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The Journal of Neuroscience, May 15, 1999, 19(10):3773-3780
Distinct Domains of the CB1 Cannabinoid Receptor Mediate
Desensitization and Internalization
Wenzhen
Jin1,
Sean
Brown2,
John P.
Roche2, 3,
Candace
Hsieh2,
Jeremy P.
Celver1,
Abraham
Kovoor1,
Charles
Chavkin1, and
Ken
Mackie2, 3
Departments of 1 Pharmacology,
2 Anesthesiology, and 3 Physiology and
Biophysics, University of Washington, Seattle, Washington
98195-6540
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ABSTRACT |
Desensitization of cannabinoid receptor signaling by a G-protein
coupled receptor kinase (GRK) was examined using the
Xenopus oocyte expression system. Application of a CB1
agonist, WIN 55,212-2, evoked a concentration-dependent increase in
K+ conductance (Kir3) in oocytes
coexpressing rat CB1 with the G-protein-gated, inwardly rectifying
K+ channels Kir3.1 and
Kir3.4. Desensitization was slight during continuous
agonist application in the absence of GRK and arrestin. However,
coexpression of GRK3 and  -arrestin 2 (-arr2) caused profound
homologous CB1 receptor desensitization, supporting the hypothesis that
GRK3 and -arr2 effectively produce CB1 receptor desensitization. To
identify the regions of the CB1 receptor responsible for GRK3- and
-arr2-mediated desensitization, we constructed several CB1 receptor
mutants. Truncation of the C-terminal tail of CB1 receptor at residue
418 ( 418) almost completely abolished desensitization but did not
affect agonist activation of Kir3. In contrast, truncation
at residues 439 and 460 did not significantly affect GRK3- and
-arr2-dependent desensitization. A deletion mutant (418-439) did
not desensitize, indicating that residues within this region are
important for GRK3- and -arr2-mediated desensitization.
Phosphorylation in this region was likely involved in desensitization,
because mutation of either of two putative phosphorylation sites (S426A
or S430A) significantly attenuated desensitization. CB1 receptors
rapidly internalize after activation by agonist. Phosphorylation of
S426 or S430 was not necessary for internalization, because the
S426A/S430A CB1 mutant internalized when stably expressed in AtT20
cells. These studies establish that CB1 desensitization can be
regulated by a GRK and that different receptor domains are involved in
GRK- and -arrestin-dependent desensitization and CB1 internalization.
Key words:
cannabinoid; desensitization; inwardly rectifying
potassium channel; G-protein-coupled receptor; -arrestin; G-protein
coupled receptor kinase; phosphorylation
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INTRODUCTION |
Cannabinoids produce their
characteristic behavioral effects as a consequence of binding to a
G-protein-coupled receptor, the CB1 cannabinoid receptor (Matsuda et
al., 1990 ; Matsuda, 1997). The abundance of these receptors and the
discovery of several endogenous ligands (Devane et al., 1992; Stella et
al., 1997) suggest that an endogenous cannabinoid neuromodulatory
system serves an important physiological role (DiMarzo et al.,
1994). Cellular consequences of CB1 receptor activation include
inhibition of adenylyl cyclase, activation of mitogen-activated
protein kinase, and modulation of ion channels (Pertwee, 1993).
The CB1 receptor activates at least two classes of potassium channels,
the voltage-dependent potassium A current and inwardly rectifying
potassium channels (GIRK or Kir3 channels) (Matsuda, 1997).
Potassium channels often control the resting membrane potential of
neurons and play a major role in determining excitability. Previous
studies from our laboratories demonstrated that CB1 receptor activated
Kir3 or GIRK channels in Xenopus oocytes (Henry
and Chavkin, 1995) and the corticotroph-like cell line AtT20 (Mackie et
al., 1995; Garcia et al., 1998), suggesting that activation of these
channels may be a key effector mechanism for cannabinoid action.
Tolerance develops rapidly during the chronic administration of
cannabinoids. Receptor desensitization or uncoupling has been consistently implicated as one of the molecular events underlying the
onset of tolerance in many systems (Appleyard et al., 1997; Kovoor et
al., 1997; Smith et al., 1988). Desensitization of G-protein-coupled receptors (GPCRs) is often associated with phosphorylation of the
receptor by G-protein-coupled receptor kinases (GRKs), followed by
binding of -arrestin (-arr) and a reduction in affinity for G-proteins (Zhang et al., 1997; Krupnick and Benovic, 1998). This sequence of events effectively attenuates signaling by the GPCR and its
ligand. Recent studies have demonstrated that GRK and -arr are
required for  opioid receptor desensitization (Kovoor et al., 1997).
-Arr also may serve other roles in the regulation of GPCR signaling.
For example, -arr has been proposed to function as an adaptor
between phosphorylated 2-adrenergic receptors and clathrin-coated endocytic pits, thus directing phosphorylated receptor
to this endocytotic pathway (Lin et al., 1997). CB1 receptor exhibits
agonist-induced receptor internalization (Hsieh et al., 1999). However,
the mechanisms underlying these phenomena for CB1 receptors have not
been elucidated. In particular, it is unknown whether -arr is
involved in CB1 internalization. In the present study we used the
Xenopus oocyte expression system and AtT20 cells stably
expressing CB1 receptors to study CB1 receptor coupling to
G-protein-gated, inwardly rectifying K+ channels.
CB1-expressing AtT20 cells were also used to study the
relationship between desensitization and internalization of the CB1
receptor. We determined that GRK3 and -arr2 were able to mediate
agonist-dependent CB1 receptor desensitization, and we found that
distinct domains of the CB1 receptor were involved in desensitization
and internalization.
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MATERIALS AND METHODS |
CB1 receptor mutagenesis. Amplicons for CB1 receptor
truncation mutants 418, 439, and 460 were produced by PCR as
follows. One nanogram of rat CB1 template (Mackie et al., 1995), 60 pmol of dNTPs (Life Technologies, Grand Island, NY), 4.0 U of
Pfu polymerase (Stratagene, La Jolla, CA), and 25 pmol of
each primer (Table 1; Life Technologies)
were combined in a final reaction volume of 100 µl. PCR was performed
in a DNA thermal cycler (model 480; Perkin-Elmer, Foster City, CA) for
28 cycles with annealing temperatures appropriate for the
oligonucleotide sequences. The CB1 sense primer (Table 1) was used for
all mutants in combination with the appropriate antisense primer (Table
1).
PCR products were ethanol-precipitated, pelleted, washed in 70%
ethanol, vacuum-dried, resuspended in 10 mM Tris and 0.1 mM EDTA, pH 8.0 (T1/10E) buffer, and gel-purified on 1%
Seaplaque agarose (FMC Bioproducts, Rockland, ME). Amplicons detected
by ethidium bromide staining were excised and purified with 0.22 µm
Micropure separators (Amicon, Beverly, MA). Eluted DNA was phenol-chloroform-extracted and ethanol-precipitated.
CB1 receptor mutants T419A, S426A, S430A, S426A/S430A and G418-N438
were produced by overlap extension of the PCR (Ho et al., 1989) using
wild-type rat CB1 as template. For each mutant, two rounds of PCR were
performed. In the first round, two separate reactions were performed.
In the first the primers were CB1 sense and the mutant antisense (Table
1). In the second the primers were the mutant sense and CB1 antisense
(Table 1). CB1 receptor mutant S426A/S430A was used as starting
template in overlap-extension of the PCR to produce CB1 receptor
mutant T419A/S426A/S430A.
Amplicon templates produced with Pfu polymerase in the first
round of PCR were joined in overlap-extension of the PCR with 2.5 U of
Taq DNA polymerase (Qiagen, Valencia, CA) using CB1 sense and antisense primers (Table 1). Purified PCR products and vector pcDNA3 (Invitrogen, Carlsbad, CA) were digested with BamHI
and EcoRI restriction endonucleases (New England Biolabs,
Beverly, MA), phenol-chloroform-extracted, ethanol-precipitated,
pelleted, washed in 70% ethanol, vacuum-dried, and resuspended in
T1/10E. The vector was dephosphorylated with shrimp alkaline
phosphatase (Boehringer Mannheim, Indianapolis, IN) and gel-purified as
above. Inserts and vector were ligated with T4 DNA ligase (New England Biolabs) overnight at 16°C. Orientation of the inserts allowed the T7
promoter to direct RNA transcripts (see below). Competent XL-1 Blue
Escherichia coli (Stratagene) was transformed and plated on
LB-amp-tet agar plates. Plasmid DNA was isolated from broth cultures
started from amp-tet-resistant bacterial colonies (Qiagen), and the
plasmid DNA was screened by BamHI and EcoRI
restriction endonuclease (New England Biolabs) digestion. Mutations
were confirmed by automated DNA sequencing (Applied Biosystems,
Perkin-Elmer).
cDNA clones and cRNA synthesis. cDNA for Kir3.1
channel was obtained from Dr. Henry Lester (California Institute of
Technology, Pasadena, CA) (GenBank accession number U01071).
Kir3.4 [clone provided by Dr. John Adelman (Vollum
Institute, Portland, OR), GenBank accession number X83584] and
-arr2 cDNA [clone provided by Dr. Robert Lefkowitz (Duke
University, Durham, NC), GenBank accession number M91590] was first
amplified by the utilization of Amplitaq DNA Polymerase (Perkin-Elmer
Cetus, Norwalk, CT) in a standard PCR using oligonucleotides designed
to add a T7 promoter region and a 45 base poly A tail. The rat GRK3
cDNA was provided by Dr. Shaun Coughlin (University of California at
San Francisco, San Francisco, CA) (Vu et al., 1991). Plasmid templates
were linearized before cRNA synthesis, and mMESSAGE MACHINE kit
(Ambion) was used to generate capped cRNA.
Oocyte culture and injection. Oocytes were prepared as
described (Kovoor et al., 1995) and were incubated in ND96 (96 mM NaCl, 2 mM KCl, 1 mM
MgCl2, 1 mM CaCl2,
and 5 mM HEPES, pH 7.5) solution supplemented with sodium
pyruvate (2.5 mM) and gentamycin (50 µg/ml). cRNA was
injected into oocytes (50 nl/oocyte) with a Drummond microinjector.
cRNAs injected into each oocyte were as follows: CB1 receptor and its
mutants, 2.5-4 ng; GRK3, 0.5 ng; -arr2, 5 ng; GIRK
Kir3.1 and Kir3.4, 0.02 ng each; and opioid
receptor, 0.4 ng. All recordings were performed 2-4 d after injection.
Oocyte electrophysiology. Oocytes were clamped at  80 mV
with two electrodes filled with 3 M KCl having resistances
of 0.5-1.5 m using a Geneclamp 500 amplifier and pCLAMP 6 software
(Axon Instruments, Foster City, CA). All data were digitally recorded (Digidata, Axon Instruments, and Intel 386PC) and filtered at 500 HZ.
Membrane current traces were also recorded using a chart recorder. To
measure inwardly K+ currents flowing through the
Kir3 channels, K+ concentration in the
oocyte saline buffer was increased from 2 to 16 mM. The
concentration of NaCl was decreased to maintain iso-osmolality.
AtT20 cell electrophysiology. Kir currents were
recorded from AtT20 cells stably expressing wild-type or 418
truncated CB1 receptors as previously described (Mackie et al., 1995;
Garcia et al., 1998). In the whole-cell configuration of the patch
clamp (Hamill et al., 1981), cells were held at 45 mV and
hyperpolarized to 100 mV for 50 msec every 5 sec. The extracellular
potassium concentration was 40 mM, and the Kir
current was defined as the component of the current blocked by 1 mM Ba2+. The average current during the
hyperpolarization was determined and plotted versus time.
Internalization. The CB1 receptor mutant S426A/S430A was
stably expressed in AtT20 cells (Mackie et al., 1995). Cells were stimulated and CB1 receptors were detected with a CB1-specific antibody
in fixed cells with confocal microscopy as previously described (Hsieh
et al., 1999).
Statistical analysis. The Student's t test was
used for comparison of independent means. Statistical significance was
defined as p < 0.05. Data from the dose-response
experiments were fitted to a simple Emax model using the nonlinear
regression analysis package NFIT to determine agonist EC50
and 95% confidence intervals (95% CI).
Chemicals. [D-Pen2,5]enkephalin
was from Peninsula Laboratories (San Carlos, CA). WIN 55,212-2 was from
Research Biochemicals International (Natick, MA). SR 141716A was from
the National Institute on Drug Abuse drug supply program (Research
Triangle Park, NC).
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RESULTS |
To characterize CB1 receptor coupling to G-protein-gated inwardly
rectifying K+ channels, cRNAs for CB1 receptor
Kir3.1 and Kir3.4 channel subunits were
injected into Xenopus oocytes. As shown in Figure
1A, basal inward
currents were observed when the KCl concentration increased from 2 to
16 mM in normal oocyte saline buffer. Superfusion of WIN
55,212-2 (WIN, 1 µM) further increased the inward
current. The current activated by WIN showed inward rectification (Fig. 1B). The WIN-evoked response was
concentration-dependent, with an EC50 of 55 nM
(45-64 nM, 95% CI; Fig. 1C). Activation of the inwardly rectifying current by WIN was completely reversed by application of the competitive antagonist SR 141716A (1 µM) (Fig. 1A). In the oocytes
expressing only CB1 receptor and Kir3.1 and Kir3.4, modest desensitization (25 ± 3.3%;
n = 9) was observed after prolonged (8 min) perfusion
of 1 µM WIN (Fig. 1A). In this study,
the amount of desensitization was defined as the percent decrease in
WIN activation of the Kir3 current after 8 min of WIN
application.
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Figure 1.
Coupling of the CB1 receptor to the
G-protein-activated inward rectifier potassium channels
Kir3.1 and Kir3.4. A,
Representative trace showing the change in current during a typical
experiment. A large inward current was apparent as the
K+ concentration was increased from 2 to 16 mM in normal oocyte saline buffer. WIN (1 µM)
in the buffer (16 mM K+) further
increased the current. To detect any change in basal current after the
agonist treatment (8 min), SR 141716A (1 µM), a CB1
receptor antagonist, was applied to displace WIN and reverse
CB1-mediated activation of the current. The interpolated decrease in
baseline current was plotted, as shown by the dashed
line. The amount of desensitization was calculated as the
percent change in response to WIN after 8 min. Current traces presented
in subsequent figures show only the agonist-activated current adjusted
for the change in baseline. B, WIN activation of
inwardly rectifying K+ channels. The
I-V relationship was generated by steps from 140 to
40 mV after subtracting the current at the same potential in 16 mM K+ buffer. C,
Concentration-response curve of WIN. Cumulatively higher
concentrations of WIN were applied to the bath followed by perfusion
with SR 141716A (1 µM). The agonist response at each
concentration was normalized as a percentage of the maximal WIN
response. Each point represents the mean response
measured in 10-14 different oocytes.
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To examine whether GRK and -arr enhanced CB1 receptor
desensitization, GRK3 and -arr2 were coexpressed with CB1 receptor and Kir3.1 and Kir3.4 channels. As shown above,
treatment with WIN for 8 min only produced a very small desensitization
in the absence of GRK3 and -arr 2 (25 ± 3.3%). Expression of
CB1 and Kir3.1 and Kir3.4 along with either
GRK3 or -arr2 alone did not significantly increase the
desensitization rate (Fig.
2A). However, coexpression both GRK3 and -arr2 caused profound agonist-dependent desensitization (63 ± 6.1%; n = 8) (Fig.
2A). These results suggested that GRK3 and -arr2
were sufficient to induce CB1 receptor desensitization. To determine
whether GRK3 and -arr2 target the CB1 receptor, we next tested
whether the GRK3- and -arr2-mediated desensitization was homologous
or heterologous. In the oocytes that were also injected with cRNA for
the opioid receptor, brief application of 1 µM DPDPE
(a opioid agonist) elicited a large inward current, 336 ± 39 nA (n = 10; Fig.
3A). During perfusion with
WIN, prominent desensitization of the CB1-mediated response was
apparent (Fig. 3A,B). However, the amplitude of the current
activated by a subsequent application of DPDPE was identical to the
DPDPE-induced current in non-WIN-55,212-2-treated oocytes (Fig.
3A,B). These data indicate that GRK3 and -arr2 mediate
homologous CB1 receptor desensitization, suggesting that they target
CB1 receptors rather than a common downstream effector, such as the
channel, G-protein, or accessory proteins.
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Figure 2.
GRK3 and -arr2 were required for CB1 receptor
desensitization. A, Representative traces showing that
the coexpression of GRK3 and -arr2 significantly increased
desensitization of Kir current activated by WIN. Top
left, Oocytes injected with mRNA for CB1 and
Kir3 show modest desensitization during an 8 min exposure
to 1 mM WIN 55,212-2. Top right, Addition of
GRK3 mRNA to the injection mix does not enhance desensitization.
Bottom left, Addition of -arr2 mRNA also does not
enhance desensitization. Bottom right, Addition of both
GRK3 and -arr2 mRNA to the injection mix significantly enhances
desensitization. The short vertical lines through the
first trace indicate the time of buffer switch from WIN
to SR 141716A perfusion. Response was adjusted by baseline subtraction.
B, Summary of data. Data are mean ± SEM;
**p < 0.05 compared with oocytes not coexpressing
GRK3 and -arr2.
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Figure 3.
GRK3 and -arr2 mediated homologous CB1 receptor
desensitization. A, Representative traces show that
current activated by WIN desensitized during prolonged treatment,
whereas the DPDPE-mediated response was unaffected. OR,
Opioid receptor; SR, SR 141716A. B,
Summary of data. Open bars, Current at initial agonist
application; filled bars, represent the current elicited
by the indicated agonist after WIN application. DPDPE-elicited current
was not affected by an intervening application of WIN, whereas the
WIN-elicited current strongly desensitizes. Data are presented as
mean ± SEM; **p < 0.05 (WIN-elicited current
at the beginning vs end of WIN application).
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It is believed that phosphorylation of serine and threonine residues in
either the third cytoplasmic loop or the C-terminal tail by GRK is the
mechanism of GRK- and arrestin-mediated desensitization of many GPCRs
(Freedman and Lefkowitz, 1996). To identify regions of the CB1 receptor
required for desensitization, we first constructed several receptor
mutants by successively shortening the CB1 receptor C-terminal tail
(Fig. 4A). Truncation
at residues 439 and 460 did not significantly affect WIN activation of
Kir3 current activation or the GRK3- and -arr2-mediated
desensitization observed (Fig. 4A,B). In a manner
similar to wild-type CB1 receptors, Kir3 activation by CB1
mutants 439 and 460 significantly desensitized (82 ± 5%;
n = 11; and 63 ± 6%; n = 8, respectively). However, truncation at residue 418 caused a dramatic
attenuation of desensitization (19 ± 3%; n = 22;
Fig. 4A,B). To determine whether the lack of desensitization in the mutant 418 was attributable to gross
alteration of receptor properties, concentration-response curves for
the 418 mutant and wild-type receptor were generated (Fig.
4C). The EC50 for WIN was similar for both
wild-type CB1 and 418 mutant (55 and 36 nM,
respectively). This result indicates that 418 mutant activation of
Kir3 currents was similar to activation by wild-type CB1
receptor. Because only slight desensitization was observed with the
418 mutant, the 20 amino acid residues between 418 and 439 are
likely to be critical for GRK- and -arr2-mediated CB1 receptor
desensitization. To test this hypothesis, we constructed a deletion
mutant (418-439) with these 20 amino acids removed. Indeed, this
deletion mutant behaved similarly to 418, showing little GRK3- and
-arr2-mediated desensitization (15 ± 6%; n = 7; Fig. 4A,B).
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Figure 4.
The region between G418 and N419 in the CB1
receptor was critical for GRK3- and -arr2-mediated CB1 receptor
desensitization. A, Schematic of the rat CB1 receptor.
Short vertical lines indicate the sites of truncation or
deletion. Representative traces show that truncation mutant 418 and
deletion mutant (418-439) eliminated GRK3- and -arr2-mediated
desensitization. B, Summary of data. Data were collected
from at least three separate experiments with three different oocyte
donors. C, Concentration response curves of WIN in
wild-type CB1 receptor and mutant (G418). Cumulatively increasing
concentrations of WIN were applied to the bath followed by perfusion
with SR 141716A. Oocytes were injected with cRNA for Kir3.1
and Kir3.4 and CB1 receptors. Each point
represents the mean response measured in seven oocytes.
wt, Wild type.
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The oocyte system offers the convenience of allowing rapid screening of
receptor mutants. However, we also wanted to determine whether
Kir current activation desensitized in an excitable cell constitutively expressing Kir channels, without exogenous
-arr2 or GRK. In addition we wished to determine whether the domains of the receptor critical for desensitization were also involved in
agonist-induced internalization (below). Because we measure internalization in AtT20 cells stably transfected with CB1 receptors, it was important to determine whether activation of Kir by
cannabinoids in these cells desensitized in a manner similar to that in
Xenopus oocytes. For both these reasons we stably expressed
CB1 and the CB1 truncation mutant 418 in AtT20 cells. We then
determined the degree of desensitization of the current during 1-2 min
of agonist exposure. Figure 5A
shows that for an individual cell expressing wild-type CB1, the current
activated by 200 nM WIN 55,212-2 rapidly desensitizes. In
contrast, the current activated in cells expressing the CB1 truncation
418 shows little desensitization (Fig. 5B). Figure 5,
C and D, shows the current amplitude before and
during WIN 55,212-2 application as a percent of the maximum current for
several cells. The current activated by WIN 55,212-2 in cells
expressing wild-type CB1 declines with a  of ~30 sec (temperature,
22-23°), whereas the current activated in cells expressing the
418 truncation shows no detectable decrease during this time. These
results suggest that similar regions in CB1 are involved in
desensitization of Kir current in AtT20 cells and oocytes.
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Figure 5.
The C terminus of CB1 is also required for
desensitization of GIRK activation in AtT20 cells. A, In
AtT20 cells stably expressing rat CB1 receptors, 200 nM WIN
55,212-2 activates an inward current that rapidly desensitizes.
B, In AtT20 cells stably expressing the CB1 truncation
mutant G418, 200 nM WIN 55,212-2 activates an inward
current, but this current shows little desensitization.
C, Aggregate data showing the rapid desensitization of
inward current activated by WIN 55,212-2 in cells expressing rat CB1
(n = 5). D, Aggregate data showing
no desensitization of the inward current activated by WIN 55,212-2 in
cells expressing the CB1 truncation G418 (n = 10).
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Was desensitization attributable to the phosphorylation of residues
between residues 418 and 439 in CB1 receptor? There are three possible
phosphorylation sites (T419, S426, and S430) in this region (Fig.
6A). To test whether
phosphorylation of any of these three sites was necessary for
desensitization, we constructed five mutants in which combinations of
the sites were mutated to alanine as diagrammed (Fig.
6A) Mutation of T419 to alanine did not prevent
desensitization (Fig. 6B), suggesting phosphorylation at this site by GRK3 is not critical. In contrast, the single mutation
of either S426A or S430A significantly attenuated the desensitization
(S426A, 23 ± 4%; S430A, 26 ± 3.5). A similar low degree of
desensitization was observed with the double mutant S426A/S430A
(25 ± 3%; Fig. 6). Our data indicate that phosphorylation of
both S426 and S430 of the CB1 receptor by GRK3 may be responsible for
its homologous desensitization by GRK3 and -arr2.
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Figure 6.
Phosphorylation of serine 426 and serine 430 in
the C terminus of CB1 likely underlies GRK3- and -arr2-mediated CB1
receptor desensitization. A, Schematic representation of
rat CB1 receptor. The amino acid sequence between G418 and N439 is
shown using single-letter amino acid abbreviations. Five mutants were
constructed based on sequentially substituting serine and threonine
residues with alanine in this region. B, Summary data
showing that single mutation of S426 or S430 but not T419 effectively
blocks GRK- and -arr-mediated desensitization. Data were collected
from at least three separate experiments with three different oocyte
donors. Data are means ± SEM.
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We next determined the role of S426/S430 in agonist-induced
internalization of the CB1 receptor. When expressed in AtT20 cells, CB1
receptors readily internalize in response to agonist (Hsieh et al.,
1999). In cells expressing wild-type CB1 receptors, the receptor is
primarily found at the cell surface (Fig.
7A). When these cells are
stimulated with 100 nM WIN 55,212-2 for 30 min, CB1
receptors internalize (Fig. 7B). Previously, we found that residues 460-464 of the receptor were required for internalization (Hsieh et al., 1999). We now wanted to determine whether
phosphorylation of S426 or S430 was also required. That is, were the
residues phosphorylated by GRK3, and presumably involved in
-arrestin 2 binding, necessary for agonist-induced internalization
of the CB1 receptor? In AtT20 cells stably expressing the CB1
S426A/S430A mutant, the receptor was found at the plasma membrane (Fig.
7C). However when these cells were stimulated with 100 nM WIN for 30 min, substantial internalization of the
receptor was evident (Fig. 7D). Thus in AtT20 cells,
phosphorylation of the CB1 receptor at S426 and S430 is not required
for its internalization. These results suggest that different receptor
domains are required for GRK/-arr-dependent desensitization and
receptor internalization.
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Figure 7.
S426 and S430 are not required for CB1 receptor
internalization. A, In unstimulated AtT20 cells stably
expressing wild-type CB1 receptor, the receptor is primarily found on
the cell surface (arrows). B, After 30 min stimulation with 100 nM WIN 55,212-2, CB1 receptors are
predominantly intracellular (arrowheads).
C, In unstimulated AtT20 cells stably expressing the
mutant CB1 receptor S426A/S430A, the receptor is primarily found on the
cell surface (arrows). After 30 min stimulation with 100 nM WIN 55,212-2, CB1 receptors are internalized, as in
cells expressing the wild-type receptor
(arrowheads).
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DISCUSSION |
The principal finding of this study is that GRK3 and -arrestin
can cause profound desensitization of CB1 cannabinoid receptor-mediated activation of Kir3 channels. Using Xenopus
oocytes and a mutagenic strategy, we defined a region (residues
418-439) of the CB1 receptor critical for GRK3- and -arr2-mediated
desensitization. It is likely that GRK3 phosphorylation of the CB1
receptor in this region underlies desensitization, because mutating
either of two serines (S426 or S430) in this region eliminates
desensitization. Furthermore, phosphorylation of this region of the
receptor does not seem to be involved in agonist-induced
internalization in AtT20 cells, because internalization proceeds
normally when both S426 and S430 are mutated to alanine. These results
also suggest that a -arrestin interaction with phosphorylated S426
and S430 is not required for CB1 internalization. The results of this
study, combined with those of our earlier work, clearly demonstrate
that distinct domains of the CB1 receptor are involved in CB1 receptor
internalization and desensitization.
Sustained administration of cannabinoids leads to rapid development of
tolerance in both animals and humans (Abood and Martin, 1992; Martin et
al., 1994). Tolerance does not involve changes in pharmacokinetics
(Dewey, 1986). It also does not correlate with changes in
receptor density (Abood and Martin, 1992; Pertwee, 1997).
Chronic administration of -(9)-tetrahydrocannabinol leads to
an uncoupling of CB1 receptors from G-proteins, as measured by a
decrease in WIN 55,212-2-stimulated GTP S binding (Sim et al., 1996).
Our results suggest a possible mechanism for this uncoupling, namely
the phosphorylation of the receptor by a G-protein-coupled receptor kinase.
At least two families of protein kinases, GRKs and second
messenger-dependent kinases, are involved in phosphorylation of GPCRs
(Freedman and Lefkowitz, 1996). G-protein-coupled receptor kinases
specifically phosphorylate agonist-activated receptors, facilitating
the binding of an inhibitory protein (arrestin) to the phosphorylated
receptor, thereby uncoupling the receptor from its G-protein(s) and
inducing receptor-specific desensitization (homologous
desensitization). Desensitization of several GPCRs [2-adrenergic receptors (AR),  1-AR,
2-AR, and m2-muscarinic, thrombin, and
opioid receptors, among others] involves GRK-mediated phosphorylation
(Inglese et al., 1993; Freedman and Lefkowitz, 1996; Kovoor et al.,
1997). When determined, the sites of phosphorylation are localized to
serines and threonines in the carboxyl-terminal tail or the third of
intracellular loop (Bouvier et al., 1988; Dohlman et al., 1987; Liggett
et al., 1992). Whether a serine or threonine is phosphorylated by a GRK
appears to depend on the overall topological structure of the activated
receptor rather than on a defined linear recognition sequence (Chen et
al., 1993). Second messenger-dependent kinases (e.g., PKA and PKC)
phosphorylate a variety of proteins. This phosphorylation may mediate a
generalized cellular hyporesponsiveness, thus sometimes causing
heterologous desensitization. (Freedman and Lefkowitz, 1996).
CB1 and opioid receptors share common features in their signal
transduction and pharmacology (Martin et al., 1994; Pertwee, 1997).
Opioid receptor desensitization requires GRKs and arrestin. Opioid
receptor desensitization was blocked by the expression of a dominant
negative GRK and enhanced by overexpression of GRKs (Raynor et al.,
1994; Pei et al., 1995). In Xenopus oocytes, homologous agonist-induced opioid receptor desensitization of GIRK activation requires coexpression of GRK3 and -arrestin 2. Phosphorylation of
serine and threonine residues in the receptor cytoplasmic tail by GRK3
underlies the desensitization (Kovoor et al., 1997). The present study
found, similarly to the opioid receptor, that desensitization of
the CB1 receptor is also GRK3- and -arrestin 2-dependent. This is
the first evidence that GRKs and arrestin may produce
desensitization of a CB1 receptor-mediated response. Furthermore,
our results indicate that phosphorylation of serines (S426 and/or S430)
in the cytoplasmic tail of the CB1 receptor may be the molecular
mechanism of homologous CB1 receptor desensitization.
Together, the results of this and our earlier study show that for the
CB1 cannabinoid receptor the processes of desensitization and
internalization can be clearly dissociated. Previously we found that
residues 460-464 of the CB1 receptor were required for internalization
(Hsieh et al., 1999). The results from the present study demonstrate
that these residues are not needed for desensitization of
Kir current activation by the CB1 receptor in
Xenopus oocytes (Fig. 4). Furthermore, because the
nondesensitizing S426A/S430A CB1 mutant internalizes normally, it is
likely that the residues phosphorylated by a GRK and involved in
-arrestin binding are not required for internalization. Additional
support that the region involved in desensitization is unimportant for internalization comes from the observation that a nondesensitizing 418-439 CB1 deletion mutant internalizes similarly to the wild-type CB1 receptor (H. Kim, A. Sorom, and K. Mackie, unpublished
observation). Internalization of the CB1 cannabinoid receptor occurs
via clathrin-coated pits (Hsieh et al. 1999) in a dynamin-dependent
manner (Kim and Mackie, unpublished observation). Thus, if -arrestin
is important for CB1 internalization, it must be interacting with
residues other than those that we have found in the current study to be critical determinants for desensitization.
In summary, our results suggest that phosphorylation of the CB1
receptor by a G-protein receptor kinase followed by binding of
-arrestin may underlie the tolerance that develops during prolonged
administration of cannabis or cannabinoids. Receptor internalization
does not seem to be involved in rapid desensitization, because either
process can proceed independently of the other.
| |
FOOTNOTES |
Received Dec. 17, 1998; revised March 4, 1999; accepted March 9, 1999.
This work was supported by National Institutes of Health Research
Grants DA08934, DA00286, DA11322, NS01588, and DA04123 (to C.C.) and
the W. M. Keck Foundation.
Correspondence should be addressed to Ken Mackie, Department of
Anesthesiology, Box 356540, University of Washington, Seattle, WA
98195-6540.
| |
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TOP
ABSTRACT
INTRODUCTION
