, confirming that signal transduction of the opioid receptors is not restricted to heterotrimeric G protein activation.
Such interactions can alter the effectiveness of agonist-driven cell signalling, determine the signals generated and alter the
trafficking, targeting, fine tuning and cellular localization of these receptors by providing a scaffold that links the
receptors to the cytoskeletal network. The current review will summarize opioid receptor interacting partners and their
role as currently understood. Increasing knowledge of the mechanisms by which these interactions are regulated is
expected to address problems related to phenomena such as pain perception, tolerance and dependence that occur upon
chronic opiate administration and define whether disruption of such interactions may contribute to the development of
novel therapeutic strategies.
Keywords: G protein-coupled receptors, G protein, cellular signalling, interacting proteins, internalization, opioid, signalling
complex, trafficking.
INTRODUCTION
The opioid receptors (µ, δ, κ), which belong to Class A of
the rhodopsin sub-family of G-protein coupled receptors
(GPCRs), mediate various functions in the nervous system
and peripheral tissues that range from modulation of pain
perception, cell proliferation and neural development to
synaptic plasticity and cell survival [1-7]. They are activated
by both endogenously produced opioid peptides and exoge-
nously administered opiate compounds, some of which are
not only among the most effective analgesics known but are
also highly addictive drugs of abuse [8-11]. All three opioid
receptor subtypes couple preferentially to Gi/o types of G
proteins and hence inhibit adenylyl cyclase and regulate a
number of other signalling intermediates, including phos-
pholipase C (PLC), mitogen-activated kinase (MAPK) and
various ion channels [12-18]. Such diverse signalling events
are mediated by various G protein subtypes in a pertussis
toxin (PTX)-sensitive or insensitive manner depending on
the system studied and the efficacy and identity of the opioid
agonists employed [15, 19-24].
Although, the first proteins shown to interact functionally
with opioid receptors were G proteins [13, 16, 20, 25, 26], to
modulate canonical G protein-dependent signalling, increas-
ing evidence in the field suggests that the situation is much
more complex [27, 28]. Indeed, observations derived from
*Address correspondence to this author at the Laboratory of Cellular
Signalling and Molecular Pharmacology, Institute of Biology, National
Centre for Scientific Research “Demokritos”, Aghia Paraskevi 15310,
Athens, Greece; Tel: 30-210-6503564, Fax: 30-210-6511767; E-mail:
iro@bio.demokritos.gr
approaches such as yeast-two hybrid screens, immunopreci-
pitation and fluorescence or bioluminescence resonance
energy transfer in living cells, suggest that a wide range of
other interacting partners can participate in the regulation of
every aspect of opioid receptor activity. Such observations
have indicated that all three opioid receptors can form multi-
component protein assemblies, that may be dynamic, invol-
ving interactions, a) between themselves or other related
GPCRs and b) with other interacting proteins. Such inter-
actions contribute to the intricate and finely tuned process of
signalling downstream of these receptors.
Substantial evidence has suggested that all three opioid
receptor subtypes are capable of forming both homo- and
hetero-dimers with themselves or less highly related GPCRs
and that these inter-receptor complexes can produce
pharmacological profiles distinct from those of monomeric
receptors [29]. Opioid receptor dimerization has been
extensively reviewed recently [30-36] and will not be the
subject of this review. However, it should be noted that a
number of observations in this area have been restricted to
studies in heterologous cell lines and require confirmation in
physiologically relevant settings [37, 38]. Instead, emphasis
will be given to novel opioid receptor interacting partners
that link the receptors to alternative signalling pathways
beyond G proteins. Fig. (1) illustrates reported interactions
for the three opioid receptor subtypes. These dynamic inter-
actions are initiated at the intracellular face of these
receptors, and serve as platforms for scaffolding proteins that
assemble various multi-protein complexes with distinct
signalling characteristics. In this review, opioid receptor-
interacting proteins that modulate signal transduction
pathways will be discussed and new ways in which opioid

Opioid Receptor- Interacting Proteins Current Drug Targets, 2012, Vol. 13, No. 1 81
receptors may regulate signal transduction pathways media-
ted by these proteins will be suggested. Such knowledge may
address fundamental questions concerning the importance
and molecular mechanisms that underlie tolerance and
dependence to opioids, and uncover factors that modulate
synaptic transmission to provide insights into a variety of
physiologic and pathophysiologic processes.
MOLECULAR DETERMINANTS OF OPIOID
RECEPTOR INTERACTIONS
A critical question in cellular signalling is what deter-
mines the specificity of signal transduction processes? There
is ample evidence for the formation of complexes maintained
by protein scaffolds that control signal specificity and this
contrasts with the classical collision-coupling model of G
protein signalling [39]. Extensive observations have demons-
trated that the cytoplasmic face of the opioid receptors and,
particularly, the third intracellular loop and the C-terminal
tail, are critical in mediating the signal transfer to G proteins
and play specific roles in mediating protein-protein inter-
actions [40-42]. The hydrophilic, cytoplasmic domains of the
three opioid receptors display high sequence similarity,
especially in the second and third intracellular loops (about
90% amino acid identity) suggesting that these receptors are
likely to interact with similar proteins. More specifically, the
third intracellular loop (i3-L) of the opioid receptor family is
comprised of some 23 aminoacids, which, with the exception
of three aminoacids, are identical between the three opioid
receptor subtypes. This i3-L loop represents one of the most
fundamental structural determinants for direct Gi/Go protein
coupling and activation [9, 12, 42-46]. Besides Gα binding,
this domain also allows direct binding of Gβγ complexes
[47] and provides contacts for other interacting proteins
(Table 1).
The C-terminal domain of GPCRs is recognized as one of
the main domains involved in regulation of GPCR function
[48, 49]. This region is also a key site for post-translational
modification, including phosphorylation, and is crucial for
receptor desensitization and trafficking. The predicted C-
terminal tails of the µ, δ and κ-opioid receptors comprise 59,
51 and 47 amino acids respectively and contain the
conserved region DENFKRCFRXXC, where X represents
amino acids that vary between the three opioid receptor
subtypes. This well conserved sequence is predicted to form
an α helix (helix VIII) that, in the currently available atomic
level structures of other GPCRs, runs parallel to the plasma
membrane and is crucial for the coupling of G proteins. This
is concluded by thioacylated cysteine residues which bear
fatty acid residues that intercalate into the lipid bilayer. The
putative helix VIII of the opioid receptors has been found to
be responsible for the direct interaction with proteins of dif-
ferent function, summarized in Table 1 that will be discussed
below. Such interactions have also been demonstrated for
other GPCRs [49-55].
Opioid receptors, upon agonist activation, interact with
members of the heterotrimeric G protein family. This type of
interaction can be dynamically influenced by a number of
other receptor-interacting proteins which can decrease the
intensity or duration of GPCR signalling by disrupting the
association of GPCR with G proteins, or by recruiting nega-
tive regulators of GPCR signalling and will be the subject of
interacting proteins interfering with G protein signalling.
Significant effort has been invested towards understanding
aspects of signalling responsible for the desensitization,
internalization and trafficking of opioid receptors. These
mechanisms are regulated by an increasing number of
scaffolding or cytoskeletal proteins, which upon receptor
interaction can alter the effectivenes of the agonist-driven
responses and, together with PDZ and chaperone containing
proteins, will be described in detail. Two additional sections
will be dedicated to the interactions affecting biogenesis and
neurotransmitter release. Finally, fine tuning of receptor
effects modulated by direct interaction with kinases and
transcription factors will also be considered.
Fig. (1). Known opioid receptor-protein interactions. Schematic representation of the known interacting partner proteins of the three
opioid receptor subtypes based on their classification throughout the text, each protein group is presented by a different color.
δ-ORμ-OR κ-OR
RGS4
Calmodulin Periplakin
PKCI
PLD2
FilaminA
hlj1
Synaptophysin
RanBPM
RPNI
M6a
RPNI
β-arrestinGASP
SNX1
EAAC1
Calnexin
RPNI
Ubiquitin
β-arrestin
GEC1
NHERF

82 Current Drug Targets, 2012, Vol. 13, No. 1 Georgoussi et al.
INTERACTING PROTEINS THAT INTERFERE
WITH G PROTEIN SIGNALLING
Calmodulin
Parts of i3-L and the proximal region of the carboxyl-
terminal tail have been shown to be important for G protein
coupling and activation [42, 44-46, 56]. The first indication
of an interaction between an opioid receptor and a protein
other than a G protein involved the µ-opioid receptor and the
Ca2+ binding protein calmodulin [57], addressed by experi-
ments using peptides derived from the µ-OR i3-L [57].
Calmodulin is a ubiquitously expressed Ca2+-sensitive regu-
latory protein but, in addition to acting as a regulator of
cytoplasmic enzymes, also modulates adenylyl cyclases,
Ca2+/CaM dependent kinases and phosphatases, Ca2+-
ATPases, ion channels and many other membrane proteins
[58]. Binding of calmodulin to the i3-L results in reduced
basal and agonist-activated [35S]-guanosine 5’-3-thio-tri-
phosphate binding in membranes expressing the µ-opioid
receptor. Given the high sequence similarity among the i3-L
of opioid receptor subtypes similar effects have also been
reported for the δ-OR [57]. Interestingly, a µ-opioid receptor
mutant K273A, displays enhanced G protein coupling
compared to wild type, but is insensitive to calmodulin [57].
These results were consistent with the prediction that
calmodulin associates competitively in sites interacting with
G protein(s) and, that in the absence of agonist, calmodulin
can limit basal signal transduction of opioid receptors. Such
results demonstrate that calmodulin competes with G protein
for binding to the opioid receptors and may serve as an
independent second messenger molecule released upon
receptor stimulation [57, 59]. This is of interest because
opioid receptors regulate aspects of cellular Ca2+ signalling.
Indeed, it was reported that activation of opioid receptors
may play a modulatory role in the fine tuning of Ca2+
signalling events [60, 61]. Regulation of Ca2+ dynamics by
neurotransmitters is a possible mechanism by which changes
in the CNS micro-environment regulate various brain
activities, including chronic pain. It was also found that
calmodulin interacts with the i3-L of the D2–dopamine
Table 1. Interacting Proteins of the Three Opioid Receptor Subtypes (µ-OR, δ-OR and κ-OR) and their Key Roles in Opioid
Function
Interacting
protein
Opioid receptor
subtype
Function
Site of interaction
References
β-Arrestin
µ-OR, δ-OR, κ-ΟR
Receptor desensitization & endocytosis
i3-L, C-terminus
[125, 126, 142,
143]
Calmodulin
µ-OR, δ-OR
Interferes with G protein activation
i3-L
[57, 59]
Calnexin
δ-OR
Plasma membrane targeting
ND
[278]
EAAC1
δ-OR
Affects neurotransmitter release
ND
[292]
EBP50/NHERF
κ-ΟR
Increase receptor recycling
C-terminus
[238]
Filamin A
µ-OR
Receptor trafficking
C-terminus
[253]
GASP
δ-OR
Receptor lysosomal targeting
C-terminus (helix VIII)
[203]
GEC1
κ-ΟR
Regulates receptor trafficking and biosynthesis
C-terminus
[244]
Glycoprotein
M6a
µ-OR, δ-OR
Receptor endocytosis and recycling
ND
[228]
hlj1
µ-OR
Receptor maturation and degradation
C-terminus
[273]
Periplakin
µ-OR
Interferes with G protein activation
C-terminus (helix VIII)
[66]
PKC1
µ-OR
Receptor desensitisation & phosphorylation
C-terminus
[158]
PLD2
µ-OR
Receptor endocytosis and recycling
ND
[170]
Protachykinin
δ-OR
Receptor sorting to LDCVs
e3-L
[267]
RanBPM
µ-OR
Reduce receptor endocytocis
ND
[185]
RGS4
µ-OR, δ-OR
Receptor signalling and scaffolding
C-terminus (helix VIII),
i3-L
[47,87]
RGS9-2
µ-OR
Receptor signalling and endocytosis
ND
[117, 120]
Ribophorin I
µ-OR,δ-OR, κ-ΟR
Regulates receptor trafficking and biosynthesis
ND
[248]
SNX1
δ-OR
Receptor lysosomal targeting
C-terminus
[204]
Spinophilin
µ-OR
Receptor internalization
ND
[199]
STAT5A/B
µ-OR, δ-OR
Interferes with transcriptional activation and protein
scaffolding
C-terminus (YXXL motif)
[15, 284]
Synaptophysin
µ-OR
Receptor signalling and trafficking
i3-L
[211]
Ubiquitin
δ-OR, κ-ΟR
Receptor degradation
C-terminus
[230, 235]

Opioid Receptor- Interacting Proteins Current Drug Targets, 2012, Vol. 13, No. 1 83
receptor [62] and enhances receptor signalling [63], with the
C-terminal tail of the metabotropic glutamate receptors 7A
and 7B (mGlu7A, 7B) [64] to alter the mode of action of
group III mGluR [64], and with the 5-HT(2C) receptor [65].
All these indicate that calmodulin, by interacting with
various GPCRs, attenuate G protein coupling and agonist-
mediated regulation of downstream effector cascades.
Periplakin
Using the C-terminal tail of the MOP-1 and MOP-1A
splice variants of the human µ-OR as bait in a yeast two-
hybrid screen against a human brain cDNA library the
predominant hit identified was periplakin [66]. Periplakin is
an actin and intermediate filament binding protein, most
widely studied in the keratinocyte cytoskeleton [67, 68].
However, it is also widely expressed in the central nervous
system. Binding of periplakin with µ-OR was confirmed
using pull-down assays employing GST fusion segments of
the C-terminal tail of MOP-1 and purified His-tagged
periplakin. The interaction of periplakin with µ-OR was
observed with both MOP-1 and MOP-1A, which differ in
their extreme C-terminus sequence, thus eliminating the
possibility that the interaction occurs within this region.
Mapping the site of this interaction using fragments of the
MOP-1 C-terminal tail demonstrated that the region close to
the seventh transmembrane domain, forming helix VIII, is
the structural determinant responsible for periplakin binding
[66]. Interaction also occurred, although less strongly, with
the equivalent region of the δ-OR.
It is known that helix VIII of rhodopsin provides contact
sites for and plays a critical role in G protein activation [69].
This region was also found to contribute to µ- and δ-OR-G
protein coupling and activation [44, 46]. A critical question
therefore arises as to whether periplakin, by interacting
within this domain, competes with G proteins for the same
sites and alters the effectiveness of G protein activation. It
was found that co-expression of periplakin with the µ-OR
splice variants MOP-1 and MOP-1A in HEK293 cells
significantly reduced the capacity of DAMGO to stimulate
[35S]GTPγS binding to G proteins in a receptor-selective
manner without interfering with agonist-mediated interna-
lization of the µ-OR [66]. It was concluded, therefore, that
periplakin interferes with opioid agonist-mediated G protein
activation. Using similar approaches evidence has also been
provided for periplakin’s interaction with the melanin
concentrating hormone-1 (MCH-1) receptor, which is
implicated in body weight regulation. The MCH-1 signals
through both Gαi and Gαq-coupled pathways. This inter-
action occurs within a segment proximal to transmembrane
helix VII, despite the fact that the MCH-1 receptor lacks the
cysteine residues in the C-terminal tail that defines the distal
end of helix VIII in many other GPCRs. Co-expression of
periplakin in HEK293 cells did not interfere with agonist-
induced internalization of the MCH-1 receptor but, as with
the µ-OR, it resulted in a large reduction of G protein acti-
vation [70, 71]. Collectively it is suggested that periplakin
limits agonist-mediated G protein activation probably by
competing with G proteins for binding to this region of
GPCRs. Recent observations demonstrate that periplakin and
neurochondrin can interact and modulate selectively the
function of a wide range of GPCRs [72].
RGS Proteins
Regulators of G protein signalling (RGS proteins) com-
prise a large family of multifunctional proteins (>30 family
members) that act as negative regulators of G proteins and
thus modify the potency and the kinetics of GPCR signalling
[73-77]. They do so by a number of mechanisms including a)
acceleration of G protein inactivation, b) direct antagonism
with G protein effectors and c) sequestration of Gα subunits
[74, 78, 79]. Growing evidence indicates that RGS proteins,
by their ability to shorten the lifetime of activated Gα, confer
selectivity for signalling pathways [79]. However, it is still
unclear to what degree G protein activation influences RGS
binding, how stable RGS-G protein association is and how
GPCRs might influence RGS-G protein association [79, 80].
It was found that RGS proteins can directly interact with
preferred receptors to regulate their function, whilst locali-
zation studies have provided evidence that RGS proteins are
recruited to the membrane in a receptor-specific manner
[80]. Direct interaction of RGS2 with the third loop of the
M1 muscarinic acetylcholine receptor [81], and the α1Α-
adrenergic receptor has been reported [82], whilst RGS4 has
been shown to bind to metabotropic glutamate receptor sub-
type 5 in rat striatum [83]. A range of studies have associated
the action of RGS proteins, mainly from the subfamilies R7,
R4 and Rz, with the regulation of signal transduction of
opioid receptors [84]. Regulation of RGS proteins by chronic
morphine treatment in rat locus coeruleus has been reported
[85] and opioid agonists have been proposed to up-regulate
RGS4 mRNA levels in PC12 cells which might contribute to
opioid desensitization [86].
RGS4 Protein
Initial observations employing pull down assays that
utilized the i3-L of the δ-OR and the C-terminal tails of the
µ-OR and δ-ΟR expressed as a glutathione-S-transferase
(GST) fusion proteins demonstrated the ability of purified
recombinant RGS4 to interact directly with both opioid
receptors [47]. RGS4 belongs to the B/R4 family of RGS
proteins and appears to be selectively expressed in the CNS
and heart [77]. RGS4 association with the µ- and δ- ORs was
confirmed by co-immunoprecipitation studies in HEK293
cells expressing tagged versions of these receptors and HA-
RGS4. This interaction occurs in the absence of agonist
stimulation, suggesting that RGS4 may be associated
constitutively with these receptors [87]. Agonist-stimulation
of µ-OR and δ-ΟR led to translocation of RGS4 to the
plasma membrane where it co-localized with both opioid
receptors [87].
Of interest is the finding that RGS4 binding occurs
within two distinct domains of δ-OR that is the C-terminal
tail and the i3-L [47]. As noted earlier, i3-L of all three
opioid receptors shares a high degree of amino-acid identity
with only three amino acid differences among them [2, 38].
Taking into account previous observations regarding the role
of i3-L in G protein coupling and activation for all ORs [44-
47], this region may also be responsible for RGS4 binding to
κ-OR. This is the first indication that an RGS protein appears
to display more than one interaction site for direct GPCR
binding and it will be of interest to explore the implications
of this. Mapping the sites of interaction within the C-
terminal tail of the µ- and δ-OR demonstrated that lack of the

84 Current Drug Targets, 2012, Vol. 13, No. 1 Georgoussi et al.
juxtamembrane domain encompassing the conserved 18
amino-acid stretch (LNPVLYAFLDENFKRCFR), abolished
binding of RGS4 to both receptors and prevented formation
of a heterotrimeric complex with RGS4 and activated Gα
[87]. This suggests that the predicted helix VIII of µ and δ-
ORs scaffolds a signalling complex consisting of the opioid
receptor, activated Gα and RGS4. The 4Box domain is res-
ponsible for the direct association of RGS4 with the acti-
vated Gα subunit and induces the binding of the RGS4/Gα
complex to µ-ΟR, while the N-terminus controls the inter-
action of RGS4 with µ-OR in the absence of Gα [47, 87].
Construction of truncated versions of RGS4 abrogated these
interactions suggesting that the regions responsible for
opioid receptor binding are those of the 4Box and the N-
terminus of RGS4 [87].
It is known that receptors promote G protein activation
and that RGS proteins tend to bind with higher affinity to
activated G proteins in vitro [88] and in intact cells [89]. One
could therefore ask whether RGS4 promotes the binding of
activated Gα, Gβγ or other proteins to the receptors. In this
respect, it was clearly shown that Gβγ and RGS4 compete
for the same binding sites of the µ-OR C-terminal tail and,
most importantly, that a signalling complex consisting of the
µ-OR, activated Gα, Gβγ and RGS4 can be formed [47].
Knowing that RGS4 and Gα subunits form in vitro a
heterotrimeric complex containing the δ- and µ-ORs, the
authors further explored whether RGS4 expression promotes
selectivity for the receptors to couple with a specific subset
of G proteins. Whilst in µ-ΟR expressing HEK293 cells
RGS4 pairs with any of the endogenous G proteins tested
[87], in contrast, RGS4 can dynamically regulate the
spontaneous selectivity of δ-OR for specific G proteins even
in the absence of agonists [87]. This was the first example of
an RGS-driven selection of coupling with a specific G
protein population depending on the presence or the
activation state of a given GPCR in living cells.
Opioid receptors stimulate ERK1/2 MAPK activity via
PTX-sensitive and insensitive G protein signalling mecha-
nisms [42, 90] and growing evidence indicates that RGS
proteins, among them RGS4, are implicated in MAPK
signalling through GPCRs [91-94]. The presence of RGS4
restricts MAPK phosphorylation by both activated µ- and δ-
ORs suggesting that RGS4 acts as an effector antagonist for
both opioid receptors [87]. It was also shown that µ-agonists
display increased potency and/or efficacy of signalling to
adenylyl cyclase in cells expressing an RGS-insensitive Gαο
compared to those expressing the wild type RGS-sensitive
Gαο protein [95]. Recent observations by the same group
demonstrate that µ-OR signalling to adenylyl cyclase is
differentially modulated by RGS proteins in permeabilized
C6 cells and this depends on the Gα proteins [96].
Opioid analgesia and development of tolerance involve
complex cellular and molecular mechanisms that, in part,
reflect loss of membrane surface opioid receptors [5, 97, 98].
Initial studies using RGS4 knockout mice have shown these
animals not to display any obvious developmental defects or
altered responses to acute morphine or withdrawal from
chronic morphine [99]. However, recent findings using
constitutive and inducible knockout mice have demonstated
that RGS4 exerts differential effects on distinct actions of
opiates in the nervous system [100]. Furthermore, distinct
studies suggest that RGS proteins may be implicated in
opioid receptor internalization. Indeed, it has been shown
that GAIP facilitates δ-ΟR internalization and recycling via
clathrin-coated vesicles [101], whereas RGS14 prevents
morphine-induced µ-OR phosphorylation and internalization
in neurons from mice [102]. Measurements of the fate of
internalized δ-OR indicated that RGS4 expression
accelerated the early kinetics of δ-OR endocytosis, an effect
that was eliminated using a ΔΝRGS4 mutant lacking the N-
terminal domain. Similarly, this ΔΝRGS4 mutant had no
effect in modulating adenylyl cyclase inhibition induced by
the activated µ-OR, thus reinforcing the functional signi-
ficance of this domain for opioid receptor signalling [87]. In
accordance with these findings the N-terminus of RGS4 was
required for Kir3 channel deactivation [103], for high affi-
nity and receptor selective inhibition [104] and for modulat-
ing pheromone signalling in yeast [105]. We can conclude,
therefore, that RGS4 is an attractive target to selectively
manipulate G protein pathways and regulate the potency,
selectivity and duration of opioid action in order to prevent
the adverse effects of tolerance and dependence. In this
respect, a number of structure-based RGS4 peptides were
designed as inhibitors of it’s GTPase activating protein
function [106]. Furthermore, small-molecule inhibitors of
RGS4 have been identified using a high-throughput flow
cytometry protein interaction assay [107]. With this screen
one compound, CCG-4986, that inhibited the GAP activity
of RGS4 and RGS4 activity on µ-ΟR was defined [107,
108]. Although currently of only moderate affinity, small
molecule inhibitors of RGS proteins may provide a first step
towards the development of novel therapeutics. It remains to
be further investigated whether these peptides can be used as
tools to potentate the effects of opioid agonists. We have just
begun to comprehend how RGS proteins are able to regulate
opioid receptor signalling events. Future studies will
undoubtedly advance our knowledge of how these proteins
could be targets for the development of novel therapeutics.
RGS9 Protein
Another RGS protein that has been shown to be involved
in opioid mediated effects is RGS9 which belongs to the
C/R7 family of these proteins [109-111]. RGS9 exists in two
forms RGS9-1 and RGS9-2 that are generated by alternative
splicing [112]. RGS9-2 is solely expressed in brain regions
and has been implicated in the selective modulation of D2
dopaminergic receptor signalling via its DEP domain [113-
115]. Negative modulation of opioid action by RGS9-2 has
been reported from in vivo studies using antisense tech-
nology and knockout mice. RGS-9 knockout mice became
tolerant to morphine and exhibited a much stronger response
to morphine withdrawal and more severe physical depend-
ence than did wild-type mice [111, 116]. RGS9-2 was also
found to co-immunoprecipitate with the µ-ΟR from memb-
ranes of mouse periaquedictal gray matter; administration of
morphine disrupted this association, probably due to the
removal of the µ-ΟR-regulated Gα subunits [117]. Indeed,
morphine activation of the µ-OR in mouse periaqueductal
gray matter membranes transferred control of Gα subunits to
RGS9-2, a phenomenon responsible for receptor desensiti-
zation [118]. The authors suggested a multi-step process
where the tolerance developed after morphine administration
is correlated with the retention or sequestration of the µ-OR

Opioid Receptor- Interacting Proteins Current Drug Targets, 2012, Vol. 13, No. 1 85
associated Gα subunits to the serine phosphorylated RGS9-2
and binding of the latter to 14-3-3 protein [118, 119]. Thus,
retention of Gα by RGS9-2 attenuates the effects of agonists
and is implicated in the development of delayed tolerance
detected after high doses of µ-opioid ligands [118, 119].
Further observations of the role of RGS9-2 in µ-ΟR
signalling were generated using PC12 cells [120]. These data
indicated that RGS9-2 interacts with HA-tagged µ-ΟR, an
interaction that was enhanced upon morphine administration.
Overexpression of RGS9-2 prevented opiate-induced
ERK1/2 phosphorylation and delayed DAMGO-induced
internalization of this HA-tagged receptor [120]. However,
following elimination of the DEP domain of RGS9-2, which
is critical for the selective targeting of this RGS to the
membrane [113, 114, 121], an acceleration of µ-OR inter-
nalization was detected [120]. However, these studies did not
provide evidence on the identity of the G protein involved or
whether lack of the DEP domain of RGS9-2 affects its
association with µ-OR.
Behavioral analysis of RGS9 knockout mice provided
evidence for the role of RGS9-2 in drug addiction, in which
the locomotor activitating actions of opioid agonists were
more pronounced [111]. Lack of RGS9 expression led to
accelerated locomotor sensitization and increased reward
sensitivity. The increased sensitivity to the rewarding actions
of morphine is attributed to the RGS9-2 actions in the
nucleus accumbens, where RGS9-2 is localized [111]. In
addition, the RGS9-2 protein levels were altered after acute
or repeated exposure to drugs of abuse [113]. All these
findings, together with the role RGS9-2 plays in striatal
cholinergic interneurons [114], establish RGS9-2 protein as
an essential molecular target in opiate action.
RGSZ2 Protein
The very first indications of an association of an RGS
protein with the opioid receptors were reported for RGSZ2,
an RGS that efficiently deactivates GαzGTP. It was
demonstrated that in membranes from periaqueductal gray
matter both RGSZ2 and RGSZ20 co-precipitated with the µ-
ORs complexed with Gz [122]. Morphine challenge reduced
the association of Gi/o/z with µ-ΟR, but enhanced their
association with RGSZ2 and RGSZ20 proteins [122]. These
studies failed, however, to indicate clearly whether a
physical interaction of RGSZ2 with the µ-OR exists. It will
be of interest to investigate whether selectivity of RGS-OR
pair formation is influenced by the nature of the opioid
agonist involved or that opioid receptors themselves have the
ability to select a specific subset of RGS proteins depending
on their abundance and the tissue in which they are
expressed.
INTERACTING PROTEINS RELATED TO OPIOID
RECEPTOR INTERNALIZATION AND DESENSI-
TIZATION
Arrestins
Activated GPCRs are substrates for G-protein receptor
kinases (GRKs) and such phosphorylated GPCRs are
binding targets for the non-visual arrestins β-arrestin 1
(arrestin 2) and β-arrestin 2 (arrestin 3). Τhe β-arrestins have
been demonstrated to be key components of GPCR-
desensitization mechanisms because they block GPCR-G-
protein interactions and promote receptor recycling by
interaction with the clathrin assembly machinery and
clathrin-dependent endocytosis [123, 124]. It was shown that
β-arrestins function as multipurpose scaffolds activating
distinct signalling cascades [124], to mediate desensitization,
internalization and signalling of a number of receptors,
including GPCRs.
Results from pull-down assays using fusion proteins and
surface plasmon resonance provided in vitro evidence that β-
arrestins (1 and 2) were able to bind to the i3-L and to the C-
terminal tail of the δ-OR [125]. Site directed mutagenesis
experiments revealed that specific serine/threonine residues
within these intracellular δ-OR domains play critical roles in
arrestin interaction. Similar studies with κ-OR have shown
that only the C-terminal tail of this receptor binds β-arrestin
1 [125, 126] in an agonist-dependent manner. Co-expression
of β-arrestin 1 with the human κ-OR in HEK293 cells
reduced U69,593-induced stimulation of [35S]GTPγS binding
and inhibition of forskolin–stimulated adenylyl cyclase
[126]. Truncation of the last 28 amino acids of the receptor
abolished the effect of the β-arrestin while mutation of
putative phosphorylation sites of the C-terminal tail (S356A/
T357G/S358G/T363A) reduced the effect of β-arrestin 1,
thus suggesting that β-arrestin 1 can also bind to the
unphosphorylated κ-OR [126]. These observations suggest
that opioid receptor phosphorylation is not an absolute
requirement for β-arrestin 1 binding. This is in agreement
with previous studies using the β2-adrenoceptor [127, 128]
and phosphorylation of GPCRs is generally considered to
enhance affinity rather than define interactions with β-
arrestins. Furthermore, the three ORs are differentially
regulated by β-arrestins, implying regulatory differences
among the three classical OR subtypes.
Kovoor et al. [129] found that co-expression of both
GRK3 and β-arrestin 2 in Xenopus oocytes lead to rapid
desensitization of K+ conductance activated by the δ-OR.
This desensitization was greatly reduced by alanine
substitution of all five serine and threonine residues within
the C-terminus of δ-OR confirming the link between recep-
tor phosphorylation, β-arrestin binding and desensitization.
β-arrestin binding in vitro has also been reported for the i3-L
of a number of GPCRs including muscarinic receptors [130],
the 5-hydroxytryptamine 2A receptor [131], the chemokine
receptor CXCR4 [132] and the dopamine D1 receptor [133].
Several groups have demonstrated that β-arrestins can
bind to non phosphorylated δ-OR and trigger receptor
desensitization and internalization [134-136]. Data obtained
from β-arrestin 1 and β-arrestin 2 RNAi experiments indicate
that both arrestins participate in phosphorylation-dependent
internalization and recycling of the δ-OR [137]. It was also
demonstrated that the post-endocytic fate of internalized δ-
OR can be regulated by GRK2-induced receptor phos-
phorylation as well as by distinct β-arrestin isoforms.
Although arrestins discriminate between active and inactive
receptors and recognize the phosphorylated state of the
receptor, the resulting differences in binding levels can vary
widely [138].
Early studies demonstrated that morphine can not
effectively internalize the µ-OR in comparison with other

86 Current Drug Targets, 2012, Vol. 13, No. 1 Georgoussi et al.
opioid ligands [139]. This was explained by the weak ability
of morphine to phosphorylate the receptor and to recruit β-
arrestins [139, 140]. Evidence suggested that G protein and
β-arrestin efficacies can differ and be modulated by “biased”
ligands [141]. Recent studies using bioluminescence
resonance energy transfer (BRET) in intact cells have shown
that β-arrestin 2 interacts with µ- and δ-opioid receptors
[142]. In the same studies the ability of twenty ligands to
promote the association of µ- and δ- receptors with G
proteins or β-arrestin 2 was tested. Marked differences of
efficacy for G proteins and the β-arrestin were found
between the two receptors. Addictive opiates such as mor-
phine and oxymorphone selectively antagonized receptor-
arrestin interactions [142]. In similar studies the ability of
several µ-OR ligands to activate G proteins was compared
with their abilities to induce receptor phosphorylation, β-
arrestin 2 association and µ-OR internalization. It was shown
that the majority of µ-OR agonists, apart from endomorphins
1 and 2, displayed similar efficacy for G protein activation,
β-arrestin 2 recruitment and receptor internalization [143].
Increasing interest has emerged on the application of
ligand bias for drug development for the opioid receptors
and strategies to separate therapeutic and side effects of
opiate drugs [144]. Among the compounds discovered,
herkinorin a novel µ-selective agonist derived from the
naturally occurring plant product salvinorin, does not
promote the recruitment of β-arrestin-2 to the µ-OR and does
not lead to receptor internalization [145]. Knockout mice
indicate that β-arrestin-2 plays an important role in the
development of morphine-induced tolerance, constipation,
and respiratory depression [146, 147]. It has therefore been
proposed that opioid agonists that do not induce receptor-β
arrestin 2 interactions or receptor internalization, which can
be achieved by selectively interrupting the binding of β-
arrestin 2, may be promising candidates for distinguishing
the therapeutic effects from the undesired side effects of
opiates and thus provide leads to therapeutics designed for
pain relief devoid of the adverse effects of opiate narcotics
[145, 148]. An alternative approach to enhance the capacity
of morphine to cause µ-OR internalization, desensitisation
and downregulation following sustained receptor occupancy
has been to treat cells with an agonist at a co-expressed
PKC-coupled receptor [149]. Morphine desensitization,
internalization and down-regulation of the µ-opioid receptor
is facilitated by serotonin 5-HT2A receptor co-activation and
this may offer a simple means to regulate opioid tolerance
and dependence using currently available therapeutics drugs
[149].
PKCI
Similar to other GPCRs, opioid receptors generally
undergo rapid desensitization within seconds to minutes after
being activated by an agonist. Receptor desensitization is
crucially important in opioid pharmacology because this
phenomenon has been associated with the development of
tolerance to and dependence on opioid agents. Depending on
the efficacy of the agonist employed, opioid receptors use a
diverse array of internalization and desensitization pathways
[150]. Desensitization is mediated by ligand dependent
phosphorylation of the receptors and subsequent binding of
arrestins. Phosphorylation of the µ-OR seems to be important
for the initiation of internalization [151]. However the
dynamic relationship between receptor phosphorylation and
receptor internalization, desensitization and down regulation
remains to be determined. A number of protein kinases have
been implicated in opioid receptor phosphorylation [138,
152]. Thus far, kinases reported to be involved include a)
members of the G protein-coupled receptor kinase (GRK)
family [152-154], b) second messenger-activated kinases
such as protein kinases A and C (PKA, PKC) [154, 155] and
c) tyrosine kinases [156, 157].
Using a yeast two-hybrid screen it was shown that a
protein kinase C-interacting protein (PKCI) also interacts
with the C-terminal tail of the human µ-OR [158]. PKCI is a
member of the histidine triad HIT family of proteins,
originally shown to inhibit in vitro bovine PKC, although
recent studies have suggested it to function as a nucleotidyl-
hydrolase [159, 160] that displays a tumor suppressor role
[161]. The interaction of murine PKCI with the µ-OR was
also confirmed in CHO cells expressing both proteins.
Ligand binding affinity of µ-OR and agonist-mediated G
protein activation were not altered by this interaction. The
association of PKCI with µ-OR reduced, however, agonist-
mediated desensitization as measured by DAMGO
stimulation of [35S]GTPγS binding following DAMGO pre-
treatment in µ-OR and PKCI co-expressing cells [158].
Moreover, PKCI suppressed PKC-related phosphorylation of
µ-OR, although it was unclear whether the inhibition was
caused by the direct attenuation of PKC activity or exerted
through other effects on PKC activity [158]. The most
interesting finding of these studies was that the analgesic
effect of morphine and the extent of tolerance were greatly
enhanced in mice lacking expression of the PKCI gene.
Recent studies have shown that PKCI-1 protein also interacts
with RGSZ1 [162]. The authors suggest that the role of
PKCI-1 is not primarily in mediating the GαzRGSZ1
interaction, but in modulating µ-OR signalling.
Recent studies demonstrated that prolonged morphine
treatment induces homologous desensitization of µ-ORs in
rat brainstem locus coeruleus (LC) neurons, both in vitro and
in vivo by a PKC-dependent mechanism, and identified
PKCα to be the isoform responsible for this desensitization
in mature LC neurons [163, 164]. During the reviewing of
the present manuscript, Ping-Yee Law’s group indicated that
morphine activation of µ-OR in HEK cells induces
translocation of a PKCε isoform from the cytosol to the lipid
raft microdomain and demonstrated an interaction between
morphine-activated µ-OR and PKCε in lipid rafts [165].
These studies support the view that µ-OR desensitization by
PKC underlies the maintenance of morphine tolerance.
PLD2
Phospholipase D (PLD) is a phospholipid-specific
phosphodiesterase that localizes in the plasma membrane
and plays important roles in processes related to cytoskeletal
organization and vesicle trafficking for secretion and
endocytosis [166-168]. Agonist-dependent activation of PLD
has been reported for many GPCRs [167]. Recent studies
suggest a role of PLD2 in the formation of endocytic vesicles
[169]. In the case of the µ-ΟR, it has been shown that the µ-
ΟR specific agonist DAMGO internalized the receptor and
stimulated PLD2 activity [170]. In contrast, morphine, which

Opioid Receptor- Interacting Proteins Current Drug Targets, 2012, Vol. 13, No. 1 87
promotes µ-OR internalization with only low efficiency, did
not stimulate PLD2 activity. Through the use of a yeast two-
hybrid screen, it was found that the rat µ-OR associates with
PLD2 [170]. This occurred in the absence of agonist and was
confirmed by co-immunoprecipitation studies in HEK293
cells expressing these proteins. DAMGO-stimulation of the
µ-OR activated PLD2 activity in an ADP-ribosylation factor
(ARF)-dependent pathway, suggesting that opioid-mediated
PLD2 stimulation may involve a direct interaction of the µ-
OR with small G proteins (e.g. ARF). This hypothesis is also
supported by the presence of a NPXXY motif, previously
demonstrated to represent an ARF binding site implicated in
PLD activation, which is highly conserved in transmembrane
domain VII of GPCRs and present in the µ-OR [168, 171].
However, ARF was only detected in the immunoprecipitates
of cells co-expressing the µ-ΟR1 and PLD2 and not of cells
expressing µ-OR1 alone [170]. Recent studies demonstrated
that overexpression of dominant negative ARF mutants or
siRNA-based ARF6 protein depletion resulted in decreased
µ-ΟR endocytosis in HEK293 cells [172]. These findings
provide new insights into the role of ARF6 for opioid
mediated PLD2 activation and the regulation of µ-OR
trafficking and signalling.
Heterologous stimulation of PLD2 by phorbol ester led to
an accelerated internalization of the µ-OR after agonist
(DAMGO, morphine) exposure, suggesting that the opioid
agonists induced a conformational change and stimulation of
PLD2 activity is required for µ-OR endocytosis. Conversely
inhibition of PLD2-mediated phosphatidic acid formation by
butan-1-ol or by overexpression of a dominant negative
PLD2 mutant prevented agonist mediated endocytosis of µ-
OR and reduced the rate of re-sensitization [170, 173].
Studies from the same group indicated that a C-terminal
splice variant of µ-OR termed MOR1D exhibited robust
endocytosis in response to both DAMGO and morphine
treatment and that MOR1D mediated a constitutive PLD2
activation facilitating agonist-induced and constitutive
receptor endocytosis [174]. Koch and colleagues also
provided evidence for an essential role of PLD2 in the
agonist-induced and constitutive endocytosis of the δ-OR
and the cannabinoid CB1 receptor [174]. Recent findings
also reveal that agonist-selective PLD2 activation plays a
key role in NADH/NADPH-mediated ROS formation by
opioids [175]. Collectively, it can be suggested that
activation of PLD2 may be a key step during the induction of
GPCR endocytosis.
RanBPM
Ran Binding Protein in the Microtubule-Organizing
Center (RanBPM) was first identified in a yeast two-hybrid
screen using the small G protein regulator of nuclear-cyto-
plasmic trafficking, Ran, as bait [176]. RanBPM, originally
identified as a 55 kDa protein, also exists as a longer full-
length form of 90 kDa and belongs to the loosely organized
family of Ran-binding proteins (RanBPs), of which there are
more than ten members [177, 178]). RanBPM is enriched in
brain and may be either cytoplasmic or membrane-bound
[177, 179, 180]. Several lines of evidence suggest that
RanBPM interacts with multiple receptors, acting as a
scaffolding protein for signal transduction processes [178].
RanBPM binds to and modulates the activity of a diverse
group of proteins, including the LFA-1 integrin receptor
[179], the cyclin-dependent kinase CDK11p46 [181], the
receptor tyrosine kinases p75NTR [182] and MET [183] and
other signalling modulators such as the de-ubiquitinating
enzyme USP11 [184].
A recent study suggests that RanBPM can also interact
with and modulate the activity of the µ-OR [185]. In this
study, co-immunoprecipitation experiments in HEK293 cells
indicated that RanBPM associates constitutively with µ-OR,
while receptor activation did not appreciably change the
extent of RanBPM binding. Functionally, RanBPM had no
effect on µ-OR-mediated inhibition of adenylyl cyclase, but
reduced agonist-induced endocytosis of µ-OR. High levels of
RanBPM altered DAMGO-stimulated trafficking of µ-OR to
resemble those reported for morphine-activated µ-OR,
namely limited β-arrestin 2 recruitment [186] and decreased
receptor internalization [139, 187]. Thus, RanBPM may play
a role in determining the effectiveness of µ-OR agonists.
Mechanistically, RanBPM interfered with β-arrestin 2-GFP
translocation upon µ-OR but not α1B-adrenergic receptor
activation, indicating selectivity of action. RanBPM, a puta-
tive scaffolding protein, is therefore a novel µ-OR interacting
partner that negatively regulates receptor internalization
without altering acute µ-OR signalling. Furthermore,
because RanBPM interacts with µ-OR constitutively, the
relative expression of RanBPM in specific neuroanatomical
areas within the CNS may be important in determining the
extent of µ-OR regulation in vivo. In line with this concept
RanBPM has been shown to bind to mGlu2 and mGlu8
metabotropic glutamate receptors known to be implicated in
modulating morphine and related opioid drug effects [188-
191]. Further investigation of the binding sites of RanBPM
in µ-OR and/or other GPCRs will contribute to the elu-
cidation of the functional consequences of these associations
and may provide novel targets for therapeutic interventions
to selectively modulate GPCR-mediated processes.
Spinophilin
Spinophilin (SPL) is a ubiquitously expressed, multi-
domain-scaffold protein, highly enriched in dendritic spines.
SPL is also called Neurabin II and bears domains for F-actin
binding, protein phosphatase 1 (PP1) binding, a single PDZ
domain involved in protein-protein interactions, a receptor
binding domain (RBD) and three coiled coil domains [192,
193]. SPL has been proposed to play an organizer role for
the actin-based cytoskeleton in dendritic spines as well as a
role in spine morphology, density regulation, synaptic
plasticity and neuronal migration [194, 195]. Recent studies
have shown that SPL interacts with multiple GPCRs to
modulate their signalling and stabilize their expression [196].
Indeed, interaction of SPL within the i3-L of the three α2-
adrenoceptor subtypes (α2A, α2B and α2C) stabilized their
surface expression in Madin-Darby canine kidney cells [194,
197]. SPL also interacts directly with the M1-muscarinic
receptor inhibiting the binding of RGS8 to the receptor and
enhancing the regulatory function of RGS8 [198].
Recent observations have shown that SPL is involved in
the acute and chronic actions of opiates and opposes the
development of tolerance and decreases sensitivity to the
rewarding actions of the drugs [199]. Indeed, deletion of the
SPL gene reduced the analgesic effects of acute morphine

88 Current Drug Targets, 2012, Vol. 13, No. 1 Georgoussi et al.
but enhanced adaptations to repeated morphine administra-
tion, including increased morphine dependence, place
conditioning and analgesic tolerance [199]. The same studies
also demonstrated that SPL interacts directly with µ-OR in
mouse striatum under basal conditions. This association was
enhanced after subcutaneous administration of either fenta-
nyl or morphine. Moreover, exposure to opiates promoted
the formation of a complex between SPL and RGS9-2.
Studies in PC12 cells have shown that SPL promotes
morphine–induced µ-OR internalization and inhibits
signalling of the receptor [199]. These observations provide
new insights as to the cellular events underlying opioid
actions and it has been suggested that spinophilin may be a
promising pharmacological target to optimize the analgesic
actions of opiate drugs with reduced side effects.
INTERACTING PROTEINS RELATED TO LYSO-
SOMAL TARGETING AND TRAFFICKING
GASP
Both δ and µ-ORs, are endocytosed via clathrin-coated
pits after agonist-induced activation, phosphorylation and
association with cytoplasmic β-arrestins [154]. However, a
number of studies have demonstrated that endocytosis
impacts differentially on the δ-OR and µ-OR [200-202]. It
has been suggested that lysosomal targeting of the δ-OR
causes proteolytic downregulation whereas recycling of µ-
ΟR to the plasma membrane after endocytosis promotes
rapid resensitization of signal transduction. Whistler and
colleagues [203] have identified a large cytosolic protein
named GPCR-associated sorting protein-1 (GASP-1) that,
among the opioid receptors, interacts selectively with the δ-
OR, and determines its degradative fate. Four clones, all of
them containing sequences from within the C-terminal 497
amino acid fragment of GASP-1, were identified using a
two-hybrid screen with the δ-OR C-terminal tail as bait.
Endogenously expressed GASP-1 co-immunoprecipitated
with δ-ΟR in HEK293 cells, thus confirming the existence of
δ-ΟR-GASP-1 interactions in cells [203].
GASP-1 appears to be a key player in lysosomal sorting
of the δ-OR and other GPCRs [203, 204]. Disrupting the
interaction between GASP and the δ-OR through receptor
mutation or overexpression of a dominant negative fragment
of GASP-1 blocked lysosomal sorting and promoted
recycling of internalized δ-ΟR to the cell surface [202].
Interestingly, GASP-1 has a high affinity for the carboxyl-
termini of GPCRs that are targeted to the degradative
pathway, and a lower affinity for heptahelical receptors that
favor the recycling pathway.
Simonin et al. [205] reported a family of at least 10
members of GASP-like proteins, although GASP-1 remains
by far the best characterized. In accordance with Whistler’s
findings, it was shown that GASP-1 associates directly with
δ-OR to determine its degradative fate. GASP-1 has also
been shown to associate in vitro with κ-OR, to a lesser extent
with the µ-OR, and with several other GPCRs. In situ
hybridizations and Northern blot analyses indicated that
GASP-1 mRNA is distributed throughout the central nervous
system, consistent with a potential for interaction with
numerous GPCRs in vivo [205]. On the other hand, GASP-2,
another member of the GASP family also binds to a diverse
array of GPCRs. However, GASP-2 does not associate with
any opioid receptor subtype suggesting the existence of
selectivity between these proteins in GPCR regulation. The
structural determinant of the GASP-1 interaction with δ-OR
was restricted to the small portion of the C-terminal tail,
corresponding to helix VIII in the three dimensional
structure of rhodopsin and, specifically, within two
conserved residues [205]. Mutation of two residues in
rhodopsin (F313 and R314) to alanine resulted in decreased
interaction between GASP-1 and GPCRs [205]. Residues in
the hydrophobic face of the putative helix VIII for the MCH-
1 [206] and oxytocin [207] support the concept of helix VIII
as an important region critical for GPCR-protein
interactions. Recent observations reveal that a mutant µ-OR
that is lacking “recycling signals” due to a deletion of a
small portion within the C-terminal tail enhances the
interaction of the receptor with GASP-1 both in vitro and in
HEK293 cells, and drives the receptor towards degradation
[208]. Thus the authors suggested that facilitating interaction
of a GPCR for GASP, either by disrupting an interaction
with a recycling protein or by enhancing the receptor’s
affinity for GASP-1 can lead to alterations of the post-
endocytic fate of the receptor. In a recent paper, dysbindin, a
cytoplasmic protein known to function in the biogenesis of
specialized organelles and to be a schizophrenia suscepti-
bility gene, has been reported to promote the sorting of
specific GPCRs to lysosomes after endocytosis [209, 210].
Dysbindin depletion up-regulated both total and surface
expression of Flag-tagged-δ-ΟR in addition to D2 dopamine
receptors, and inhibited proteolytic down regulation of the
Flag-δ-ΟR. It was therefore suggested that dysbindin might
also play a significant role in controlling the post-endocytic
sorting of various GPCRs in a number of cells [210].
Synaptophysin
Using the full length and truncated versions of the µ-OR
as bait in a yeast-two-hybrid screen, synaptophysin was
identified as a novel µ-OR interacting protein [211]. Synap-
tophysin is a major integral membrane glycoprotein of
synaptic vesicles and is also found in brain vesicles, presu-
mably as a result of receptor trafficking [212]. Synapto-
physin interacts with various nerve terminal proteins such as
dynamin I, adaptor protein 1 (AP-1), the v-SNARE vesicle-
associated membrane protein 2/synaptobrevin II (VAMP2),
the vesicular proton pump V-ATPase and myosin V [213].
Synaptophysin is involved in the regulation of SNARE
assembly, formation of the fusion pore and is essential for
the biogenesis of synaptic-like microvesicles [214]. The
structural determinant of synaptophysin binding in the µ-OR
is located in the receptor’s i3-L. Co-immunoprecipitation
and BRET assays using tagged versions of the receptor and
synaptophysin in HEK293 cells confirmed the constitutive
interaction of µ-OR with synaptophysin [211]. Subsequent
GST pull-down assays verified that synaptophysin speci-
fically interacts with the i3-L of µ-OR [211]. Binding of
synaptophysin to µ-OR did not alter the binding charac-
teristics of the receptor, nor did it affect agonist-mediated
inhibition of adenylate cyclase.
It is believed that µ-OR endocytosis is mediated via a
dynamin-dependent clathrin-coated vesicle pathway [140],
and existing evidence suggests that synaptophysin interacts

Opioid Receptor- Interacting Proteins Current Drug Targets, 2012, Vol. 13, No. 1 89
with dynamin I [215]. Dynamin I is responsible for the rapid
fission of budding vesicles and acts as a molecular scissor in
sequestering clathrin-coated vesicles away from the plasma
membrane [216, 217]. Confocal microscopy of HEK293
cells and primary cultures of neurons has shown that
synaptophysin co-localizes with µ-OR at the plasma
membrane and the cytosol and that overexpression of
synaptophysin enhances µ-ΟR endocytosis. One explanation
for the observed effects is that synaptophysin recruits
dynamin to the plasma membrane, facilitating fission of
clathrin-coated vesicles. Breakage of the interaction between
synaptophysin and dynamin prevents agonist-mediated µ-OR
endocytosis. In addition, the synaptophysin-mediated
increase in µ-OR trafficking leads to an attenuated agonist-
induced receptor desensitization and a faster receptor
resensitization [211]. Synaptophysin interacts with the AP1
adaptor complex and provides adaptor sites for microtubule-
based vesicle transport to axons and nerve terminals [218].
Thus, synaptophysin may play multiple functional roles in µ-
ΟR regulation. Since µ-OR desensitization contributes to the
development of opiate tolerance [147] and synaptophysin-
augmented µ-OR trafficking leads to decreased agonist-
induced receptor desensitization, it is suggested that
synaptophysin may attenuate the development of opiate
tolerance.
Sorting Nexin 1
Another protein proposed to contribute to the endosomal/
lysosomal targeting οf δ-OR is Sorting Nexin-1 (SNX1).
SNX1 is a member of a relatively large family of sorting
nexins. Sorting nexins are cellular trafficking proteins having
a phospholipid binding domain and a strong predisposition
for protein-protein interactions mainly through coil-coil
formation [219]. Interestingly, it was reported that SNX1 has
a low affinity for GPCRs that prefer the recycling pathway.
SNX1 is ubiquitously expressed in endosomes together with
SNX2. Using a library of C-terminal tail-fusion proteins
from representative GPCRs it was demonstrated in pull
down assays that δ-ΟR binds SNX1 [204]. Further analysis
by surface plasmon resonance (SPR) measurements, which
quantify the affinity of the interaction, have shown that
SNX1 binds to the C-terminal tail with high affinity, dis-
playing a Kd value of 20 nM [204]. More experimental
evidence is, however, required to determine the regulatory
role of this interaction in δ-OR endocytic sorting and
signalling. Trejo and colleagues [220] showed that the
sorting of activated Protease Activated Receptor-1 (PAR1)
from endosomes to lysosomes is regulated by SNX1. SNX1
co-localizes with internalized PAR1 on early endosomes and
is also found associated with activated PAR1 in cellular
lysates. Strikingly, depletion of endogenous SNX1 by
siRNA markedly inhibited agonist-induced PAR1 degrada-
tion, whereas expression of a SNX1 siRNA resistant mutant
protein restored agonist promoted PAR1 degradation in cells
lacking endogenous SNX1, indicating that SNX1 is nece-
ssary for lysosomal degradation of PAR1 [221]. However,
none of these studies has demonstrated a direct interaction
between the PAR1 C-terminal tail and SNX1. Whether
SNX-1 or other SNX proteins mediate the endosomal to
lysosomal sorting of opioid receptors in vivo remains to be
determined.
Glycoprotein M6a
Glycoprotein M6a is a member of the proteolipid protein
(PLP) family of tetraspan membrane proteins and mainly
expressed in neurons [222-224]. It is generally believed that
glycoproteins of the PLP family function as structural
proteins for myelination. M6a, however, has also been
suggested to play a role as a modulator for neurite outgrowth
[225] and spine formation [226] and as nerve growth factor-
gated Ca2+ channel in neuronal differentiation [227].
Using a yeast two-hybrid screen and the full-length rat µ-
OR as bait, M6a was identified to associate with µ-OR [228].
BRET and co-immunoprecipitation experiments confirmed
agonist-independent µ-OR-M6a interactions in HEK293
cells co-expressing the receptor and the glycoprotein. Co-
expression of µ-OR with M6a, but not with other members
of the PLP family, occurs in many brain regions, further
suggesting specific interactions between them in vivo. The
transmembrane domains IV, V, and VI of µ-OR and the
protein stretch/domain including transmembrane domains 3
and 4 of M6a are important regions for this interaction. M6a
did not affect the expression or agonist binding to the µ-OR
but enhanced the constitutive and agonist-dependent
internalization as well as the recycling rate of the receptor;
whereas a dominant negative of M6a prevented agonist-
induced µ-OR internalization [228]. Further investigation of
the role of M6a in the post-endocytotic sorting of µ-OR
indicated that µ-OR and M6a are primarily targeted to
recycling endosomes after endocytosis [229]. This enhanced
post-endocytotic sorting of µ-OR into the recycling pathway
was accompanied by a decrease in agonist-induced receptor
down-regulation of M6a in HEK293 co-expressing cells. The
physiological relevance of these findings was further tested
in primary cultures of cortical neurons, where co-expression
of M6a significantly increased the translocation of µ-OR
from the plasma membrane to intracellular vesicles at steady
state and markedly augmented both agonist-induced and
constitutive receptor endocytosis [229].
M6a was also found to interact with the δ-OR, the canna-
binoid CB1 receptor and the somatostatin sst2A receptor,
which share substantial homology within transmembrane
domains V and VI suggesting that M6a might play a general
role in the regulation of certain GPCRs [228]. Furthermore,
co-expression of M6a enhanced the post-endocytotic sorting
of δ-OR into the recycling pathway, indicating that M6a
might have a more universal role in opioid receptor post-
endocytotic sorting [229].
Taken together these observations demonstrate a new
function for the M6a PLP member, namely a scaffolding role
in the intracellular trafficking of certain GPCRs. The
detailed molecular mechanisms of M6a-mediated receptor
trafficking merits further investigation. However, based on
the fact that µ-OR endocytocis counteracts the development
of opioid tolerance by inducing rapid recycling/ resensi-
tization of the receptor, M6a-enhanced µ-OR endocytosis
and recycling rate could be implicated in the attenuation of
opioid tolerance.
Ubiquitin
Ubiquitination is an essential procedure for the endocytic
sorting of various GPCRs to lysosomes. Ubiquitin is a 76

90 Current Drug Targets, 2012, Vol. 13, No. 1 Georgoussi et al.
amino acid polypeptide that binds covalently to GPCRs via
the epsilon-amino group of lysine residues and targets them
for proteasomal degradation (lysine48-ubiquitination) or
down-regulation in lysosomes (lysine63-ubiquitination). In
the case of δ-ΟR, the misfolded or incompletely folded
portion of newly synthesized receptors is transported to the
cytoplasmic site of the endoplasmic reticulum (ER)
membrane via the Sec61 translocon. There, receptors are
deglycosylated and conjugated with ubiquitin prior to
degradation by the cytoplasmic 26 S proteasome [230]. As
far as ligand activated plasma membrane δ-OR is concerned
its endocytosis and post-endocytic trafficking to lysosomes
is ubiquitin independent as shown using inhibitors of
proteasomal degradation or mutations of cytoplasmic lysine
residues [231]. After δ-OR is sorted to lysosomes the E3
ubiquitin ligase AIP4 specifically controls its down-regula-
tion by directly ubiquitinating wild type receptors. Thus δ-
OR’s proteolytic fate is controlled by two deubiquitinating
enzymes, which are localized to late endosome/lysosome
membranes containing internalized δ-ORs [232]. The
activity of the proteasome was also found to affect δ-OR
internalization because proteasome inhibitors reduced δ-OR
endocytosis, in contrast with lysosomal protease inhibitors,
that lead to polyubiquitinated δ-OR accumulation [233].
In the case of µ-OR, agonist-induced internalization is
followed by rapid recycling to the plasma membrane without
any interaction with ubiquitin. µ-OR’s recycling is directed
by a specific 17-amino acid sequence in the C-terminal tail
that specifically promotes the sorting of receptors into a
rapid recycling pathway [234]. On the other hand, κ-OR is
targeted to lysosome for degradation upon polyubiquitination
at its C-terminal tail [235].
PDZ DOMAIN CONTAINING PROTEINS THAT
MODULATE TRAFFICKING
EBP50/NHERF
κ-OR has been demonstrated to undergo U50,488H-
induced internalization and down regulation via a GRK, β-
arrestin and dynamin-dependent process involving clathrin
coated vesicles [236, 237]. In an effort to determine the
signals for trafficking of internalized κ-OR to the recycling
or down regulation pathway the possible involvement of
Ezrin-radixin-moesin [ERM]-binding phosphoprotein 50
(EBP50/ Na+/H+ exchanger regulatory factor (NHERF) asso-
ciation with this receptor was investigated [238]. EBP50/
NHERF is a 55kDa multidomain protein that has multiple
phosphorylation sites, two tandem PDZ domains at its N-
terminus and an ezrin/radixin/moesin (ERM) binding its C-
terminus which allows NHERF to interact with a family of
actin-binding proteins. This phosphoprotein is widely
distributed in tissues and particularly enriched in polarized
epithelia. PDZ domains were originally identified in the
postsynaptic density protein-95 as three repeats of 90
residues containing the conserved motif Gly-Leu-Gly-Phe.
EBP50/NHERF co-immunoprecipitated with κ-ΟR in CHO
cells stably expressing the human Flag-tagged κ-ΟR.
Generation of two mutants in the extreme C-terminus of the
Flag-tagged human κ-OR which did not immunoprecipitate
with the EBP50/NHERF indicated that the free C-terminus
of κ-OR is critical for the interaction [238]. The PDZ domain
I, but not the PDZ domain II of EBP50/NHERF was
involved in this interaction. EBP50/NHERF binds at the
extreme C-terminal domain NKPV, which is distinctly
different from the sequence D(S/T)XL of the optimal C-
terminal motif in the β2-adrenoceptor [239-241]. EBP50/
NHERF did not have any effect on U50,488H induced
internalization of the κ-OR, but it caused an apparent
increase in the recycling rate of internalized receptors [238].
Expression of EBP50/NHERF did not affect U50, 488H
binding affinity and U50,488H-stimulated [35S]GTPγS bind-
ing or p42/p44 MAP kinase activation. However, agonist
treatment enhanced EBP50/NHERF-human κ-ΟR interac-
tions and expression of EBP50/NHERF but not a truncated
form lacking the ERM-binding domain, abolished agonist-
induced down regulation of the κ-OR. These results demons-
trate that binding of EBP50/NHERF to the cortical actin
cytoskeleton is critical for its inhibitory effect on down-
regulation. It was therefore concluded that blunting down-
regulation by EBP50/NHERF may be due either to inhibition
of κ-OR internalization, or to an increased recycling rate of
the internalized receptor by limited lysosomal targeting.
GST-C-terminal tails of the µ-OR or δ-ORs did not bind
purified EBP50/NHERF-1 [238, 242]. On the other hand, the
association of κ-OR with EBP50/NHERF plays an important
role in accelerating Na+/H+ exchange via an effect that is
mediated in a receptor dependent but a G protein-independ-
ent manner [242]. It was also shown that EBP50/NHERF
association with the β2-adrenoceptor induces stimulation of
Na+/H+ exchanger in a manner that is independent of G
proteins [240]. NHERF interaction with the β2-adrenoceptor
promotes recycling of the internalized receptor whereas loss
of this interaction results in a more efficient targeting of the
β2-adrenoceptor to lysosomes. Interestingly, NHERF interac-
tion with β2-adrenoceptor can be selectively disrupted by
GRK5 phosphorylation of Ser411 in the PDZ binding
domain [241]. By contrast, the association of NHERF with
the parathyroid hormone PTH1 receptor via a PDZ domain
interaction leads only to a redistribution of signalling with
the formation of molecular complexes that enhance Gαi/o-
mediated PLC activation [243].
PROTEINS THAT MODULATE TRAFFICKING IN
THE BIOSYNTHETIC PATHWAY
GEC1
By yeast two-hybrid screening of a human brain cDNA
library with the C-terminal tail of the human κ-OR as the
bait a truncated form of GEC1-(38-117) was identified.
GEC1 selectively interacts with κ-OR but not with the C-
terminal domain of the human µ-OR, or δ-OR [244]. GEC1
is a 117-amino-acid protein [245], which is highly homo-
logous to GABARAP, GATE-16 and Apg8/aut7, members
of the microtubule associated protein family. GST-tagged
GEC1 interacted with the full length κ-OR and tubulin. It
was demonstrated that GEC1 facilitates trafficking of Flag-
tagged human κ-OR from the endoplasmic reticulum/Golgi
to plasma membranes. Immunoprecipitation and pull-downs
have also indicated that GEC1 binds directly to N-ethyl-
maleimide–sensitive factor (NSF), a protein critical for intra-
cellular membrane-trafficking [244]. Electron microscopy
studies in rat brain have also shown that GEC1 is associated
with ER, Golgi apparatus and plasma membranes as well as

Opioid Receptor- Interacting Proteins Current Drug Targets, 2012, Vol. 13, No. 1 91
being scattered in the cytoplasm of neurons [246]. Employ-
ing a yeast two hybrid technique the same group recently
determined the amino-acid residues in both GEC1 and
human κ-ΟR involved in this interaction [247]. Generation
of a molecular model of GEC1, based on the x-ray crystal
structure of GABARAP identified that the residues involved
in human κ-OR binding form curved hydrophobic patches on
the exterior surface of GEC1, which interacts within the
FPXXM motif in the C-terminal tail of κ-OR [247].
However, the authors also suggest that GEC1 is likely to
bind to other molecules by hydrophobic interactions and not
specific amino acid residues, thus supporting an expanding
functional role of GEC1 with chaperone like effects.
Ribophorin I
Using a targeted proteomic approach and mass spectro-
metry analysis, Law’s group identified ribophorin I as a
novel µ-OR-interacting protein [248]. The interaction
between ribophorin I and µ-OR was confirmed by both co-
precipitation studies from transiently transfected N2A cells
and direct gel overlay studies. Ribophorin I is an integral
component of rough microsomal membranes [249].
Ribophorin I is accepted as a member of the oligosaccharide
transferase (OST) family and is assumed to act as a
chaperone or as an escort to facilitate the N-glycosylation of
selected substrates [250]. Ribophorin I has also been shown
to have a multifunctional role and facilitate additional
processes, such as ER quality control [251, 252].
By means of both confocal microscopy and FACS
analysis, it was demonstrated that the expression level of µ-
OR could be regulated by ribophorin I levels. Ribophorin I
seemed to mediate µ-OR exocytotic activity, which was
clearly related to its OST activity. More specifically,
ribophorin I stimulated the transport of an export-deficient µ-
OR mutant (C2), lacking a motif, 344KFCTR348, at the
proximal carboxyl tail, from the ER to the cell surface, but
not the export of the glycosylation-deficient µ-OR mutant-µ-
OR5ND. This phenomenon suggests that ribophorin I not
only is involved in biosynthesis of nascent polypeptides as
previously reported, but also plays a pivotal role in their
maturation and plasma membrane expression. Additionally,
the fact that overexpression of ribophorin I could enhance
cell surface expression of C2 might reflect ribophorin I -
associated ER quality control processes to facilitate export of
the C2 mutant from the ER other than the common calnexin
pathway [248]. Ribophorin I was also shown to interact with
other highly glycosylated GPCRs such as α1A-adrenoceptor
and α2C- adrenoceptor but not α2B- adrenoceptor as well as
with δ-OR and κ-OR [248]. Although ribophorin I acted as a
chaperone for δ-OR and κ-ΟR, it exhibited differential
effects on their expression at the plasma membrane. Over-
expression of ribophorin I and FACS analysis indicated that
ribophorin I significantly increased the δ-OR level on the
cell surface. On the other hand, κ-OR has fewer N-glyco-
sylation sites, and overexpression of ribophorin I had mini-
mal effect on κ-OR plasma membrane expression. Knocking
down ribophorin I levels with siRNA resulted in a decrease
of κ-OR plasma membrane expression, suggesting that the
function of ribophorin I depends not only on the glyco-
sylation state of the receptor but on the number of glyco-
sylation sites [248]. Ribophorin I interaction with δ-ΟR and
µ-ΟR supports the notion that ribophorin I is a key regula-
tory component that serves as a chaperone or controller to
transport these receptors from the ER to the cell surface.
INTERACTIONS THAT LINK OPIOID RECEPTORS
TO THE CYTOSKELETON
Filamin A
Another protein that has been identified as a direct
binding partner for human µ-OR is filamin A [253]. Filamin
A (ABP-280) is a large cytoskeleton protein known to
couple membrane proteins to actin filaments and maintain
the integrity of the cytoskeleton. Filamin binds actin using its
N-terminus and homodimerizes with another filamin mole-
cule using its C-terminus. Filamin A is also known to bind to
different intracellular signalling molecules such as SEK-1,
an activator for stress activated protein [254], transmem-
brane molecules such as β-integrins [255] presenilins [256]
and caveolin, a scaffolding protein.
Using a yeast two-hybrid system it was demonstrated that
the human µ-OR associates within its C-terminus with
filamin A. This interaction was verified by co-immuno-
precipitation studies in HEK293 cells expressing the µ-OR
and filamin A and by in vitro pull downs using a GST fusion
peptide encompassing the C-terminus of the µ-OR [253]. A
number of studies have also shown the association of filamin
with other GPCRs including the D2 and D3 dopamine
receptors [257, 258], the calcium sensing receptor [259,
260], the metabotropic glutamate receptor 7b splice variant
[261] and the calcitonin receptor [262]. Interestingly, the
sites of interaction and the functional effects mediated by
filamin differ significantly among the various GPCRs
examined.
DAMGO-induced down-regulation of µ-ΟR and func-
tional desensitization of the receptor were abolished in cells
lacking filamin A. In addition, the level of internalized µ-OR
following agonist administration was greatly attenuated,
suggesting that filamin A plays a central role in controlling
µ-ΟR trafficking [253]. Simon’s group reported recently that
in cells lacking filamin A, chronic morphine treatment leads
to up-regulation of the µ-ΟR, due to enhanced coupling of
the receptor with the G proteins [263]. A possible explana-
tion for that could be that filamin A may be important for the
proper receptor conformation or positioning of the µ-OR in
the cell membrane. Other studies using a mutant filamin A
lacking the actin-binding domain suggest that filamin A does
not act via the actin cytoskeleton but that other functions
may require functional binding of the µ-OR- filamin A
complex to actin [264].
Recent findings using organotypic striatal slice cultures,
suggest that filamin interacts with ultra-low dose naloxone
and naltrexone treatment to prevent chronic and acute mor-
phine-induced µ-OR-Gs coupling, possibly by preventing
interaction of µ-OR with filamin [265, 266]. The authors
report the identification of a specific C-terminal region of
filamin A as the high affinity binding site of naloxone and
naltrexone in their suppression of µ-ΟR signaling. Wang et
al., [265] proposed that repeated µ-OR stimulation leads to
particular µ-OR-filamin formation that weakens Gi/o-µ-OR-
filamin complexes allowing µ-OR to interact with Gs upon

92 Current Drug Targets, 2012, Vol. 13, No. 1 Georgoussi et al.
subsequent morphine stimulation. Naloxone and its ana-
logues, by binding to filamin A, prevent this altered µ-OR-
filamin A interaction, thus preventing the switch to Gs
coupling that may contribute differently to effects such as
analgesic tolerance and physical dependence produced upon
prolonged exposure to opioids. These observations give an
opportunity to formulate a new generation of pain ther-
apeutics that may provide long-lasting analgesic effects
devoid of undesired effects.
INTERACTING PROTEINS INVOLVED IN SORTING
INTO LARGE DENSE-CORE VESICLES
Protachykinin
Results from immunofluorescence, electron microscopy
and co-immunoprecipitation studies have demonstrated that
the δ-OR interacts with protachykinin, a content protein of
secretory granules [267]. Protachykinin is found in large
dense-core vesicles (LDCVs) of small dorsal root ganglion
neurons (DRG) and is the precursor of substance P [268],
which, in turn, is a pronociceptive neuropeptide that is
released after tissue damage or in response to nociceptive
stimuli [269]. Protachykinin undergoes cleavage and other
processing events in LDCVs to produce substance P and
other mature peptides while LDCVs are transported to the
nerve terminals for secretion. In their study Guan et al. [267]
found that protachykinin directly interacts with δ-OR and
that this interaction is responsible for sorting δ-OR into
LDCVs. This novel interaction of protachykinin allows
regulated insertion of δ-OR in C fibers upon nociceptive
stimulation, enabling δ-OR-mediated spinal analgesia. This
interaction is mediated by the substance P domain of
protachykinin and the third extracellular domain of δ-OR.
Deletion of the preprotachykinin A gene reduced stimulus-
induced surface insertion of δ-OR and abolished δ-OR-
mediated spinal analgesia. Studies have demonstated that δ-
OR interacts with µ-OR in the spinal cord and that blockade
of δ-ORs enhances µ-OR mediated analgesia [270]. Guan et
al. [267] have shown that in preprotachykinin A knockout
mice spinal morphine analgesia was enhanced, suggesting
that µ-ΟR functions may be modulated by δ-OR insertion
into the plasma membrane. Moreover, they found that
morphine tolerance did not develop when stimulus-induced
surface insertion of δ-ORs was eliminated in preprota-
chykinin A knockout mice. Thus the authors propose that
protachykinin is also involved in the regulation of µ-OR-
mediated analgesia and the development of morphine
tolerance [267]. This was the first study linking the two
regulatory systems of pain transmission in the spinal cord,
tachykinin and opioid receptors. These observations provide
the background for further analysis of yet another aspect of
opioid-mediated analgesia. A recent review provides insights
into the molecular mechanisms that determine the sorting of
neuropeptide and neuropeptide receptors into secretory
pathways and provide the background for further analysis of
opioid-mediated analgesia [271].
In contrast to the above studies elegant work by Basbaum
and collaborators [272] demonstrated recently that the δ-OR
is trafficked to the cell surface under resting conditions,
independently of substance P and is internalized upon δ-OR
activation by agonists. They found that µ-ΟR is expressed in
peptidergic pain fibers and the δ-OR in myelinated and
nonpeptidergic afferents. Use of a δ-OR-eGFP reporter
knockin mouse provided for the first time evidence for a
different view regarding δ-and µ-OR function and relation-
ship to the control of mechanical and heat pain messages.
CHAPERONE POSSESSING PROTEINS
Hlj1, a Member of the Heat Shock Protein 40 Family
Recent observations from work that also employed a
yeast two-hybrid screen using as bait the C-terminal tail of
the human µ-OR along with a few amino acids of the seventh
transmembrane domain, and a human brain cDNA library as
prey, indicated that the C-terminal portion of Hlj1 (aa 227-
337) interacts with µ-OR [273]. Hlj1 is a member of the heat
shock protein 40 (HSP40) family [274]. Results from co-
immunoprecipitation studies carried out with HEK293 cell
lysates, confirmed the interaction between these proteins. In
addition, immunofluorescent studies showed significant co-
localization between Hlj1 and the human µ-OR in HEK293
cell membranes.
Molecular chaperones including heat shock proteins have
been shown to be involved in the maturation and degradation
of receptors at the ER [275, 276]. The interaction of a
HSP40 family member with µ-OR could be important for
correct receptor conformation and for positioning the
receptor on the cell membrane, trafficking and/or regulation
of µ-OR. However, the functional significance of Hlj1
interaction with µ-OR and the physiological relevance of this
interaction in the brain has still to be determined.
In the course of purifying the κ-OR, the heat shock 70
protein (HSP70) was recently identified as another opioid
receptor interacting protein [277]. HSP70 is expressed in all
cells and is involved in protein folding and translocation.
The functional significance of this interaction with respect to
proteasome-degradation of κ-ΟR is not yet defined. Further
studies are required to determine and clarify the biological
significance and the involvement of heat shock and other
chaperone related proteins in the internalization and
desensitization pathways mediated by opioid receptors.
Calnexin
Calnexin is an abundant integral membrane phospho-
protein of the ER of eukaryotic cells. Its role is to facilitate
and direct the proper folding and maturation of newly
synthesized receptors residing in the ER and help them to
avoid proteasomal degradation. Recent observations have
demonstrated that human flag-δ-OR interacts with calnexin
in the ER. The amount of calnexin co-precipitated with the
flag-δ-ΟR in HEK293 cells was decreased upon antagonist
treatment [278]. The ability of opioid ligands to dissociate δ-
ΟR precursors from calnexin and enhance their processing
and maturation suggests that the receptors are inappro-
priately retained in the ER and may be targeted to degrada-
tion prematurely. These observations support previous
findings that GPCR biosynthesis can be pharmacologically
modulated at the ER level, revealing a new site of regulation
that could be also targeted to control cellular responsiveness
in therapeutic settings [279].

Opioid Receptor- Interacting Proteins Current Drug Targets, 2012, Vol. 13, No. 1 93
INTERACTIONS WITH PROTEINS POSSESSING
TRANSCRIPTIONAL ACTIVITY
STAT5 Proteins
Several studies have shown that traditional signalling
pathways activated by cytokine or growth factor receptors
are also shared by heptahelical receptors [280, 281]. For
example, angiotensin receptor activates the Jak2 tyrosine
kinase following stimulation with angiotensin II [282], while
Signal Transducer and Activator of Transcription 5 (STAT5)
interacts directly with the consensus motif YXXL of this
receptor, a sequence which is also found in several other
GPCRs [283], including all three opioid receptor subtypes.
Pull-down experiments employing GST-fusion proteins
encompassing the C-terminal regions of the µ-OR (µ-CT) or
δ-OR (δ-CT) and cellular extracts expressing STAT5A or
STAT5B, respectively, revealed direct interactions of
STAT5A and STAT5B with the µ-CT and the δ-CT,
respectively [15, 284]. Co-immunoprecipitation studies in
cells co-expressing either the µ-OR or the δ-OR with
STAT5A/B confirmed the association of these proteins in a
cellular context. Deletion of the YXXL motif from either
opioid receptor abolished STAT5A/B binding, thus defining
this conserved tetrapeptide as the structural determinant
within the µ-CT and the δ-CT responsible for STAT5A and
STAT5B binding respectively. The amount of STAT5A co-
precipitated with the µ-OR is independent of morphine or
DAMGO stimulation [15]. In contrast, STAT5B associates
constitutively with the δ-OR, while upon agonist administra-
tion the STAT5B-δ-OR interaction is abolished [284]. The
differential pattern of STAT5 association between the two
opioid receptors can be explained by the different signalling
pathways these receptors mediate upon their activation [47].
STAT5A and STAT5B are tyrosine-phosphorylated by c-
Src in various cell lines upon µ-OR and δ-OR stimulation
with morphine and other selective opioid peptides. Addi-
tionally, activation of these receptors results in the transcrip-
tional activation of a STAT5-responsive reporter gene [15,
284]. Considering previous observations confirming
complex formation between the Src/Jak tyrosine kinases and
GPCRs [285-287], a question that arises is whether multi-
meric signalling complexes of the opioid receptors are
formed. A series of co-precipitation studies indicated that
this is indeed the case and that STAT5B forms pairs with
selective Gα subunits and Gβγ in HEK293 cells [284]. A
novel signalling pathway mediated by a multicomponent
complex (signalosome) initiated at the δ-CT, which serves as
a protein platform, is proposed and shown in Fig. (2). The
biological consequences of the µ, δ-opioid receptors-
STAT5A/B pathways are unclear but STAT transcription
factors have been shown to have crucial roles in the
regulation of neuronal survival and neurite outgrowth [288,
289].
Binding of STAT5A/B to the intracellular parts of µ and
δ-ORs supports the concept that GPCRs can physically
associate with transcription factors, creating signalling com-
plexes mechanistically analogous to those observed for
growth factor and cytokine receptors. Taking into account
that opioid receptors belong to the list of cell surface
Fig. (2). A putative signalling complex between the δ-OR, STAT5B and G protein subunit(s) leading to STAT5B activation. STAT5B
associates constitutively with the C-terminal tail of δ-OR, Gα and Gβγ subunits of G proteins. c-Src interacts with Gβγ, which acting as a
scaffold brings c-Src close to the δ-receptor. Upon DSLET activation of the flag-δ-OR, STAT5B dissociates from the receptor, and is
phosphorylated by c-Src in a Gαi/o protein-dependent manner. Activated STAT5B forms a complex consisting of Gβγ, selective active Gαi/o
subunits and c-Src. Subsequently, phosphorylated STAT5B dimerizes and translocates to the nucleus where it binds to specific DNA target
sequences and alters gene transcription.
NH
2
COOH
Activation
Transcription
Gβγ
Agonist
COOH
Gβγ
P
P
P
P
STAT5B
STAT5B
P
P
STAT5B
STAT5B

94 Current Drug Targets, 2012, Vol. 13, No. 1 Georgoussi et al.
receptors that homodimerize or hetero-oligomerize [30-36],
it is possible that traditional signalling pathways activated by
cytokine or growth factor receptors are also shared by these
different receptor types. This type of mechanism of trans-
criptional activation by direct association of a transcription
factor with a GPCR is currently unique to the opioid recep-
tors and may play a role in the alteration of gene expression
in specific target neurons. Identification of multimeric
protein complexes and characterization of interaction
networks is highly relevant to modern drug design. The fact
that a STAT5B-δ-OR signalling complex occurs sponta-
neously supports the notion that stable constitutive associa-
tions of receptors, G proteins and accessory proteins can
function as membrane entities to be targeted for the
development of new therapeutic agents.
INTERACTIONS AFFECTING NEUROTRANSMIT-
TER RELEASE
Excitatory Amino-Acid Carrier 1
Initial observations have demonstrated that co-expression
in Xenopus oocytes of the δ-OR with the excitatory amino-
acid carrier 1 (EAAC1), a neuronal-specific transporter,
down-regulates EAAC1 function [290, 291]. In addition it
was demonstrated that DPDPE-activated δ-OR counteracts
the down regulation of EAAC1-mediated uptake [292]. In
the same studies, co-immunoprecipitation and immuno-fluo-
rescence microscopy in both oocytes and rat hippocampal
neurons indicated co-localization of these proteins and
reported for the first time direct interaction between the δ-
OR and EAAC1 [292]. It is therefore proposed that the δ-OR
can reduce EAAC1 function by direct protein-protein
interaction and that activation of the δ-OR disrupts this
inhibitory interaction. The modulation of EAAC1 function
was specific for the δ-OR because co-expression of µ-OR or
a Na+/K+ pump did not influence the EAAC1-mediated
signal [292]. Previous observations have also shown that
uptake via the glial GLT-1 glutamate transporter was
influenced by δ-OR stimulation in astroglial cultures [293],
while other groups have demonstrated that glutamate
transporters play a critical role in the development of mor-
phine tolerance, abnormal pain sensitivity and withdrawal
syndrome [294-296]. It is therefore plausible to suggest that
glutamate transporters may contribute to the neural
mechanisms of opiate abuse, however, the question of how
the activity of glutamate transporters in brain is regulated
during opiate misuse remains to be investigated.
CONCLUDING REMARKS
Opioids, through the action of their receptors, control
several aspects of neurotransmitter signalling in both the
central nervous system and the periphery. Opioid receptors
play a pivotal role in phenomena related to analgesia,
however, chronic opioid administration results in a number
of cellular neuroadaptations ranging from tolerance, with-
drawal and finally addiction. To understand these molecular
events the discovery of novel targets that interfere with
opioid receptor function is obligatory. This can be achieved
by identification of novel receptor accessory and regulatory
proteins that upon receptor activation alter signal responses
and effectiveness. As described, various laboratories have
identified numerous accessory proteins which are able to
interact with these receptors. Some of these proteins rep-
resent novel receptor partners while others have already been
identified as interacting proteins for other GPCRs. The
functional roles of such receptor interacting proteins are
beginning to be dissected out. The physiological significance
and relevance of the identified interactions to phenomena
such as tolerance and dependence are beginning to emerge
but it is clear that these interactions contribute to every
aspect of opioid receptor function, from ligand binding,
signalling and endocytosis to transport to the site of action,
proper anchoring and trafficking. We anticipate that an
accelerated pace toward the discovery of new interacting
proteins using proteomic strategies will ensue. As we
continue identifying more previously unanticipated interac-
tions and the existence of additional multicomponent
complexes, we should become better equipped to propose
functional roles for the observed associations, to identify
novel therapeutic targets and propose more effective stra-
tegies for encountering pathological situations where the
functionality of opioid receptors is relevant.
ACKNOWLEDGMENTS
This work was supported by the EU grant
“NORMOLIFE” (LSHC-CT2006-037733) to Z.G and
Biotechnology and Biosciences Research Council (grant
BB/E006302/1) to G.M. Many thanks should be mentioned
to M-P. Papakonstantinou (N.C.S.R “Demokritos) for her
assistance.
ABBREVIATIONS
AC = Adenylyl cyclase
ACB = Nucleus accumbens
AON = Anterior olfactory nucleus
AP-1 = Adaptor protein 1
AR = Adrenergic receptor
ATFx = Activating transcription factor x
BRET = Bioluminescence resonance energy transfer
CaM = Calmodulin
cAMP = Cyclic 3',5'-adenosine monophosphate
CB = Cannabinoid receptor
COS = African green monkey kidney
CREB = cAMP Response Element Binding Protein
DAG = Diacylglycerol
DSLET = [D-Ser2]-Leucine Enkephaline – Thr
DAMGO = [D-Ala2,N-MePhe4,Gly-ol5]-enkephalin
δ-ΟR = δ-opioid receptor
DPDPE = (D-Pen2,D-Pen5]-enkephalin
EAAC1 = Excitatory amino-acid glutamate
transporter 1
EBP50 = Ezrin-radixin-moesin [ERM]-binding
phosphoprotein 50

Opioid Receptor- Interacting Proteins Current Drug Targets, 2012, Vol. 13, No. 1 95
ER = Endoplasmic reticulum
ERK = Extracellular signal-regulated protein kinase
ERM = Ezrin/radixin/moesin
GABA = γ-amino butyric acid
GASP = G protein-coupled receptor associated
sorting protein
Gaxβyγz = Heterotrimetric G protein composed of x, y
and z subunits of α, β and γ subunits
respectively
GPCR = G protein-coupled receptors
G proteins = Guanine nucleotide binding proteins
GRK = G protein-coupled receptor kinase
GTPγS = Guanosine 5΄-Ο-(3-thiotriphosphate)
GST = Glutathione S-transferase
HA = Hemaglutinin
HSP40 = Heat shock protein 40
HSC73 = Heat shock cognate protein 73
HEK293 = Human embryonic kidney
IP3 = Inositol triphosphate
JAK = JANUS tyrosine kinase
JNK = c-Jun N-terminal kinase
κ-ΟR = κ-opioid receptor
LDCVs = Large dense-core vesicles
M1 AChR = M1 muscarinic cholinergic receptor
µ-ΟR = µ-opioid receptor
MAP = Mitogen activated protein
MAPK = Mitogen activated protein kinase
µ-CT = µ-opioid receptor carboxyl-terminal tail
NHERF = Na+/H+ exchanger regulatory factor
NG108-15 = SOMATIC cell hybrid mouse neuroblastoma
and rat glioma
NSF = N-ethylmaleimide –sensitive factor
ND = Non determined
OTU = Olfactory tubercle
PAR1 = Protease activated receptor-1
PDGF = Platelet derived growth factor
PDZ = PSD-95/ Drosophila Discs-Large septate
junction protein/ epithelial tight junction
protein ZO-1 (a domain that binds the C-
terminal X-Ser/Thr-X-Val/Leu sequence)
PIP2 = Phosphatidylinositol biphosphate
PKA = Protein kinase A
PKC = Protein kinase C
PLC = Phospholipase C
PLD = Phospholipase D
PTX = Bordetella pertussis toxin
RGS = Regulator of G protein signalling
SNX1 = Sorting nexin 1
STAT = Signal Transducer and Activator
of Transcription
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Received: April 09, 2010 Revised: March 23, 2011 Accepted: March 26, 2011
PMID: 21777181