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INTRODUCTION |
Phosphorylation of membrane receptors, such as tyrosine kinase
receptors or G-protein coupled receptors
(GPCRs),1 leads to an
alteration in the receptor activities (1). While autophosphorylation of
the tyrosine kinase receptors by the associated kinases results in the
initiation of the signal cascades, agonist-induced phosphorylation of
the GPCRs by protein kinases, recruited to the vicinity of the
receptors, blunts the signal cascades. Rapid phosphorylation of these
GPCRs, by specific protein kinases, i.e. G-protein-coupled
receptor kinases, GRKs, and the subsequent binding of arrestins, has
been considered to be a mechanism for the agonist-induced homologous
desensitization of these receptors (2, 3).
From the cloning of the receptors, it is clear that µ-,
-, and
-opioid receptors belong to the superfamily of GPCRs (4, 5). As with
other GPCRs, prolonged agonist treatment results in an attenuation of
effector responses and down-regulation of the receptor, both in clonal
cell lines that express the opioid receptor endogenously (6, 7) or in
cell lines that stably express cloned opioid receptors (8, 9). Chronic
exposure to opioid agonist results in a reduced receptor affinity for
the agonist, as well as diminished ability of the agonist to either inhibit cAMP accumulation (10-12), or to open inwardly rectifying potassium channels (GIRK-1) (13, 14). This reduction in the opioid
agonist activity has been suggested to involve receptor phosphorylation. Pei and colleagues (15) demonstrated phosphorylation of the
-opioid receptor that appeared to parallel the loss in agonist inhibition of adenylyl cyclase activity in HEK293 cells. Transient transfection of the dominant negative mutant of
ARK1 into
these cells reduced the degree of receptor phosphorylation which
suggested the involvement of GRKs in DOR1TAG phosphorylation. Phosphorylation of the µ-opioid receptor in the presence of agonist has been demonstrated both in HEK293 (16) and in Chinese hamster ovary
cells (13, 17). Furthermore, a parallel time course was reported
between µ-opioid receptor phosphorylation in Chinese hamster ovary
cells and the loss in µ-opioid receptor regulation of GIRK-1 in
Xenopus oocytes (13). Co-injection of the cDNAs of
DOR1TAG,
-arrestin 2,
ARK2, and GIRKs has been shown to result in
an increase in the rate of agonist-induced homologous desensitization of the
-opioid receptor (14). These data would tend to support the
hypothesis that phosphorylation of the opioid receptor is the probable
mechanism for the observed receptor desensitization.
There are some inconsistencies, however, within the reported data on
opioid receptor desensitization and phosphorylation. Although the
phosphorylation of the opioid receptor has been reported to be very
rapid, the agonist-induced loss of receptor activity as measured by
either adenylyl cyclase activity or GIRK-1 channel activity, especially
in the case of µ-opioid receptor, occurs rather slowly (6, 14).
Comparing the rates of desensitization of the two splice variants of
the µ-opioid receptor, MOR-1 and the shorter isoform MOR-1B
receptors, Zimprich and colleagues (18) showed that MOR-1B desensitized
more slowly than MOR-1. The authors attributed this difference to the
absence of Thr394 in the splice variant MOR-1B.
Subsequently, Pak et al. (19), reported that the mutation of
Thr394 in the wild-type MOR-1 to Ala resulted in the
complete blockade of the agonist-induced receptor desensitization.
These data are in contrast with those reported in which the agonist
could induce receptor desensitization in a µ-opioid receptor mutant
in which all the Ser/Thr residues of the third intracellular loop and
the carboxyl tail are mutated to alanine (20). It should be noted, however, that in neither of these studies was receptor phosphorylation determined and therefore, it is uncertain whether the reported mutations have affected the overall phosphorylation of the receptor. Furthermore, the observed µ-opioid receptor phosphorylation in Chinese hamster ovary cells was not blocked by protein kinase C (PKC)
inhibitors, thus suggesting the involvement of GRKs (21). However,
opioid receptor regulation of phospholipase C in Xenopus oocytes appeared to involve PKC (13), and µ-opioid receptor desensitization in Xenopus oocytes, as measured with GIRK-1
regulation, was potentiated by PKC activation (13, 21). Consequently, the manner by which these data contribute to the role of receptor phosphorylation in the loss of opioid receptor activity remains to be demonstrated.
In light of these discrepancies, we chose to investigate both receptor
phosphorylation and desensitization in the same system. Exposure of
HEK293 or neuro2A cells stably expressing the µ-opioid receptor to
the opioid agonist DAMGO resulted in a rapid phosphorylation of the
receptor protein, which is time- and agonist
concentration-dependent. However, the phosphorylation of
the receptor does not correlate directly to the loss of the µ-opioid
receptor mediated inhibition of adenylyl cyclase activity. Furthermore,
alteration in the phosphorylation state of the
- and µ-opioid
receptors, expressed in the same system, induced differential
regulation of these two receptor subtypes. These data suggest a pathway
in which µ-opioid receptor phosphorylation may not be the major
determinant in the eventual loss of agonist response.
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EXPERIMENTAL PROCEDURES |
Materials--
Expression vector pCDNA3 was from Invitrogen
(San Diego, CA). DMEM, Met/Cys-free DMEM, phosphate-free DMEM, and
Geniticin (G-418) were purchased from Life Technologies, Inc. (Grand
Island, NY). [3H]Diprenorphine (39.0 Ci/mmol),
[3H]adenine (15-25 Ci/mmol), and
[35S]Met/Cys (5.0 Ci/mmol) were supplied by Amersham.
[32P]Orthophosphate (400-800 mCi/ml) was supplied by ICN
(Costa Mesa, CA). 125I-Acetylated cAMP was purchased from
Linco Research (St. Charles, MO). Polyclonal antibodies for the cAMP
radioimmunoassay were a generous gift from Dr. T. Gettys (Medical
University of South Carolina, SC). The polyclonal antibodies to
-arrestin were a generous gift from Dr. R. J. Lefkowitz (Howard
Hughes Medical Institute, Durham, NC). cDNAs for the bovine
ARK2
(GRK3) and
ARK1K220R (GRK2K220R) were generous gifts from Dr. J. Benovic (Thomas Jefferson University Medical School, Philadephia, PA) and were subcloned into the expression vector pCDNA3. cDNA
encoding the
-arrestin 2 was the generous gift of Dr. S. G. Ferguson (Dept. of Cell Biology, Duke University Medical Center,
Durham, NC). Forskolin was purchased from Calbiochem (La Jolla, CA).
DAMGO and other opioid ligands were supplied by NIDA, National
Institutes of Health. All other chemicals were purchased from Sigma.
Cell Culture and Transfections--
Human embryonic kidney
HEK293 cells and neuroblastoma neuro2A cells were stably transfected
with the rat µ-opioid receptor cDNA, MOR-1, subcloned into the
EcoRI/XbaI sites of the expression vector
pCDNA3 by the calcium phosphate precipitation method (22). In order
to facilitate the identification of the µ-opioid receptor with both
polyclonal and monoclonal antibodies, a hemagglutinin (HA) epitope tag
(YPYDVPDYA), recognized by the 12CA5 or 3F10 monoclonal antibodies
(Boehringer Mannheim), was spliced into the NH2-terminal
immediately after the initial methionine codon as described earlier
(23). Cell colonies stably expressing the µ-opioid receptor were
isolated by selection in the presence of 1 mg/ml Geniticin (G418) for
10 to 14 days, and confirmation of µ-opioid receptor expression was
determined by whole cell binding using [3H]diprenorphine
(specific activity 39.0 Ci/mmol) in 25 mM HEPES buffer, pH
7.6. Specific binding is defined as the difference between the
radioactivity bound to the cells in the presence and absence of 100 µM naloxone. The selected clones, identified as MOR1TAGID2 (for neuroblastoma neuro2A cells) or HEKMT (for HEK293 cells), were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum, 100 µg/ml streptomycin, 100 IU/ml penicillin, and 250 µg/ml G418 under humidified atmosphere with
10% CO2. For transient transfection, the cDNA
was introduced into the cells also by the calcium phosphate method and
the assays were performed 48 h after transfection.
Opioid Inhibition of Intracellular cAMP Level--
Two separate
methods were used in the determination of the opioid agonist effect on
the intracellular cAMP. The experiments in which neuro2A MOR1TAGID2
cells were used, the effect of DAMGO on cAMP levels was determined by
measuring the conversion of the [3H]adenine-labeled ATP
pools to [3H]cAMP as described (6). In the experiments
when HEKMT were used, the intracellular cAMP level was determined by
radioimmunoassay using 125I-acetylated cAMP and rabbit
polyclonal antibodies which recognize the acetylated cAMP. In either
method, cells were seeded in 24-well plates (2 × 105
cells/well) 48 h prior to experiments. In the
[3H]adenine assay, the cells were prelabeled for 2 h
in 0.5 ml of DMEM supplemented with 29.3 mM
NaHCO3, 15.3 mM glucose, 15.4 mM NaCl, 2.5 µCi of [3H]adenine, and 0.25 mM
3-isobutyl-1-methylxanthine. Immediately before challenging the cells,
in both methods, the plates were placed on ice, the radioactive or
growth media was removed and replaced by 0.5 ml of ice-cold
Krebs-Ringer-HEPES buffer (KRHB: 110 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 25 mM glucose, 55 mM sucrose, 10 mM HEPES, pH 7.4) containing
0.25 mM 3-isobutyl-1-methylxanthine, 5 µM
forskolin (or no forskolin in case of basal determination) with the
indicated concentrations of DAMGO. The plates were subsequently incubated at 37 °C for 15 min and the reaction terminated by
addition of 50 µl of 3.3 N perchloric acid. In the case
of the [3H]adenine assay, [32P]cAMP was
added as an internal standard and the amount of [3H]cAMP
synthesized was then separated from the other 3H-labeled
nucleotides by double column chromatography as described by White and
Karr (24). In the case of radioimmunoassay, the perchloric acid in each
well was neutralized with 120 µl containing 2 M KOH, 1 M Tris, and 60 mM EGTA. The amount of cAMP in
each well was determined by comparing the ability of the diluted
samples to compete for 125I-acetylated cAMP binding to the
antibodies with that of standard concentrations of acetylated cAMP.
In the experiments in which the rate and extent of desensitization were
determined, cells were pretreated with the indicated concentrations of
DAMGO for various time intervals (30 min to 4 h). The same
concentration of DAMGO was included during the 2-h labeling of the ATP
pools with [3H]adenine. After the incubation period, the
media was removed and replaced with 0.5 ml of KRHB containing
forskolin, 3-isobutyl-1-methylxanthine, and DAMGO. The level of
inhibition using 1 µM or 20 nM DAMGO in these
chronic agonist-treated cells was determined by averaging the results
from at least 3 passages of cells.
Receptor Phosphorylation--
Neuro2A (MOR1TAGID2) or HEK293
(HEKMT) cells were seeded at 70-80% confluence in 100-mm plates
24 h prior to receptor phosphorylation assay. On the day of the
assay (90-100% confluence), cells were washed twice with
phosphate-free DMEM and incubated in 4 ml of the same medium for 1 h at 37 °C and 10% CO2. Then, the cells were incubated
with 100 µCi/ml [32P]orthophosphate for 2 h at
37 °C and 10% CO2. Agonists and other compounds were
added as indicated. The reaction was stopped by putting the plates on
ice, at which point the labeling medium was rapidly removed, the cells
were washed twice with ice-cold phosphate-buffered saline and harvested
with 1 ml of lysis buffer (25 mM HEPES, pH 7.4, 1%, v/v,
Triton X-100, 5 mM EDTA, with 100 µg/ml bacitracin, 10 µg/ml leupeptin, 0.1 mM phenylmethylsulfonyl fluoride,
100 µg/ml soybean trypsin inhibitor, 10 µg/ml pepstatin A, and 20 µg/ml benzamidine as protease inhibitors and 50 mM sodium fluoride, 10 mM sodium pyrophosphate, and 0.1 mM sodium vanadate as phosphatase inhibitors) in
microcentrifuge tubes. The receptor was solubilized by rotating the
samples for 1 h at 4 °C and the insoluble cellular debris was
then removed by centrifuging the samples at 14,000 × g
for 15 min at 4 °C. Supernatants were diluted to twice the volume
with lysis buffer (without Triton X-100) before loading onto 1-ml wheat
germ lectin affinity columns pre-equilibrated with Buffer A (25 mM HEPES, pH 7.4, 100 mM NaCl, and 0.1% Triton X-100). The columns were then washed with 10 ml of Buffer A to remove
non-bound radioactive proteins and the µ-opioid receptor-containing fraction was eluted with 3 ml of Buffer A containing 0.5 M
N-acetylglucosamine, and the protease/phosphatase inhibitors
as indicated above. The samples were incubated in the presence of
either 2.5 µg of HA-monoclonal antibody or 5 µl of polyclonal
antisera (552G) developed against the COOH-terminal of the receptor
(23), and 60 µl of slurry (50%) of prewashed immunopure protein
A-agarose (or protein G-agarose) beads (Pierce) overnight at 4 °C.
The beads were subsequently washed twice with Buffer A and three times
with Buffer A without NaCl. Afterward, receptor protein was dissociated
from the beads by adding 50 µl of SDS-PAGE sample buffer (62.5 mM Tris buffer, pH 6.8, 2% SDS, 3 M urea, 10%
glycerol, 5% 2-mercaptoethanol, and 0.001% bromphenol blue). The
samples were heated at 42 °C for 1 h and then separated on a
10% SDS-PAGE. After electrophoresis, gels were dried and the
phosphorylated proteins were visualized and quantified by using the
PhosphorImager Storm 840 system (Molecular Dynamics, Sunnyvale, CA).
Data Analysis--
Saturation binding data were analyzed using
the computer program Ligand, providing estimates of receptor density
(Bmax) and agonist affinity
(Kd). Mean values from individual treatment groups
were statistically analyzed by a one-way analysis of variance (ANOVA)
with subsequent comparisons among treatment groups and from their
control by the Student's t test.
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RESULTS |
Time-dependent Loss of the Agonist Inhibition of
Forskolin-stimulated Intracellular [3H]cAMP Production
upon DAMGO Pretreatment--
In cell lines stably expressing the
µ-opioid receptors, prolonged treatment with agonists resulted in a
gradual loss in the receptor activities (7). This loss in agonist
activity has been attributed to the desensitization and down-regulation
of the µ-opioid receptor. In order to investigate the role of
receptor phosphorylation in such cellular adaptation processes, a
hemagglutinin (HA) epitope was introduced at the NH2
terminus of µ-opioid receptor so as to facilitate the
immunoprecipitation of the receptor with antibodies other than the
polyclonal antibodies developed against the last 15 amino acids of the
carboxyl tail sequence (23, 25). For HEK 293 clone (HEKMT), the
Kd and Bmax values for [3H]diprenorphine were determined to be 1.3 ± 0.16 nM (n = 2) and 13.1 ± 0.54 pmol/mg of
protein (n = 2),
respectively.2 In previous
studies, the Kd and Bmax
values with the neuro2A clone (MOR1TAGID2) were determined to be
0.33 ± 0.02 nM (n = 3) and 1.9 pmol/mg of protein (n = 3), respectively (26). There
was no observable difference between epitope-tagged (MOR1TAG) or
wild-type (MOR1 WT) receptor in the DAMGO affinity for the receptor and
the potency of DAMGO to inhibit forskolin-stimulated cAMP production in
the neuro2A cells (25). The EC50 to inhibit adenylyl
cyclase activity and Kd of DAMGO in HEKMT cells have
a value of 7.5 ± 1.6 nM (n = 3) and
4.2 ± 0.51 nM (n = 3), respectively,2 and compare favorably with those obtained
with the MOR1TAGID2 cells.
In the HEK 293 cells expressing the wild-type µ-opioid receptor,
prolonged exposure to DAMGO resulted in the loss of the agonist ability
to inhibit forskolin-stimulated cAMP production. The kinetics of the
loss in agonist activity were monophasic and the rates were relatively
slow. The receptor was completely desensitized after 24 h of DAMGO
pretreatment (Fig. 1A).
Similar loss in the µ-opioid receptor activity in the neuro2A
MOR1TAGID2 cells was observed (25). For our studies, we tested the
ability of 20 nM and 1 µM DAMGO to inhibit
adenylyl cyclase activity. These concentrations correspond to the
sigmoidal and the asymptotic region of the log dose-response curve,
respectively (25). As shown in Fig. 1B, there was a
time-dependent decrease in the ability of DAMGO to inhibit
the intracellular cAMP production after 5 µM DAMGO
pretreatment in both the MOR1TAGID2 and the HEKMT cells. The
desensitization rate was very slow in both cell lines. It is not
surprising that the loss of 1 µM DAMGO response was
significantly slower than that in the loss of 20 nM DAMGO
response, after 4 h of DAMGO pretreatment. However, it is
surprising, that even the decrease in the activity of 20 nM
DAMGO took a relatively long period of time (Fig. 1B).

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Fig. 1.
Time-dependent loss in the
agonist inhibition of forskolin-stimulated intracellular cAMP
production upon DAMGO pretreatment. Neuro2A or HEK293 cells stably
expressing MOR1TAG were pretreated with 5 µM DAMGO for
the indicated time intervals. A and B, HEKMT were
challenged with 1 µM DAMGO ( ) and internal cAMP was
measured by radioimmunoassay method as described. B, Neuro2A
MOR1TAGID2 cells were challenged with 1 µM ( ) or 20 nM ( ) DAMGO and accumulation of cAMP was measured by
column chromatography assay as described under "Experimental
Procedures." *, p < 0.05. Data shown represent
mean ± S.E. of at least three independent experiments each
performed in triplicate.
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Phosphorylation of HA Epitope-tagged µ-Opioid Receptor in the
Presence of DAMGO--
In order to investigate the phosphorylation of
the µ-opioid receptor in either the MOR1TAGID2 or HEKMT cells,
immunoprecipitation of the receptor proteins with either the polyclonal
antisera (552G), which recognize the TAPLP epitope of the carboxyl
terminus of the µ-opioid receptor (23), or the monoclonal antibody
(12CA5 or 3F10), which recognizes the HA epitope, was carried out. The problem of nonspecific phosphorylated proteins being immunoprecipitated by these antibodies was overcome by partial purification of the µ-opioid receptor. This partial purification was carried out by the
adsorption of the receptor and other glycoproteins onto a wheat germ
agglutinin column, followed by subsequent elution of the receptor
from the columns with N-acetylglucosamine. Thus, in all our
receptor phosphorylation studies, the Triton X-100 extracts of the
total cellular proteins were partially purified with the wheat germ
agglutinin columns as described under "Experimental Procedures" prior to immunoprecipitation.
When the MOR1TAGID2 cells were radiolabeled with
[32P]orthophosphate and treated with 5 µM
DAMGO, purified receptor was resolved on SDS-PAGE and revealed a
diffused phosphoprotein band migrating at approximately 65-75 kDa
(Fig. 2, second
lane). The migration of this phosphoprotein band in the
SDS-PAGE corresponded to that of the HA epitope-tagged µ-opioid
receptor when Western blot analysis was carried out with either
polyclonal antiserum 552G or monoclonal antibody 12CA5 (23). The
incorporation of [32Pi] into the
immunoprecipitated epitope-tagged µ-opioid receptor in the presence
of DAMGO was dramatically increased compared with the basal control
level. This increase in the phosphorylation can be demonstrated to be
associated with the µ-opioid receptor from the following
observations: (a) the antagonist naloxone can completely
block the DAMGO-induced increase in phosphorylation (Fig. 2,
third lane), and (b) in the
untransfected neuro2A control cells, no phosphoprotein with these
molecular weights was immunoprecipitated from cells treated with DAMGO
for the same period of time (Fig. 2, fourth lane).

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Fig. 2.
DAMGO-induced phosphorylation of
MOR1TAG. Untransfected neuro2A cells (N2A) or stably
transfected with MOR1TAG (MOR1TAGID2) were labeled with
[32Pi] and then treated with 5 µM DAMGO with or without 25 µM naloxone for
30 min at 37 °C as indicated. Phosphoprotein bands were resolved on
10% SDS-PAGE. The phosphoreceptor band corresponding to MOR1TAG is
indicated by an arrow.
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DAMGO-induced µ-opioid receptor phosphorylation was rapid, occurring
within the first few minutes of agonist addition and reaching a maximum
within 20 min of DAMGO exposure (Fig. 3,
A and B). The low levels of basal receptor
phosphorylation in the control (without agonist treatment) were
dramatically increased when the cells were treated with 5 µM DAMGO for 20 min. A similar level of increase was
observed when the HEKMT cells were treated with DAMGO for 30 min (Fig.
3C). The amount of phosphorylated receptor stayed at the
maximum level during the first hour of DAMGO treatment. During this
time period, the DAMGO-induced phosphorylation was
concentration-dependent (Fig.
4). After 30 min of cell exposure to 100 nM DAMGO, there was a detectable increase in
phosphorylation. This increase reached a plateau at 3 µM,
giving an EC50 of 450 ± 120 nM (Fig.
4B). The EC50 value for DAMGO to induce 50% of maximal phosphorylated µ-opioid receptor was markedly higher than the
3.1 ± 0.9 nM required to elicit 50% of maximal
adenylyl cyclase inhibition, under identical conditions.

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Fig. 3.
Time course of DAMGO-dependent
MOR1TAG phosphorylation. After [32Pi]
labeling, the cells were treated with 5 µM DAMGO for the
indicated time intervals as described under "Experimental
Procedures." The samples were resolved on 10% SDS-PAGE and the
phosphoreceptor bands were quantified and analyzed by PhosphorImager
Storm (Molecular Dynamics). A, extracted phosphoreceptor
from MOR1TAGID2, corresponding to different DAMGO treatment period.
Pictured is the result of a single experiment representative of at
least three performed. B, receptor phosphorylation bands
were quantitatively analyzed with PhosphorImager and band intensities
expressed as a percentage of the maximum phosphorylation (20 min). Data
shown are mean ± S.E. *, p < 0.05; **,
p < 0.01 compared with the maximum phosphorylation.
C, quantitation analysis of time course of DAMGO-induced
MOR1TAG phosphorylation in HEKMT. Data shown are mean ± S.E. of
three independent experiments. **, p < 0.01.
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Fig. 4.
MOR1TAG phosphorylation is agonist
concentration dependent, reaching a plateau at 3 µM with an EC50 of 450 nM. A, MOR1TAGID2 cells were radiolabeled
and treated for 30 min with increasing concentrations of DAMGO.
Lanes 1-8 correspond to 1, 10, 100, 300, 1,000, 3,000, 10,000, and 30,000 nM, DAMGO, respectively. Pictured are
the results of a single experiment representative of at least three
performed. B, phosphoreceptor band was quantitatively
analyzed with PhosphorImager and band densities expressed as percentage
of maximal phosphorylation. Data shown are mean ± S.E. of at
least three separate experiments.
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After 1 h, there was a progressive decrease in the amount of
phosphorylated µ-opioid receptor in the MOR1TAGID2 cells (Fig. 3,
A and B). The phosphorylation level decreased to
58 ± 19% after 4 h of DAMGO treatment when compared with
that after 20 min of DAMGO exposure, p < 0.01 (Fig.
3B). A similar decrease (70 ± 8.5%) was observed in
the HEKMT cells (Fig. 3C). This observed phosphorylation decrease could be a result of (a) loss of receptor
molecules, (b) decrease in the affinity of 552G for the
TAPLP epitope due to conformational changes, or (c)
dephosphorylation of the phosphorylated receptor molecules. A decrease
in the affinity of antiserum 552G for the phosphorylated receptor was
ruled out by using anti-HA monoclonal antibody for immunoprecipitation
of the µ-opioid receptor. Using 12CA5 to immunoprecipitate the
receptor, receptor phosphorylation shows a decrease to 61 ± 19%
(n = 3) after 4 h of agonist treatment (data not
shown) which was similar to that observed when 552G was used.
The decrease in the overall cellular receptor level during chronic
agonist treatment as the mechanism for the observed decrease in
µ-opioid receptor phosphorylation can be eliminated by determining the receptor level by either [3H]diprenorphine binding or
the 35S-labeled Met/Cys labeling of the receptor. After
4 h of DAMGO treatment, [3H]diprenorphine binding
was decreased by 19 ± 4% (n = 3) and the 35S-Met/Cys labeled receptor level was decreased by 26 ± 2.6% (n = 3) as quantitated by the radioactivity
incorporated into the 65-75-kDa band in SDS-PAGE after
immunoprecipitation (data not shown). Thus, the level of
µ-opioid receptor being down-regulated was insufficient to explain
the phosphorylation decrease during the prolonged agonist
treatment (58 ± 19%), and was most likely due to the
dephosphorylation of the receptor (Fig. 3).
Modulation Effect of Phosphorylation on µ-Opioid Receptor
Desensitization--
Although the DAMGO-induced phosphorylation of the
receptor reached its maximal level by 20 min, there was a minimal loss
in the ability of either 20 nM or 1 µM DAMGO
to inhibit adenylyl cyclase activity after 5 µM DAMGO
treatment (Fig. 1B). If the receptor phosphorylation is a
determinant in the loss of activity, then an alteration in the
phosphorylation level of the receptor should change the extent of the
receptor desensitization. Thus, we sought to modify the level of the
µ-opioid receptor phosphorylation either by the inhibition of
receptor dephosphorylation or by enhancement of phosphorylation by
overexpression of the GRKs.
Since there was a time-dependent decrease in the level of
receptor phosphorylation which could not be explained completely by the
decrease in the receptor protein level, then it is possible that the
change indicates an alteration in the equilibrium between the
activities of the protein kinase(s) and the phosphatase(s). Thus,
calyculin A, a potent inhibitor of phosphatase I and II was included in
the phosphorylation experiments in order to increase the phosphorylated
receptor level. When HEKMT cells were pretreated with 20 nM
calyculin A for 30 min prior to the addition of DAMGO, the
phosphorylated receptor level was dramatically increased (Fig. 5A). The concentration of
DAMGO used for this experiment (0.5 µM) corresponds to
the EC50 of the maximal phosphorylation level (Fig.
4B). A similar increase was observed with MOR1TAGID2 cells were pretreated with calyculin A (data not shown). Interestingly this
calyculin A-induced increase in phosphorylation did not alter the
degree of receptor desensitization. The ability of DAMGO to inhibit the
adenylyl cyclase activity was not altered significantly after 30 min of
0.5 µM DAMGO treatment in the presence of 20 nM calyculin A in both HEKMT (Fig. 5B) and
MOR1TAGID2 cells (data not shown). Higher concentrations of calyculin A
could not be used because of its relative toxicity toward the cell
lines used. Nevertheless, as summarized in Fig. 5, 20 nM
calyculin A, which had a pronounced effect on the magnitude of receptor
phosphorylation, did not significantly alter the acute or chronic
effects of DAMGO.

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Fig. 5.
Effects of calyculin A on MOR1TAG
phosphorylation and desensitization. HEKMT cells were treated, as
indicated, with 0.5 µM DAMGO in the presence or absence
(control) of 20 nM calyculin A. A, effects of
calyculin A on DAMGO-induced MOR1TAG phosphorylation. Pictured are the
results of a single experiment representative of three performed.
B, effects of calyculin A on DAMGO-induced MOR1TAG
desensitization. HEKMT cells either untreated (dark bars) or
treated with 0.5 µM DAMGO for 30 min (hatched
bars) were challenged with 0.5 µM DAMGO in the
presence of 5 µM forskolin. 20 nM calyculin A
or vehicle was added 30 min prior to pretreatment with DAMGO. The
results were normalized as a percentage of maximal inhibition of
forskolin-stimulated adenylyl cyclase activity (the DAMGO-mediated
inhibition in untreated cells). Data shown represent mean ± S.E.
of three separate experiments performed in triplicate.
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From our own studies and those reported (13), it is clear that protein
kinase C is not involved in the DAMGO-induced phosphorylation of the
µ-opioid receptor. Similar to previous studies performed with DOR1TAG
(15), depletion of PKC by pretreatment with the phorbol ester, PMA, did
not affect DAMGO-induced phosphorylation but attenuated the PMA-induced
MOR1TAG phosphorylation, p < 0.005 (n = 3) (Fig. 6). Furthermore, the inclusion
of a PKC-specific inhibitor, chelerythrine chloride (10 µM), inhibited the PMA-induced, but not the
DAMGO-induced, phosphorylation of the µ-opioid receptor, p < 0.05 (n = 3) (Fig. 6). Since
activation of the opioid receptor resulted in a decrease in the
intracellular cAMP level, DAMGO-induced phosphorylation must
involve protein kinases other than PKC and the
cAMP-dependent kinase, PKA. The presence of GRK consensus sites within the opioid receptor amino acid sequence, and the implication of GRKs in the phosphorylation of GPCRs, suggests that the
observed results could be due to the GRK phosphorylation of the
agonist-activated µ-opioid receptor. More specifically, the
implication of
ARK2 in phosphorylation of several GPCRs, and the
ability of co-injected
ARK2 to accelerate
-opioid receptor desensitization in the Xenopus oocytes (14) argues for a
probable involvement of this protein kinase in µ-opioid receptor
phosphorylation.

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Fig. 6.
Activated PKC can phosphorylate MOR1TAG but
is not involved in DAMGO-induced phosphorylation. MOR1TAGID2 cells
were pretreated without (control) or with (PKC-depleted) 1 µM PMA for 24 h before labeling with
[32Pi]. The cells were then stimulated for 30 min with 5 µM DAMGO (hatched bars), 1 µM PMA (dark bars), or vehicule (basal)
(white bars). PKC inhibitor, chelerythrine chloride (10 µM), was added 30 min before adding phosphorylation
stimuli. Receptors were then purified and resolved on SDS-PAGE as
described under "Experimental Procedures." Phosphoreceptor bands
were quantitatively analyzed with a PhosphorImager and expressed as a
percentage of the basal phosphorylation level seen in control cells.
Data shown represent mean ± S.E. of three separate experiments.
*, p < 0.05; **, p < 0.005 compared
with the control cell value.
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As mentioned above, another approach to alter the receptor
phosphorylation level would be to overexpress
ARK2 in HEKMT cells. After 30 min treatment with 0.5 µM DAMGO, the
phosphorylated receptor level was increased compared with the basal
phosphorylation level (Fig.
7A). The expression of
ARK2
significantly elevated the phosphorylation level in an
agonist-dependent manner, while the co-expression of the
mutant of
ARKs,
ARK1K220R, abolished the phosphorylation effect
of
ARK2 (Fig. 7A). The expression of this mutant alone
does not affect dramatically the endogenous phosphorylation level. In the same cell line, under the same conditions, a
minimal loss in DAMGO-induced inhibition of forskolin-stimulated
adenylyl cyclase activity was detected after 30 min of DAMGO
pretreatment. Overexpression of
ARK2 alone, or with
ARK1K220R
mutant, did not alter DAMGO-induced MOR1TAG desensitization (Fig. 7B).
The same results were obtained when increasing amounts of
ARK2 or
ARK1K220R cDNAs (up to 30 µg of cDNA) were used (data not
shown).

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Fig. 7.
Effects of overexpressing
ARK2 on DAMGO-induced phosphorylation and
desensitization of µ-opioid receptor.
HEKMT cells were transiently transfected with 2.5 µg of ARK2
and/or 10 µg of ARK1K220R mutant cDNA as indicated.
A, after [32Pi] labeling, the
cells were treated with 0.5 µM DAMGO, as indicated, for
30 min and phosphoreceptor bands were resolved on SDS-PAGE as described
under "Experimental Procedures." B, DAMGO-mediated
MOR1TAG desensitization was carried out in HEKMT cells, in the absence
(control) or presence of either ARK2 or ARK2 and ARK1K220R
mutant ( ARK2/Mutant). Untreated ( ) or treated ( ) cells with
0.5 µM DAMGO for 1 h were challenged with 0.5 µM DAMGO in the presence of 5 µM forskolin.
The basal level ( ) (control in the absence of agonist and forskolin)
and the forskolin stimulated level ( ) of cAMP were measured for each
transfection condition.
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One of the consequences of GPCR phosphorylation is the enhancement of
-arrestin binding to the protein, resulting in further uncoupling of
the receptor from the G-proteins (2). The failure to observe any
receptor desensitization after maximal receptor phosphorylation could
be attributed to an insufficient endogenous
-arrestin level in the
HEKMT cells. Studies with other GPCRs (27, 28) and the
-opioid
receptor desensitization in Xenopus oocytes (14) have
suggested that overexpression of
-arrestin can potentiate receptor
desensitization. Hence,
-arrestin 2 was overexpressed in HEKMT cells
in order to investigate whether an increase in the level of this
protein would accelerate the loss of the µ-opioid receptor-mediated
inhibition of adenylyl cyclase activity. As shown in Fig.
8A, Western blotting revealed
that HEKMT cells do endogenously express
-arrestin 2. The expression level of
-arrestin 2 was increased when HEKMT cells were transfected with 0.01 or 0.1 µg of
-arrestin 2 cDNA. However, even in
presence of overexpressed
-arrestin 2, the desensitization of
MOR1TAG after 1 h treatment with DAMGO was not altered as compared
with the vehicle-transfected HEKMT cells (Fig. 8B). As a
result of the failure to observe any effect of
ARK2 or
-arrestin
2 on DAMGO-induced MOR1TAG desensitization, we considered the
functional activity of these two proteins in our system. Thus, we chose
-opioid receptor as an internal positive control.

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Fig. 8.
Effects of overexpressing
-arrestin 2 on DAMGO inhibition of
forskolin-stimulated adenylyl cyclase activity. A,
HEKMT cells were transiently transfected with increasing amounts of
-arrestin 2 cDNA and samples from whole cell extract were loaded
on 10% SDS-PAGE. Immunoblotting using polyclonal anti- -arrestin 2 antibodies revealed increasing band intensities corresponding to
-arrestin 2. B, under the same transfection conditions,
HEKMT cells were subjected to adenylyl cyclase assays by measuring
DAMGO inhibition of cAMP production. Untreated ( ) or treated ( )
cells with 0.5 µM DAMGO for 1 h were challenged with
0.5 µM DAMGO in the presence of 5 µM
forskolin. The basal level ( ) (control in the absence of agonist and
forskolin) and the forskolin stimulated level ( ) of cAMP were
measured for each -arrestin 2 transfection condition.
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To eliminate any transfection artifacts, HEKMT cells, which stably
express MOR1TAG were transfected with HA-tagged
-opioid receptor
(DOR1TAG). The expression level of DOR1TAG in HEKMT was 1.42 ± 0.16 (n = 6) pmol/mg of protein. The inserted
hemagglutinin epitope at the NH2 terminus of both receptor
subtypes enabled us to purify and immunoprecipitate both receptors with
the same monoclonal antibody. After 30 min treatment with 0.5 µM DAMGO and 1 µM DPDPE, phosphorylated
receptor proteins were resolved on 12% SDS-PAGE and revealed two
diffuse bands corresponding to MOR1TAG (65-75 kDa) and DOR1TAG (50-60
kDa). There was a small, although not significant, increase in basal
phosphorylation of DOR1TAG in the presence of overexpressed
ARK2.
However, as expected, DPDPE-induced DOR1TAG phosphorylation was
markedly increased when
ARK2 was overexpressed to a level about
3-fold of that in the absence of agonist (p < 0.01)
(Fig. 9A). These data would
argue that the
ARK2-mediated potentiation of phosphorylation is
agonist dependent. Co-expression of
ARK2 and
ARK1K220R
mutant significantly reduced the DOR1TAG phosphorylation level
(p < 0.01).

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Fig. 9.
Effects of overexpressing
ARK2 and -arrestin 2 on
agonist-induced phosphorylation and desensitization of MOR1TAG and
DOR1TAG. A, HEKMT cells stably expressing MOR1TAG
were transiently co-transfected with 10 µg of DOR1TAG, 2.5 µg of
ARK2, and 10 µg of ARK1K220R mutant cDNA as indicated.
After [32Pi] labeling, the cells were treated
with 0.5 µM DAMGO and 1 µM DPDPE for 30 min
and phosphoreceptor bands corresponding to MOR1TAG and DOR1TAG were
resolved on 12% SDS-PAGE as described under "Experimental
Procedures." The graph shown represents mean ± S.E. for MOR1TAG
(gray bars) and DOR1TAG (dark bars)
phosphorylation of at least three independent experiments quantified by
PhosphorImager analysis. Data were normalized to the corresponding
basal phosphorylation level in the absence of agonist treatment and
kinase overexpression. **, p < 0.01; *,
p < 0.05 compared with the corresponding controls
without kinase overexpression or to the sample co-expressing ARK2
and ARK1K220R. B, HEKMT cells transiently
transfected with DOR1TAG ( , ) or with DOR1TAG, ARK2, and
-arrestin 2 ( , ) were treated with 0.5 µM DAMGO
( , ) or with 1 µM DPDPE ( , ) for the
indicated time intervals. The same agonist and concentration were used
for the challenge in the presence of 5 µM forskolin as
described under "Experimental Procedures." Data shown represent
mean ± S.E. of three to five separate experiments performed each
in triplicate. *, p < 0.05.
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Similar to the HEK 293 cells expressing MOR1TAG alone (Fig. 7), a
small, but significant increase (p < 0.05), was
observed when DAMGO-induced MOR1TAG phosphorylation was determined in
the presence of
ARK2 and DOR1TAG. This increase was abolished in the
presence of
ARK1K220R mutant. Overexpression of the
ARK1K220R mutant alone did not have any significant effect on the phosphorylation level of either receptor, compared with the corresponding controls (Fig. 9A). Consistent with data reported above (Fig.
7A), this result indicates that the transient expression of
this mutant might be unable to compete with the endogenous GRKs.
Alternatively, these data suggest the involvement of different kinases,
other than
ARKs, in opioid agonist-induced receptor phosphorylation.
In the same system expressing both receptors, agonist-induced receptor
desensitization was carried out by measuring agonist-induced inhibition
of forskolin-stimulated adenylyl cyclase activity. After pretreatment
with 0.5 µM DAMGO or 1 µM DPDPE, Fig.
9B shows agonist-mediated time-dependent
desensitization for µ- and
-opioid receptors. As expected, the
rate and magnitude of DPDPE-induced DOR1TAG desensitization were faster
than those for DAMGO-induced MOR1TAG desensitization. The co-expression
of
ARK2 and
-arrestin 2 with DOR1TAG in the HEKMT cells
significantly enhanced both the rate and magnitude of the DPDPE-induced
DOR1TAG desensitization (Fig. 9B). After 2 h of 1 µM DPDPE treatment, the extent of the loss in
-opioid
receptor was significantly increased from 37 ± 4% in the control
cells (HEKMT/DOR1TAG) to 68 ± 12% in HEKMT cells transfected
with DOR1TAG/
ARK2/
-arrestin 2 (p < 0.05) (Fig. 9B). Intermediate responses in DPDPE-induced receptor
desensitization were obtained when
ARK2 (or
-arrestin 2) alone
was expressed in HEKMT cells in the presence of DOR1TAG (data not
shown). The failure to observe a complete DOR1TAG desensitization
within the 2-h agonist treatment may be due to the
recycling/resensitization of the sequestered receptors or to newly
synthesized receptors. However, there was no observable effect on
DAMGO-induced MOR1TAG desensitization when
ARK2 and
-arrestin 2 were co-expressed (Fig. 9B). These results show that µ-
and
-opioid receptors are under differential regulation by the
ARK2 and
-arrestin 2. It is clear also that modulation of
receptor phosphorylation does not affect DAMGO-induced MOR1TAG
desensitization, as measured by agonist inhibition of adenylyl cyclase activity.
 |
DISCUSSION |
The evidence in support of the phosphorylation of GPCRs as the
essential step in the agonist-mediated desensitization of these receptors is compelling. Direct phosphorylation experiments with various GPCRs, especially those with the
2-adrenergic
receptor, clearly indicate a direct correlation between the degree of
receptor phosphorylation and the extent of desensitization (29, 30). The dephosphorylation of the
2-adrenergic receptor with
phosphatase treatment resulted in resensitization of
receptor-stimulated adenylyl cyclase activity (31). These data were
further supported by the experiments in which the
2-adrenergic receptor desensitization could be
attenuated by Ser/Thr mutations (32). In addition, the identification
of a family of receptor kinases,
ARKs, which phosphorylate only the
agonist-activated receptor, as well as the ability of a inactive mutant
of the
ARKs to block receptor phosphorylation and subsequently
receptor desensitization, indicate unequivocally the important role of
phosphorylation of GPCRs in the agonist-induced receptor
desensitization, particularly for receptors such as the
2-adrenergic receptor (33, 34).
As opioid receptors belong to the GPCR family (1, 5), it is logical to
hypothesize that the loss of opioid responses during chronic treatment
follows a mechanism similar to that reported with the
2-adrenergic receptor and other GPCRs. Previous reports have demonstrated a direct relationship between receptor
phosphorylation and agonist-induced opioid receptor desensitization
(11, 13, 15, 16). Receptor phosphorylation as the mechanism for
µ-opioid receptor desensitization was supported also by a recent
report (19) in which mutation of Thr394 or
Glu393 to Ala was shown to result in the complete block of
agonist induced loss of receptor activity. Since GRKs are acidokinases
(34), the results reported by Pak et al. (19) suggest that
phosphorylation of the receptor by GRKs is the mechanism of µ-opioid
receptor desensitization.
In the present work, we provide evidence in contrast to a direct
correlation between µ-opioid receptor phosphorylation and desensitization. By measuring, under identical conditions in the same
system, the loss of opioid receptor activities and receptor phosphorylation, we were able to demonstrate a difference in the time
course of these processes. The rate of a loss of DAMGO inhibition of
forskolin-stimulated adenylyl cyclase activity in either MOR1TAGID2 or
HEKMT cells was relatively slow in comparison with the agonist-induced receptor phosphorylation in both cell lines (Figs. 1 and 3). Since both
neuro2A and HEK293 cells stably expressing the µ-opioid receptor have
similar rates, this relatively slow rate of µ-opioid receptor desensitization could not be attributed to the phenotypic differences between the present observations and those previously reported. Furthermore, this slow desensitization rate was also observed with the
human neuroblastoma SH-SY5Y cells endogenously expressing both the µ-
and
-opioid receptors (7). The difference in rates of receptor
phosphorylation and desensitization could also be demonstrated with
endogenously expressed receptors. Maximal phosphorylated µ-opioid
receptor in human neuroblastoma SH-SY5Y cells can be obtained by
incubating these cells in the presence of 1 µM DAMGO for
30 min (data not shown).
The absence of a direct correlation between µ-opioid receptor
phosphorylation and desensitization was further demonstrated by
altering the level of receptor phosphorylation. The inclusion of
calyculin A or the overexpression of
ARK2 significantly increase the
level of MOR1TAG phosphorylation. However, this increase did not affect
the DAMGO-induced receptor desensitization (Figs. 5 and 7). The lack of
an effect due to overexpression of
ARK2 on µ-opioid receptor
desensitization does not exclude the possible involvement of other
known GRKs. Different effects of the overexpression of various GRKs in
other GPCRs such as dopamine (35),
-adrenergic (27), and chemokine
(CCR-5) (36) receptor desensitization have been reported. With the
presence of multiple putative GRK sites within the carboxyl tail of
µ-opioid receptor alone, the sites in the receptor that are being
phosphorylated in the presence of agonist alone could be different from
those in the presence of the overexpressed
ARK2. Overexpression of
other GRKs could have a potentiating effect on the DAMGO-induced
receptor desensitization. However, in an effort to identify a probable
mechanism of phosphorylation and desensitization, our studies continued
to evaluate the effects of
-arrestin. This approach was used because
an increase in GPCR phosphorylation by GRKs directly enhances the
affinity of
-arrestin for the receptor and thus, promoting a further
uncoupling of the receptor from the G-proteins (2, 37). Agonist-induced
receptor desensitization can be potentiated subsequently by
overexpression of the
-arrestin in the system (27, 28). Our present
study shows that co-expression of
ARK2 and
-arrestin potentiated
the desensitization rate of DOR1TAG but not of MOR1TAG. These data are
consistent with the homologous desensitization of the G-protein-coupled inward rectifying K+ channels reported by Kovoor et
al. (14, 15), and indicate differential regulation of these two
opioid receptor subtypes, by
ARK2 and
-arrestin 2. Since both
µ- and
-opioid receptors were expressed in the same cells that
were co-transfected with
ARK2 and
-arrestin 2, the differences
between the regulation patterns of these two opioid receptors could not
be attributed to phenotypic or transfection artifacts. From the
carboxyl tail truncation mutant studies with the
-opioid receptor
(38) and our own studies (data not shown), it is apparent that
agonist-induced phosphorylation of the opioid receptors occurs at the
carboxyl tail domain. Thus, the difference in the regulation of these
two opioid receptor subtypes can be attributed to the difference in intrinsic domains of the receptor protein, specifically, the varying COOH termini of these two receptors. Furthermore, for µ-opioid receptor, the magnitude of phosphorylation induced by various opioid
ligands appeared to correlate with the ability of these ligands to
modulate the response, by measuring adenylyl cyclase activity (17) or
receptor internalization (39). As high concentrations of agonists were
used in these studies, the observed difference in the overall
phosphorylation is most likely due to the agonist-induced phosphorylation of specific sites on µ-opioid receptor. This
observation indicates that different agonist-activated conformations of
µ-opioid receptor may exist. Subsequently, the receptor can modulate
the intracellular responses by this specific phosphorylation.
Identification of phosphorylated sites will help to elucidate the
phosphorylation mechanism(s) and to understand the role of
phosphorylation in µ-opioid receptor signal transduction.
As for µ-opioid receptor and other GPCRs (40), the lack of
correlation between receptor phosphorylation and desensitization does
not imply that receptor phosphorylation does not have a role in the
receptor desensitization. The differences between the time course of
receptor desensitization and phosphorylation, and the inability of the
alteration in the phosphorylation state of the receptor to influence
µ-opioid agonist-induced receptor desensitization, only suggests that
an alternative mechanism(s) is involved in the cellular adaptation
processes during chronic DAMGO treatment. One probable receptor
desensitization mechanism is receptor internalization/sequestration. MOR1B, a splice variant of MOR-1, has been reported to desensitize at a
slower rate than MOR-1 (41). The disappearance of the MOR-1B receptor
in the presence of agonist also occurred at a slower rate than MOR-1.
However, both the rates of desensitization and internalization of
MOR-1B could be reverted to that of MOR1 if monensin was included
during the incubation, suggesting the rapid recycling of the receptor
proteins (41). Since the regulation of the adenylyl cyclase by
µ-opioid agonist has been reported to be highly dependent on the
receptor density (19), the internalization/sequestration of the
µ-opioid receptor could be a critical factor in the observed loss of
response. Such a mechanism is suggested by the fact that the rate of
overall dephosphorylation of the µ-opioid receptor parallels that of
receptor desensitization (Figs. 1 and 3). Dephosphorylation of GPCRs in
sequestered vesicles has been suggested, although dephosphorylation has
been proposed to be the resensitization mechanism (42). The overall
phosphorylation state of all cellular µ-opioid receptors should
reflect the trafficking of the receptors into the sequestered vesicles.
However, whether the sequestration/internalization of the
µ-opioid receptor is the alternative mechanism for the observed loss
in DAMGO inhibition of adenylyl cyclase activity remains to be
demonstrated. Nevertheless, our current studies clearly demonstrate
that phosphorylation of the µ-opioid receptor is not the obligatory
event for the DAMGO-induced receptor desensitization, as measured
by the regulation of adenylyl cyclase activity.