CB1 Cannabinoid Receptors and Their Associated Proteins PDF

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Current Medicinal Chemistry, 2010, 17, 1382-1393
CB1 Cannabinoid Receptors and their Associated Proteins

Allyn C. Howlett*, Lawrence C. Blume and George D. Dalton
Department of Physiology and Pharmacology, Wake Forest University Health Sciences, One Medical Center Blvd.,
Winston-Salem, NC 27157, USA
Abstract: CB1 receptors are G-protein coupled receptors (GPCRs) abundant in neurons, in which they modulate neurotransmission. The CB1 receptor influence on memory and learning is well recognized, and disease states associated with
CB1 receptors are observed in addiction disorders, motor dysfunction, schizophrenia, and in bipolar, depression, and anxiety disorders. Beyond the brain, CB1 receptors also function in liver and adipose tissues, vascular as well as cardiac tissue,
reproductive tissues and bone. Signal transduction by CB1 receptors occurs through interaction with Gi/o proteins to inhibit adenylyl cyclase, activate mitogen-activated protein kinases (MAPK), inhibit voltage-gated Ca2+ channels, activate
K+ currents (Kir), and influence nitric oxide (NO) signaling. CB1 receptors are observed in internal organelles as well as
plasma membrane. -Arrestins, adaptor protein AP-3, and G-protein receptor-associated sorting protein 1 (GASP1) modulate cellular trafficking. Cannabinoid Receptor Interacting Protein1a (CRIP1a) is an accessory protein whose function has
not been delineated. Factor Associated with Neutral sphingomyelinase (FAN) regulates ceramide signaling. Such diversity
in cellular signaling and modulation by interacting proteins suggests that agonists and allosteric modulators could be developed to specifically regulate unique, cell type-specific responses.
Keywords: Anandamide (arachidonylethanolamide), AP-3, 2-arachidonoylglycerol (2-AG), -arrestin, CP55940, CRIP-1a,

endocannabinoids, G-protein coupled receptors (GPCRs), GASP, rimonabant (SR141716), 9-tetrahydrocannabinol (THC),
WIN55212-2.
CB1 CANNABINOID RECEPTOR PHYSIOLOGY,
PATHOLOGY AND PHARMACOLOGY
CB1 receptors are G-protein coupled receptors (GPCRs)
initially described as having such great abundance in brain
tissue and neuronal cells, that the much lower levels in other
tissues appeared to be of lesser significance [1]. In the early
years of cannabinoid receptor characterization, pharmacological investigation of antinociception was emphasized because of the potential for a non-opioid analgesic, and pharmaceutical industry drug development programs were based
upon this therapeutic opportunity [2,3]. This clinical application was thwarted as a result of the untoward side effects of
memory impairment, cognitive dysfunction, and sedation
[4,5], but has recently been reconsidered [6]. Although
pharmaceutical development included treatment of nausea in
cancer chemotherapy [7], the only CB1 agonists to succeed to
market have been dronabinol (also known as 9tetrahydrocannabinol (THC)) and nabilone [8-10].
Current research has made significant progress in clarifying the role of CB1 receptors in such important aspects of
cognition as reversal learning by which memory traces can
be attenuated in the process of developing new patterns in
response to novel relevant stimuli [11]. Highly coupled to
memory and learning, CB1 receptors play a role in addiction
processes that extend from reinforcement of high-fat, sweet
food intake [12] to dependence on reinforcing drugs and
alcohol [13-15]. CB1 receptors abundant in the basal ganglia
are implicated in motor dysfunction including Huntingtons
disease and amyotrophic lateral sclerosis [16]. Other central
nervous system diseases that might involve a CB1 receptor
component include schizophrenia, and bipolar, depression,
and anxiety disorders [17,18]. To understand the mecha*Address correspondence to this author at the Department of Physiology
and Pharmacology, Wake Forest University Health Sciences, One Medical
Center Blvd., Winston-Salem, NC 27157, USA;
E-mail: ahowlett@wfubmc.edu
0929-8673/10 $55.00+.00
nisms underlying these behavioral responses, we now know

that CB1 receptors are not only involved in neuronal synaptic
remodeling as learning takes place, but also in neurogenesis,
neuronal migration and appropriate axonal targeting and
synaptogenesis as the brain develops [19-21].
The existence of CB1 receptors in liver and adipose tissues was not appreciated until clinical trials of the CB1 antagonist rimonabant (also known as SR141716A) for the
treatment of obesity and dyslipidemias [22,23] uncovered the
anomaly that decreases in adiposity in humans could not be
entirely explained by a central nervous system effect of the
drug to curtail food intake [24,25]. Current studies are focusing on both CB1 and CB2 receptors in hepatic metabolism
and pathologies [26,27]. The interest in metabolic syndrome
led to the expansion of studies of CB1 receptors on vascular
as well as cardiac tissue [28-30]. CB1 receptors are also relevant to many other organ systems including reproductive
tissues [31], bone [32], and skin [33].
To summarize the relevant pharmacology, synthetic analogs of THC include classical cannabinoid agonists such as
the highly potent and efficacious HU210, and the nonclassical highly potent and efficacious agonists which have
a modified ring structure, such as CP55940 and CP55244
[34,35]. The aminoalkylindole CB1 agonists include the full
agonist WIN55212-2. The endogenous ligands for the CB1
receptor are a family of eicosanoid derivatives referred to as
endocannabinoids, most notably 2-arachidonoylglycerol (2AG) which behaves as a full agonist, and anandamide (arachidonylethanolamide), which behaves as a partial agonist.
These CB1 agonists have been reviewed previously [34-37],
as well as in the present series (Mechoulam, 2010; Pertwee,
2010). CB1 antagonists include the high-affinity aryl pyrazole, rimonabant, formerly known as SR141716, as well as
other CB1-selective antagonists of similar structure including
AM251, taranabant, VCHSR and AM4113 [35,38-42].
LY320135 is a CB1 antagonist having a structure that resembles the aminoalkylindole class of antagonists [43].
2010 Bentham Science Publishers Ltd.
CB1 RECEPTOR CELLULAR SIGNALING VIA GPROTEINS

Domains of the CB1 receptor that selectively interact with
Gi/o proteins have been identified [44] and appear to be coupled to Gi/o proteins in the absence of exogenous agonists
[45-47]. The juxtamembrane domain, which extends from
transmembrane (TM) 7 to the cys palmitoylation site, forms
a helix referred to as H8 [48-51]. This domain is able to trigger G-protein activation [44,48,52] via Go or Gi3
[46,47,53]. CB1 receptor mutants truncated at the membrane
surface of the TM7 were not able to inhibit Ca2+ channels in
response to CB1 agonists [54]. The H8 amphipathic helix
possesses cationic charges facing the G-protein surface that
are important for agonist efficacy [48,50], with hydrophobic
residues of the helix being critical for proper structural folding and positioning within the membrane bilayer [55]. A
conformational change in the TM7 domain could displace
the TM7-H8 elbow (see [51]. To test this, point mutations
that replaced Leu at the elbow region with Phe or Ile were
compared with the wild-type CB1 receptor [56]. Human Embryonic Kidney 293 (HEK293) cells heterologously expressing the mutant receptors exhibited reduced agoniststimulated [35S]GTPS binding to G-proteins, and the CB1
receptor mutants were dissociated from Gi3 although they
could still bind Gi1 and Gi2 [56]. These CB1 receptor
mutants exhibited alterations in internalization in response to
agonists [56]. The mutant receptors also exhibited significantly reduced inhibition of the Ca2+ channels but a more
rapid time course in response to WIN55212-2, which could
be overcome by providing an abundance of the preferred Gprotein, Go A [56].
There is evidence to suggest that the CB1 receptor IL3 facilitates association with Gi1 or Gi2 [44,46,47,53]. Helical structures at the N-terminal side of IL3 possessing a
BBXXB-like motif characterize the potential for this domain
to interact with G-proteins [57]. The IL3 helical region near
H6 is believed to interact with Gi1 [53]. A double mutation
of Leu-Ala in the IL3-H6 junction switched the helical domain to a single turn structure, thereby converting the CB1
receptor from interacting with Gi to interacting with Gs
[53,58].
Signal transduction pathways utilized by CB1 receptors
have often been associated with interaction with Gi/o class of
G-proteins as demonstrated by their sensitivity to pertussis
toxin [37,59]. Inhibition of adenylyl cyclase, activation of
the mitogen activated protein kinase (MAPK) family of
kinases, inhibition of voltage-gated Ca2+ channels, and activation of inwardly rectifying K+ currents (Kir) are all signaling pathways that are pertussis toxin-sensitive (see reviews
[34,37]. Regulation of ion channels by CB1 receptors are the
direct result of G release upon Gi/o activation [60-64]. Ntype voltage-gated Ca2+ currents were inhibited in differentiated neuroblastoma cells [62,63,65-67] and P/Q-type Ca2+
currents were inhibited in rat cortical and cerebellar neurons
and cultured AtT-20 pituitary cells heterologously expressing CB1 receptors [68,69]. L-type Ca2+ currents in cat brain
arterial smooth muscle cells were inhibited by CB1 agonists,
although the mechanism may not directly involve G [70].
Current Medicinal Chemistry, 2010 Vol. 17, No. 14
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cAMP-PROTEIN KINASE A (PKA) SIGNALING

Inhibition of adenylyl cyclase isoforms 5 and 6 via Gi/o
is the best characterized CB1 signaling pathway, as it results
in reduced cyclic AMP-stimulated protein kinase A (PKA)
activity. Reduced PKA activity impacts important cellular
signaling events including voltage-dependent current flow at
A-type K+ channels [71] and tyrosine phosphorylation of
focal adhesion kinase (pp125FAK) and FAK-related nonkinase (FRNK) [72-74]. In hippocampal neurons, the excitotoxic sequalae to NMDA-mediated Ca2+ release was attenuated by CB1-mediated decreases in PKA phosphorylation of
ryanodine channels [75].
If the ability of Gi/o proteins to interact with CB1 receptors is compromised by pertussis toxin treatment, interaction
with Gs becomes possible [43,76,77]. CB1 receptor agonists
increase cyclic AMP accumulation in globus pallidus
[77,78], due to either augmentation of adenylyl cyclase 2/4/7
activation via G derived from Gi/o [79] or the sequestration of Gi/o proteins by other Gi/o-coupled receptors
thereby facilitating coupling to Gs [61, 80]. A CB1-mediated
activation of PKA has been shown to promote Ca2+ influx
into neuroblastoma cells [81], phosphorylate DARPP-32 in
corpus striatum neurons [82,83], and regulate K-type K+
channels in hippocampal neurons [84].
MAPK SIGNALING
MAPK activation by CB1 agonists has been observed almost universally in cells expressing both endogenous and
recombinant CB1 receptors [85-88]. In CHO cells heterologously expressing CB1 receptors, extracellular signalregulated kinase 1/2 (ERK1/2; also known as p42/p44
MAPK) required Gi/o as an initial step, presumably via the
G subunits or else via CB1 agonist-stimulated association
with -arrestin [85]. There are multiple pathways that could
result in CB1-mediated ERK1/2 phosphorylation and activation, and these may vary depending upon the cell type and
the stimulus. Astrocytoma cells (and CHO cells expressing
recombinant CB1 receptors) utilized a pathway involving
phosphatidylinositol-3-kinase (PI3K) and protein kinase B
(PKB, also known as Akt), which then activates Raf-1,
MAP-ERK Kinase (MEK), and ERK1/2 [87,89,90]. In CHO
cells heterologously expressing CB1 receptors, MAPK activation by insulin receptors or insulin-like growth factor
(IGF) receptors could be blocked by SR141716-mediated
sequestration of Gi/o proteins [91,92], suggesting some
mechanism by which CB1 receptors could be directly involved in the transactivation of these receptor tyrosine
kinases. Both glioblastoma or lung carcinoma cells employed cannabinoid-mediated transactivation of epidermal
growth factor (EGF) receptors in the PKB/Akt and ERK
activation pathway [93].
N1E-115 neuroblastoma cells and hippocampal neurons
utilized a pathway involving CB1-mediated attenuation of
PKA activity and reduction of c-Raf phosphorylation, which
facilitates MEK activation [94,95]. N18TG2 neuroblastoma
cells appear to utilize a mechanism that involves PKC, a
Ca2+-calmodulin-stimulated enzyme, a matrix metalloprote-
1384 Current Medicinal Chemistry, 2010 Vol. 17, No. 14
ase and the activity of a vascular endothelial growth factor

(VEGF)-like receptor. This ERK1/2 activation was associated with Ca2+ influx as determined by cellular uptake of
radioisotopic Ca2+ [96-98] In cultured cortical interneurons,
endocannabinoids stimulated TrkB receptor tyrosine phosphorylation [99]. In PC12 cells heterologously expressing
both TrkB receptors and CB1 receptors, co-immunoprecipitation studies implicated a complex formation in response
to cannabinoid stimulation [99]. However, in nerve growth
factor (NGF)-stimulated PC12 cells, anandamide was reported to inhibit TrkA receptor-induced Rap1/B-Raf/ERK
activation [100]. CB1 receptors and Gi/o proteins could also
regulate ERK1/2 activation in PC12 cells via a non-receptor
Src or Fyn tyrosine phosphorylation.
p38 MAPK was activated by cannabinoid receptor agonists in human vein endothelial cells [101], mouse hippocampal slices [102], and CHO cells heterologously expressing CB1 receptors [103]. Jun N-terminal kinase (JNK1 and
JNK2) was activated by CB1 agonists via Gi/o, PI3K and Ras
in CHO cells heterologously expressing CB1 receptors [103].
Those studies implicated a transactivation of platelet-derived
growth factor (PDGF) receptors in the cannabinoid agonistmediated activation of JNK [103]. In cultured neuroblastoma
cells, the CB1 receptor-mediated stimulation of JNK required
Gi/o, activation of Rap1, Ral and Rac, and phosphorylation
of Src tyrosine kinases [104-106]. Src and JNK phosphorylation led to activation of the transcription factor Stat3, resulting in gene expression involved in neurite growth and elongation [104-106].
NITRIC OXIDE (NO) SIGNALING
CB1 receptor-mediated production and release of NO via
neuronal NO synthase (nNOS) or endothelial NOS (eNOS)
has been demonstrated for a variety of tissues including saphenous vein segments [107], endothelial cells [108-110],
monocytes [111], brain slice preparations [112] and
neuroblastoma cells [113, 114]. Cannabinoid stimulation of
NO-sensitive guanylyl cyclase and cyclic GMP production
was detected in neuroblastoma cells [113, 115, 116]. The
observation that CB1 agonists promote the translocation of
NO-sensitive guanylyl cyclase from the cytosol to a
membranous organelle compartment suggests that interactions between these proteins may occur in neuronal cells
in the form of a receptor-effector complex [113].
In studies in which cannabinoid agonists reduced NO
production in response to an excitotoxic stimulus in cerebellar granule cells, the CB1 receptor-mediated inhibition of
voltage-gated Ca2+ channels was shown to be responsible
[116]. Similarly, in mouse cortical neurons undergoing excitotoxic degeneration in response to prolonged NMDA, CB1
receptor stimulation ameliorated NO generation by a mechanism that involved reduction in PKA activity and a change in
the phosphorylation of nNOS [117-119]. NO production by
inducible NOS (iNOS) in response to an inflammatory stimulus in saphenous vein endothelial cells [107], RAW264.7
macrophages [120], microglial cells [121], and astrocytes
[122-124] was also reduced by cannabinoid receptor stimulation and the reduction in cyclic AMP [107,120,123,124].
Howlett et al.
CB1 RECEPTOR INTERACTION WITH PROTEINS

OTHER THAN G-PROTEINS
The diverse intracellular localization of endogenouslyexpressed CB1 receptors was reported in studies using antibodies that recognize an N-terminal epitope of the receptor
[125]. Immunocytochemical localization and sub-cellular
fractionation studies indicated that a predominance of
endogenously expressed CB1 receptor in neuroblastoma cells
reside in internal organelles, particularly along the perinuclear membranes [125]. Constitutively synthesized CB1
receptors that had been pulse-chase labeled with [35S]-amino
acids were degraded via at least two different mechanisms
having elimination half-lives of approximately 5 hr (70% of
receptors) and >24 hr (30% of receptors) in cells that had not
been exposed to agonist ligands, suggesting the presence of
multiple pools of functional receptors within neuronal cells
[125]. Cellular CB1 receptors heterologously expressed in
CHO-CB1 cells were recognized by FACS and confocal microscopy to transit from internal stores to the plasma membrane in response to the antagonist rimonabant and from
plasma membrane to intracellular compartments in response
to agonist CP55940 [126]. Pretreatment with CP 55940 resulted in rapid desensitization of the CB1 receptor agonistmediated inhibition of forskolin-stimulated adenylyl cyclase
or MAPK activation. Pretreatment of CHO-CB1 cells with
rimonabant resulted in significant augmentation of CP55940mediated activation of MAPK, but had no significant effect
on CP55940-mediated inhibition of forskolin-stimulated
adenylyl cyclase [126]. Internalization of CB1 receptors from
neuronal plasma membranes at the soma has been proposed
to be a mechanism by which these receptors can be diverted
to plasma membrane loci along neuronal extensions [127129]. To add to the complexity of interpreting data, many
studies have been performed using exogenously expressed
CB1 receptors that are fusion proteins with a fluorescent protein at the C-terminal tail, or else localization was determined using antibodies against epitopes in the C-terminal
tail. Methods of detection that depend upon recognition of
the C-terminal differ in their results when compared with
endogenously expressed receptors having an accessible Cterminal for interaction with accessory proteins (see diversity
of responses reported in [130]). Thus, it appears that domains
along the CB1 receptor C-terminal have the potential to alter
the dynamics of cellular trafficking and signal transduction
(summarized in Table 1). CB1 receptors located at internal
organelles may be of particular importance in autocrine regulation of cellular functions, particularly as sites of anandamide synthesis and degradation may be localized to intracellular membranous organelles (enzymes reviewed by
[131]). Both cellular localization and function may be directed by CB1 receptor association with a variety of interacting proteins.
INTERACTION OF THE CB1 RECEPTOR WITH ARRESTINS
The level and duration of GPCR signaling activity is
regulated by the two distinct processes of desensitization and
endocytosis (see [132]). GPCR desensitization begins within
seconds of agonist exposure and involves phosphorylation of
agonist-activated receptors by GPCR kinases (GRKs). -
Table 1.
1385
Role of CB1 Receptor-Associated Proteins in Protein Trafficking and Signal Transduction
CB1-Associated
Protein
Site of CB1 interaction
Role in Protein Trafficking
Gi1
C-terminal side of Intracellular Loop 3
--
Gi2
N-terminal side of Intracellular Loop 3
--
Gi3 and Go
C-terminal Helix 8
--
Gs
C-terminal side of Intracellular Loop 3
--
Role in Signal Transduction
References
cAMP inhibition
MAPK activation
[44,46,53,58,
85-93]
cAMP inhibition
[44,46,
MAPK activation
85-93]
cAMP inhibition
[44-56,
Ca2+ channel inhibition
60-69,
MAPK activation
85-93]
cAMP stimulation
[43,53,58,61,
76,77,80]
-Arrestin
Mid-C-terminal tail;
C-terminal distal 20 amino acids
Plasma membrane internalization
Desensitization of Kir3
[133, 135, 137,
Kinetics of MAPK
138-139]
Gi- and ERK-associated
[130]
--
[145-146]
AP-3
Leu-Leu or Tyr signals
Late endosome budding to

vesicles, or lysosomes
GASP1
C-terminal Helix 8
Targeting to lysosome
2+
CRIP1a and
CRIP1b
Distal C-terminal tail
--
Ca channel inhibition
[150-152]
FAN
Mid-C-terminal tail
--
Ceramide-mediated Raf1-MEKERK; stimulation of metabolic

processes
[86-87, 162163, 167-168]
arrestin1 and -arrestin2 immediately bind to the agonistoccupied, phosphorylated GPCR which prevents G-proteinmediated signal transduction, while the GPCR-arrestin complex associates with clathrin to initiate GPCR endocytosis. In
addition to attenuating GPCR signaling and mediating
GPCR internalization, -arrestins function as scaffolds in
GPCR-mediated endosome-based signaling pathways.
CB1 receptor desensitization by GRKs and -arrestins
was first demonstrated using Xenopus laevis oocytes and
CB1 receptor mutants [133]. CB1 receptor-mediated activation of G-protein gated inwardly rectifying potassium channels (Kir3, also known as GIRK) was significantly desensitized in oocytes that co-expressed GRK3, -arrestin 2, Kir3
channels, and CB1. A deletion CB1 receptor mutant with 20
amino acids (residues 418-439) removed from the C-terminal
tail of the receptor did not exhibit GRK3 and -arrestin 2mediated CB1 receptor desensitization in oocytes, which
indicated that residues located in this region of the receptor
are required for CB1 receptor desensitization by GRK3 and
-arrestin 2. Furthermore, the mutation of two possible
GRK3 phosphorylation sites (S426A, S430A) significantly
attenuated GRK3 and -arrestin 2-mediated CB1 receptor
desensitization in oocytes, which indicated that GRK3mediated phosphorylation of S426 and S430 is necessary for
CB1 receptor desensitization. Finally, these studies demonstrated S426A/S430A CB1 receptor mutants that were stably
expressed in AtT20 cells retained their ability to be internalized. This finding suggested that distinct domains of the CB1
receptor are involved in GRK/-arrestin-dependent CB1 receptor desensitization versus internalization. Later studies
provided corroborative evidence supporting this seminal
study that CB1 receptor desensitization is -arrestin 2dependent. The presynaptic expression of dominant negative
GRK2 or -arrestin 2 reduced desensitization of CB1 receptor-mediated presynaptic inhibition of glutamatergic neurotransmission in rat hippocampal neurons [134].
The S426A/S430A CB1 receptor desensitization-deficient
mutant has also been used to demonstrate that CB1 receptor
phosphorylation determines the time course of CB1 receptor
agonist-mediated ERK1/2 activity [135]. The CB1 receptor
agonist CP55940 transiently activated ERK1/2 in human
embryonic kidney 293 (HEK293) cells stably expressing
wild-type (WT) CB1 receptors, with a peak of ERK1/2 phosphorylation at 5 min followed by a rapid dephosphorylation.
In contrast, the duration of S426A/S430A CB1 receptormediated activation of ERK1/2 was significantly prolonged
compared with WT, and was dynamically reversed by rimonabant in HEK293 cells. Thus, the time-course of CB1
receptor-mediated ERK1/2 and Kir3 activation both appear to
be regulated by the same distinct domains of the CB1 receptor that are directly phosphorylated. During agonist treatment, -arrestin was recruited to the plasma membrane in the
S426A/S430A CB1 receptor mutant with the same kinetics as
WT CB1 receptors, which suggests that phosphorylation of
S426 and S430 is not required for -arrestin recruitment.
Nevertheless, a di-phosphorylated peptide created to mimic
this domain was able to bind to -arrestin 2 [136]. Based on
the observation that the S426A/S430A CB1 receptor mutant
could be internalized in HEK293 cells, Daigle and colleagues postulated that -arrestin recruited to nonphosphorylated S426A/S430A CB1 receptors following agonist treatment retained its scaffolding functions [135].
CB1 receptor mutants have also been utilized to correlate
CB1 receptor internalization with -arrestin recruitment
[137]. The extreme carboxy terminal tail (amino acid resi-
dues 460-473) of the rat CB1 receptor contains a cluster of

six serine (S) and threonine (T) residues that are potential
sites for phosphorylation. HEK293 cells were stably transfected with CB1 receptors that were truncated at amino acid
residue 460 (V460Z) or mutated at putative GRK phosphorylation sites (T461A/S463A, S465A/T466A, T468A/S469A,
T461A-T466A, T461A-S469A) [137]. CB1 receptor internalization proceeded normally in HEK293 cells when the
CB1 receptor was truncated at residue 460 (V460Z) or when
any two phosphorylation sites were mutated (T461A/S463A,
S465A/T466A, T468A/S469A). However, CB1 receptor internalization was reduced when four sites were mutated
(T461A through T466A), and was abolished when all six
sites were mutated (T461A through S469A). In HEK293
cells in which the CB1 receptor internalized, -arrestin was
recruited to the plasma membrane and co-localized with CB1
receptors in response to agonist treatment, which suggests arrestin mediates CB1 receptor internalization. -arrestin was
recruited to internalization-competent mutant CB1 receptors
to the same maximal extent as to WT-CB1 receptors. However, the rate of -arrestin recruitment to internalizationcompetent
(V460Z,
T461A/S463A,
S465A/T466A,
T468A/S469A) CB1 receptors was approximately 3- to 6times slower compared with WT-CB1 receptors. Moreover,
-arrestin was not recruited to internalization-incompetent
mutant CB1 receptors (T461A through T466A, T461A
through S469A). These results suggest that -arrestin recruitment is coupled to the ability of the CB1 receptor to internalize, inasmuch as -arrestin recruitment was attenuated
when CB1 receptor internalization was reduced. Furthermore, -arrestin recruitment is not solely a function of CB1
receptor phosphorylation, as -arrestin was recruited to the
V460Z CB1 receptor mutant.
In vivo studies have also shown that -arrestins can regulate CB1 receptor activity. The role of -arrestin 2 in cannabinoid-mediated behavioral effects was investigated in arrestin 2 (-/-) and -arrestin 2 (+/+) mice [138]. No differences in CB1 receptor levels were observed in cerebellum,
cortex or hippocampus of -arrestin 2 (-/-) and -arrestin 2
(+/+) mice. However, 9-THC produced greater antinociception and hypothermia in -arrestin 2 (-/-) mice compared to
-arrestin 2 (+/+) mice, while no differences were observed
in either assay for other CB1 receptor agonists (e.g.,
CP55940, methanandamide). The finding that only THC
activity was influenced by deletion of -arrestin 2 suggests
THC may activate CB1 receptors in a manner that leads to
the recruitment of -arrestin 2 and not -arrestin 1. In another study, mice were chronically treated with THC to induce tolerance to the behavioral effects of THC and the relationship between THC-induced changes in CB1 receptor activity and the levels of GRKs and -arrestins in specific
mouse brain regions were investigated [139]. THC upregulated GRK2, GRK4, and -arrestin 1 levels in striatum,
which suggested these proteins contribute to CB1 receptor
desensitization and endocytosis in this brain region. Chronic
THC treatment increased GRK4 and -arrestin 2 in cerebellum, whereas this treatment increased GRK2 and -arrestin 2
levels in hippocampus. In the striatum and cerebellum, THCinduced up-regulation of GRKs and -arrestins were ERKdependent, because they were prevented in genetic (RasGRF1 knockout mice) and pharmacological (SL327-
Howlett et al.
pretreated mice) models of ERK dysfunction. However, in

the hippocampus, changes in GRKs and -arrestins were
ERK-independent. The findings in cerebellum and striatum
suggest THC-induced ERK activation may play an important
role in CB1 receptor desensitization and the expression of
cannabinoid tolerance.
INTERACTION OF THE CB1 RECEPTOR WITH
ADAPTOR PROTEIN AP-3 AND GASP
Other interacting proteins are also involved in the regulation of CB1 receptor trafficking between plasma membranes
and internal organelles. In a study of CB1 receptors endogenously expressed in Neuro2A neuroblastoma cells [130], a
C-terminal antibody recognized CB1 receptors in intracellular compartments that merged with markers for late endosomes and lysosomes [130]. CB1 receptors co-localized
with the adaptor protein AP-3 as detected in merged confocal images as well as co-immunoprecipitation studies [130].
AP-3 complex (comprised of 3A and B (important for
clathrin binding and sorting of LeuLeu signals), , 3A and
B, and subunits, with B isoforms being brain-specific) is a
ubiquitous adaptor protein complex that responds to acidic
Leu-Leu or Tyr-mediated sorting signals, and is intrinsic to
vesicle budding from late endosomes [140]. No association
of CB1 receptors was observed with AP-2 [130], which is
associated with clathrin-dependent vesicle endocytosis from
plasma membranes trafficking to early endosomes. Subcellular fractionation of Neuro2A cells by differential centrifugation as well as merged confocal micrographs identified CB1
receptors in the same compartment as endosomal markers
AP-3 and Rab7. These data suggested that nascent CB1 receptors might bypass the plasma membrane and be delivered
to the endosomal/lysosomal compartments under certain
circumstances. Following depletion of cellular AP-3 by
siRNA from Neuro2A or cultured hippocampal cells, a significantly greater fraction of CB1 receptors appeared on
punctate domains along the plasma membrane surface, suggesting that one function of the AP-3 complex might be to
divert CB1 receptors away from plasma membrane localization.
It is interesting to speculate on the role that intracellular
CB1 receptors might play in neuronal regulation. Subcellular
fractionation of Neuro2A cells by differential centrifugation
as well as merged confocal micrographs suggested that endosomal CB1 receptors co-localized with Gi and phosphoERK1/2, indicating their potential for functional signal
transduction [130]. At presynaptic terminals, synaptic vesicle
life cycle includes stages of birth by budding from endosomes, neurotransmitter filling of nascent vesicles, docking of filled vesicles at the synaptic active zone, priming,
Ca2+ stimulated fusion and neurotransmitter release, followed by clathrin-dependent endocytosis of fused vesicle
membrane and incorporation into early endosomes for recycling (reviewed by [140]). AP-3B is enriched in brain
clathrin-coated vesicles and endosomes, and is associated
with budding profiles on early-endosomes that traffic to late
endocytic/lysosomal compartments. In neurons, AP-3B is
found in neuronal soma and axonal terminals in both soluble
and membrane-bound forms, where it might serve in selective sorting and transport of cargo from the trans-golgi net-
work to the synapse. Using a PC12 pheochromocytoma cell

model of synaptic-like micro-vesicle formation from endosomes, AP-3 functioned in a brefeldin-A-sensitive, ARF1
(ADP-ribosylation factor binding-protein 1)-dependent, and
clathrin- and dynamin-independent manner to sort AP-3
cargo [141-144]. Thus, one could speculate that the delivery
of CB1 receptors via endosome-derived synaptic vesicles
might serve as a mechanism to place functional CB1 receptors at peri-synaptic membranes in correlation with the rate
of synaptic vesicle release.
G-protein receptor-associated sorting protein 1 (GASP1)
can regulate post-endocytic targeting of the CB1 receptor to
the lysosome for degradation [145,146]. GASP1 could promote trafficking of those CB1 receptors that had been internalized in response to agonist ligands [145], suggesting that
the plasma membrane receptors would have previously been
coupled to heteromeric G-proteins as part of the G-protein
signaling cycle. The association between GASP1 and the
CB1 receptor was demonstrated by co-immunoprecipitation
studies as well as pull-down assays using a glutathione-Stransferase (GST)-CB1-H8 peptide fusion protein [145]. Prolonged (>1 hr) agonist treatment (100 nM WIN55212-2) of
HEK293 cells stably expressing EGFPN-term-CB1 receptors,
resulted in confocal microscopic images of CB1-EGFP that
merged with GASP1 as well as with Lyso-tracker dye, indicating the co-localization of these proteins at the lysosome
[146]. When cultured neurons from neonatal rat spinal cord
were treated with high concentrations of agonist (1.5 M
WIN55212-2), CB1 receptor elimination from soma and neuritic extensions was detectable at 6 hr, continued over the
ensuing 24 hr, and was sustained at about 50% depletion for
the next 24 hr of treatment [146]. This response was attenuated after the spinal neurons had been transduced by viral
delivery of dominant-negative cGASP1-AAV, demonstrating
a role for GASP1 in the down-regulation of CB1 receptors
from their plasma membrane compartments in conditions of
prolonged activation [146].
In in vivo studies in mice, the development of analgesic
tolerance observed after four days of WIN55212-2-treatment
could be attenuated by transduction with the dominant negative cGASP1-AAV, leading these researchers to suggest that
GASP1 functions primarily to promote transit to the
lysosome in conditions of tolerance [146]. A requirement for
GASP1 for down-regulation of CB1 receptors in the dorsal
horn of the mouse spinal cord was observed as a reduction
from about 14% [3H]-CP55940 autoradiographic density
after 7-day treatments with WIN55212-2 to only 5% reduction in the cGASP1-AAV-transduced mice [146]. Additional
evidence from brain striatum has suggested that the role of
GASP1 may be more extensive and complex than simply
delivery of GPCRs to the lysosome for degradation. A
GASP1-knock out mouse on a C57Bl/6 background was
created, and the Bmax for D1-like, D2-like dopamine receptors and muscarinic-like receptors were determined after
cocaine-sensitization or cocaine self-administration protocols
that would increase exposure of these receptors to their endogenous agonists for long periods of time [147]. In contrast
to what would be predicted if GASP1 regulated transit to
lysosomes, the genetic deletion of GASP1 resulted in a decrease in receptor binding maxima in the self-
1387
administering animals, and no differences from WT in the

control and cocaine-sensitized groups [147]. These data coupled with behavioral findings call into question the role that
GASP1 may play in directing these receptors to be degraded,
and suggest that GASP1 may have greater diversity in its
cellular actions. GASP1 interacts with conserved FR residues within H8 in diverse GPCRs [148], many of which interact with G-proteins via the IL3 (as the CB1 receptor does
with Gi1 and Gi2). Because Gi3 and Go utilize the CB1 receptor H8 domain for G-protein-mediated signal transduction, one would expect a competition to exist between
GASP1 versus these G-proteins at the H8 domain of the CB1
receptor.
CANNABINOID
RECEPTOR
INTERACTING
PROTEIN1a (CRIP1a)
The CB1 receptor has recently been shown to interact
with a novel protein, the cannabinoid receptor interactingprotein, CRIP1a/b, within its C-terminus domain. CRIP1a/b
were discovered by the Lewis laboratory, who reported that
deletion of the CB1 receptor C-terminal tail slowed the time
to peak Ca2+ current inhibition, augmented the tonic inhibition of Ca2+ currents, and promoted the ability of the CB1
receptor to sequester G-proteins [54,149]. They hypothesized
that the C-terminal tail could serve an auto-inhibitory function. In seeking an accessory protein to regulate this activity,
they used the CB1 receptor distal C-terminal as bait in a yeast
two-hybrid screen to identify a pair of splice variant proteins,
CRIP1a and CRIP1b [150]. CRIP1a could bind to a GSTCB1-C-terminal tail fusion protein, and could be coimmunoprecipitated with the CB1 receptor [150].
The gene for CRIP1a and CRIP1b, is located on human
chromosome two and consists of 3 coding exons. Both
CRIP1a and CRIP1b have highly conserved sequence homology due to the fact that both proteins are encoded by
exons 1 and 2. However, alternative splicing of exon 3 leads
to the sequence variation between these two isoforms: exon 3
encodes for amino acids 111-128 in CRIP1b and 111-164 in
CRIP1a. Currently, both CRIP isoforms have been cloned
and sequenced for human, rat and mouse, with significant
homology seen between the rodent species. However, an
alternative splicing variation has been found in mouse cerebellar tissue, which like CRIP1a/b is encoded for by exons 2,
which includes an extension of the 3 prime end of exon 2
[150]. Genomic database searching has identified CRIP1a in
all vertebrates, but CRIP1b is only found in primates indicating recent evolutionary processing of this gene [150]. In addition, CRIP1a distribution in mouse brain reveals coexpression with CB1 in excitatory glutamatergic neurons, but
not in inhibitory GABAergic interneurons [151,152].
CB1 receptor mutagenesis studies show that the last 9
amino acids on the C-terminus of CB1 are the minimum residues required for CRIP1a/b binding [150]. Furthermore, both
isoforms require amino acids 34-110 (comprising exons 1
and 2) to interact with the CB1. Because neither of the CRIP
proteins interacts with the CB2 receptor, they possess a
unique and specific means for modulating the physiological
effects mediated through the CB1 receptor. Alternative splicing isoforms that bind to CB2 have yet to be discovered, and
if identified may serve to selectively and differentially alter
CB1 and CB2 signaling.
To date, little is known about the functional relevance of
the CRIP1 family of proteins due to the lack of a threedimensional model and insufficient sequence homology to
other known proteins. A major difference between CRIP1a
and CRIP1b is that CRIP1a contains a palmitoylation site
and a C-terminus PDZ class I ligand [150]. Site specific
palmitoylation is a known modification that many adaptor
and scaffolding molecules undergo and serves to regulate the
function and trafficking of G-proteins, GPCRs and Src family kinases [153]. Proteins that contain PDZ ligands are believed to have a role in determining the cellular distribution
of proteins that possess PDZ domains that recognize them.
The functional relevance of CRIP1a having a PDZ ligand is
intriguing, as it may allow CRIP1a to 1) interact with other
proteins and act as a scaffolding site to establish variations in
signal transduction, 2) enable the formation of
homo/heterodimerization between CB1 and/or other receptors, and 3) modulate CB1 trafficking events such as localization, desensitization, or internalization.
Studies using superior cervical ganglion neurons (SCG)
stably transfected with CB1 receptors have shown that the
subunits of the trimeric G-protein complex can directly interact with N-type Ca2+ channels to inhibit Ca2+ influx [60].
This effect can be reversed by the antagonist/inverse agonist
rimonabant, indicating that CB1 receptors release G
subunits to suppress N-type Ca2+ channel activity. CRIP1a,
but not CRIP1b, attenuated the inhibitory effects of CB1 on
N-type Ca2+ channels [150]. However, WIN55212-2 stimulation of CB1-mediated inhibition of N-type Ca2+ channels was
unaltered by CRIP1a. These results taken together suggest
that CRIP1a/b may functionally modulate CB1 signal transduction in an agonist-independent manner. Additional research to define the effects of CRIP1a/b on other cannabinoid agonist and antagonist-regulated signal transduction
will further delineate these mechanisms.
Many GPCRs can initiate agonist-independent regulation
of signal transduction pathways, in which case ancillary proteins that internally bind to GPCRs can be key modulators of
receptor-mediated events. There is also great variation in the
C-termini of GPCRs, allowing for diverse and differential
interactions, post-translational modifications, and trafficking
seen within this superfamily of transmembrane proteins. Additionally, protein-protein interactions positively modulate
GPCR signaling by influencing ligand-binding affinity and
specificity, coupling between receptors, G-proteins and effectors, or targeting to specific subcellular locations. Receptor-interacting-proteins like Homers and dopamine receptor
interacting proteins (DRIPs) regulate the intracellular activity of GPCRs using various mechanisms. For example, metabotropic glutamate receptors (mGluRs) are known to interact via their C-terminus with a family of proteins called
Homers. Homer proteins have been implicated in a variety of
roles including trafficking of type I mGluRs and receptormediated signal localization [154]. This class of proteins is
subdivided into three families (1, 2, 3), and similar to what is
seen in CRIP1a/b, families 1 and 2 are splice variations of
Howlett et al.
the same gene. For the most part, Homer proteins are constitutively expressed and have two functional sites: an aminoterminal Enabled/Vasodilator-stimulated phosphoprotein
(VASP) homology 1 (EVH1) domain and a coiled-coil
leucine zipper carboxy-terminus. In addition to these two
functional sites, Homers possess a PDZ domain that has been
shown to bind with PSD-95, GKAP and Shank1 and 3 [155].
One important aspect on mGluR receptors is their ability to
function as dimers, and therefore, the Homer family of proteins may participate in this process [156]. Homers are also
known to form a complex between mGluRs and IP3 receptors [154]. Thus, if CRIPs exhibit functional homology to
Homer proteins, then CRIPs may play a pivotal role in localization of the CB1 receptor to its signaling partners, such as
voltage-gated Ca2+ channels and G-protein linked K+ channels. However, unlike CB1 receptors, the majority of
mGluRs are located at post synaptic density areas where
Homers can be up-regulated by neuronal activity [154]. An
example of this is that expression of Homer 1a is induced
upon neuronal excitation [157], and due to the lack of a binding domain in its C-tail, can act as a dominate-negative protein by disrupting interactions between mGluR receptors and
other proteins. In HEK293 cells, expression of Homer1a, but
not its isoform Homer-1b, resulted in increased mGluR
translocation to the plasma membrane. Studies suggest that
Homer 1b influences mGluR 1 and 5 retention in intracellular stores until the up-regulation of Homer 1a evokes transit
of receptors to the plasma membrane [158]. Because Homer
1a and CRIP1b both lack functional domains in their C-tail,
they may perform similar functions as dominant negative
regulators.
Like CB1 receptors, D2 dopamine receptors are GPCRs
that are predominately expressed on presynaptic terminals,
couple to Gi/o proteins, and initiate similar signal transduction pathways. D2 receptors associate with interactingproteins (DRIPs) that bind to the C-terminus of the receptor.
The D2 receptor DRIP neuronal Ca2+ sensor 1 (NCS-1) inhibits D2 receptor desensitization in a Ca2+-dependent fashion by binding to GRK2 and preventing its phosphorylation
of the D2 receptor [159]. Because CRIP1a/b bind to a domain
near the CB1 receptor internalization site, CRIP may serve a
similar function as NCS-1. To date, other dopamine receptor
interacting-proteins (calcyon and DRIP78) have also been
identified. The endoplasmic reticulum membrane-bound
protein DRIP78 regulates the transport of many GPCRs (D1,
M2, AT1), via their C-tail, to the plasma membrane [160].
Both sequestration and overexpression of DRIP78 results in
D1 and 2AR localization in the endoplasmic reticulum,
along with reduced ligand binding and receptor glycosylation
[161].
The further identification of CRIP proteins as well as
their functional importance will provide a greater understanding into the regulation and neurotransmission of cannabinoid receptors. Because CRIP1a colocalizes with CB1
signaling at excitatory synapses, it is a novel target for the
treatment of disorders associated with excessive excitatory
transmission, such as epilepsy [152]. Thus it will be important to continue research into the CRIP proteins to elucidate
the mechanisms involved in CRIP1a/b modulation of CB1
receptors and the opportunity that this offers in the development of cannabinoid based drugs.

FACTOR ASSOCIATED WITH NEUTRAL SPHINGOMYELINASE (FAN)
Signal transduction via regulation of the second messenger ceramide can be regulated by CB1 receptors either via
sphingomyelin hydrolysis or by de novo synthesis of ceramide (see review [162,163]). To summarize, transient increases in intracellular ceramide are regulated by the interaction of the CB1 receptor with neutral sphingomyelinase via
the interacting protein FAN. Once released into the cell, this
short-lived production of ceramide can regulate cellular
metabolic processes. The enzymatic production of ceramide
de novo is under the CB1 receptor regulation of serine palmitoyltransferase or ceramide synthase, and initiates signaling
pathways leading to cyclooxygenase-2 expression and apoptosis in cells that are susceptible to this mediator [164-166].
FAN directs the agonist-stimulated CB1 receptor to the
regulation of sphingomyelin hydrolysis, leading to the release of ceramide [167]. In an astrocyte model, CB1 agonists
promoted a complex of FAN with CB1 receptors, and this
could be blocked by the CB1 antagonist rimonabant, but not
by pertussis toxin treatment, indicating that Gi/o proteins
were not intrinsic to the process [167]. The requirement for
FAN is based upon the attenuation of the ceramide generation in cells that express a dominant negative form of this
protein [167]. CB1 receptors and FAN could coimmunprecipitate, demonstrating their interaction as a complex. Studies based upon FAN interactions with other proteins suggest that the C-terminal of the CB1 receptor is the
site of a putative neutral sphingomyelinase activation domain
[167]. Interestingly, FAN is a WD40 repeat protein, as is the
G subunit of G-proteins and many other adaptor proteins
[163]).
Sphingomyelin hydrolysis in cultured astrocytes or
glioma cells yielded an increase in intracellular ceramide
within 15 min [87,168]. CB1-mediated release of ceramide
activates the Raf-1-MEK-ERK pathway to regulate glucose
metabolism [86]. The Guzman laboratory has speculated that
this ceramide-signaling pathway may serve an important
function of astrocytes to supply metabolic substrates to neurons for oxidative metabolism at synapses and other sites
having rapid biosynthetic requirements [163].
PERSPECTIVES
The majority of the data regarding cellular mechanisms
for CB1 receptor actions have been based upon experiments
performed in the brain or in neuronal models (Table 1). As
we now are learning about CB1 receptors in many other cell
types, it is important to recognize that cellular signal transduction for highly specialized cells may deviate from what
we have learned regarding neurons. Neurons are excitable
cells, and are structurally unique in their cell-cell interactions
(i.e., they are polarized in the structure of axons and dendrites, and form unique sites of interaction, the synapse).
Other cell types possess their unique abilities to respond to
hormones and other mediators to regulate their specific functions within a tissue or organ. While we can use our present
knowledge as a guide, we certainly cannot expect that all cell
types will respond to CB1 receptor stimulation via activation
1389
of the same signaling pathways. This notion of diversity

leads us to speculate that agonists and allosteric modulators
might be discovered or developed that can trigger the regulation of unique, cell-specific responses.
CONFLICT OF INTEREST
The authors have not received financial contributions to
the work being reported, and do not report any conflict of
interest. ACH has served on the speakers bureau for SanofiAventis within the last three years.
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Received: December 01, 2009
Revised: February 16, 2010
Accepted: February 18, 2010

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1382

Current Medicinal Chemistry, 2010, 17, 1382-1393

CB1 Cannabinoid Receptors and their Associated Proteins

Keywords: Anandamide (arachidonylethanolamide), AP-3, 2-arachidonoylglycerol (2-AG), -arrestin, CP55940, CRIP-1a,

nisms underlying these behavioral responses, we now know

CB1 Cannabinoid Receptors and their Associated Proteins

CB1 RECEPTOR CELLULAR SIGNALING VIA GPROTEINS

Current Medicinal Chemistry, 2010 Vol. 17, No. 14

cAMP-PROTEIN KINASE A (PKA) SIGNALING

1384 Current Medicinal Chemistry, 2010 Vol. 17, No. 14

ase and the activity of a vascular endothelial growth factor

CB1 RECEPTOR INTERACTION WITH PROTEINS

CB1 Cannabinoid Receptors and their Associated Proteins

Current Medicinal Chemistry, 2010 Vol. 17, No. 14

Role of CB1 Receptor-Associated Proteins in Protein Trafficking and Signal Transduction

Site of CB1 interaction

Role in Protein Trafficking

C-terminal side of Intracellular Loop 3

N-terminal side of Intracellular Loop 3

C-terminal side of Intracellular Loop 3

Role in Signal Transduction

Ca2+ channel inhibition

Plasma membrane internalization

[133, 135, 137,

Gi- and ERK-associated

Leu-Leu or Tyr signals

Late endosome budding to

Distal C-terminal tail

Ceramide-mediated Raf1-MEKERK; stimulation of metabolic

[86-87, 162163, 167-168]

1386 Current Medicinal Chemistry, 2010 Vol. 17, No. 14

dues 460-473) of the rat CB1 receptor contains a cluster of

pretreated mice) models of ERK dysfunction. However, in

CB1 Cannabinoid Receptors and their Associated Proteins

work to the synapse. Using a PC12 pheochromocytoma cell

Current Medicinal Chemistry, 2010 Vol. 17, No. 14

administering animals, and no differences from WT in the

1388 Current Medicinal Chemistry, 2010 Vol. 17, No. 14

CB1 Cannabinoid Receptors and their Associated Proteins

INTERACTION OF THE CB1 RECEPTOR WITH

Current Medicinal Chemistry, 2010 Vol. 17, No. 14

of the same signaling pathways. This notion of diversity

Howlett, A. C.; Bidaut-Russell, M.; Devane, W. A.; Melvin, L. S.;

1390 Current Medicinal Chemistry, 2010 Vol. 17, No. 14

CB1 Cannabinoid Receptors and their Associated Proteins

Current Medicinal Chemistry, 2010 Vol. 17, No. 14

1392 Current Medicinal Chemistry, 2010 Vol. 17, No. 14

migration and morphogenesis by transactivating the TrkB receptor.

Rameau, G. A.; Tukey, D. S.; Garcin-Hosfield, E. D.; Titcombe, R.

CB1 Cannabinoid Receptors and their Associated Proteins

tively enhanced in beta-arrestin2 -/- mice. Behav. Pharmacol.,

Received: December 01, 2009

Revised: February 16, 2010

Accepted: February 18, 2010

Current Medicinal Chemistry, 2010 Vol. 17, No. 14

Resh, M. D. Palmitoylation of ligands, receptors, and intracellular

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