(Received for publication, October 11, 1995; and in revised form, December 11, 1995)
From the
Prolonged exposure to abused drugs such as opiates causes
decreased response to the drug; this reduced sensitivity is thought to
be due to the loss of receptors, or down-regulation. The molecular
mechanism of the opiate receptor down-regulation is not known. In order
to address this, we generated a number of mutants of the opiate
receptor COOH-terminal tail. When expressed in the Chinese hamster
ovary cells, both the wild type and the receptor with a deletion of 37
COOH-terminal residues bind diprenorphine with comparable affinities
and show similar decreases in cAMP levels in response to D-Ala
, D-Leu
, enkephalin
(DADLE). However, the truncated receptor does not show down-regulation
from the cell surface upon prolonged exposure (2-48 h) to DADLE.
In contrast, both the wild type receptor and the receptor with the
deletion of only 15 COOH-terminal residues show substantial
down-regulation upon long term DADLE treatment. These results suggest
that the region located between 15 and 37 residues from the COOH
terminus is involved in the receptor down-regulation. In order to
identify residues that play a key role in down-regulation, point
mutations of residues within this region were examined for their
ability to modulate receptor down-regulation. The receptor with a
mutation of Thr
to Ala does not down-regulate, whereas
the receptor with a mutation of Ser
to Gly down-regulates
with a time course similar to that of the wild type receptor. Taken
together, these results suggest that the COOH-terminal tail is not
essential for functional coupling but is necessary for down-regulation
and that Thr
is critical for the agonist-mediated
down-regulation of the
opiate receptor.
It has been well established that chronic exposure to opiates such as morphine causes a decrease in the sensitivity to opiates(1) . This reduced sensitivity is thought to be due to the loss of receptors from the cell's surface. This phenomenon, termed ``down-regulation,'' has been implicated in the opiate tolerance/dependence that is seen in narcotic addicts. The molecular basis of the down-regulation phenomenon is not known.
Cell lines
that express high levels of opiate receptors have been used for studies
with agonist-mediated receptor function such as
down-regulation(2, 3, 4) . A neuroblastoma
glioma hybrid cell line (NG108-15) has been widely used to
characterize the
subtype of the opiate
receptor(2, 3, 4) . Upon exposure to agonist
the receptor number on the NG108-15 cells rapidly decreases (as
detected with radioligand binding assays). Treatment for up to 24 h
leads to a decrease of about 50-80% of the cell surface
receptors(2, 3) . This receptor down-regulation is
thought to be due to internalization of the ligand-receptor complex
followed by degradation of the receptor (4) .
The primary
structure of the opiate receptors as deduced from the cDNA has revealed
that all three subtypes of the opiate receptors are members of the
G-protein-coupled receptor family(5, 6, 7) .
Many structural features that are conserved in other G-protein-coupled
receptors are found in the opioid receptors; these include the
consensus N-linked glycosylation sites near the amino
terminus, a palmitoylation site in the COOH-terminal tail, and sites
for phosphorylation in the first and third intracellular domain and in
the COOH-terminal tail(5, 6, 7) . Extensive
studies have been carried out to examine the role of COOH-terminal tail
in modulating receptor-mediated events in the case of adrenergic
receptors(8) . In contrast, relatively few studies have
examined the role of COOH-terminal tail in modulating such events by
other neuropeptide receptors. We have therefore examined the role of
COOH-terminal tail in agonist-mediated down-regulation of the opiate
receptor using mutations of the COOH-terminal tail. We find that a
portion of the COOH-terminal tail is necessary for the agonist-mediated
down-regulation and that Thr plays an important role in
the down-regulation of the
opiate receptor.
Figure 1:
Schematic representation of the
structure of the full-length mouse opiate receptor, WT. The
putative glycosylation sites are shown as branched chains, and
the putative palmitoylation site is shown as a beaded line near the COOH-terminal tail. The COOH-terminal residues
333-372 are shown by filled circles. The flag
epitope-tagged receptor (F-WT) contains additional amino acid sequence
shown in capital letters near the NH
terminus. The lower panel shows the COOH-terminal tail residues
333-372 of the wild type receptor in single-letter amino acid
code. The asterisks point to the putative protein kinase C
phosphorylation sites, and the numbers indicate the amino acid
positions; the numbering is according to Evans et al.(6) . The amino acid sequence of the mutants identical to
the wild type is represented by a line, and the changes are as
indicated.
Approximately 3
10
Chinese hamster ovary (CHO) (
)cells were
transfected with 5 µg of Qiagen-purified plasmid DNA using
Lipofectin reagent (Life Technologies Inc.). Colonies with stable
expression were selected in medium containing 500 µg/ml of
Geneticin (Life Technologies Inc.). 24-48 colonies were tested
for receptor expression by binding assay using
[
H]diprenorphine(10) . Specific binding
is defined as the difference between the radioactivity bound to the
cells in the presence and absence of 10 µM diprenorphine.
Expression of the receptor was also confirmed by Western blotting of
the membranes from the stably expressing cells using a flag
tag-specific antibody, M1 (IBI/Kodak).
Figure 2:
Functional coupling of the wild type (WT,
), flag-tagged wild type (F-WT,
), or mutant lacking the
COOH-terminal 37 amino acids (
C37,
) receptors. The
functional coupling was examined by changes in cAMP levels as a measure
of the inhibition of adenylate cyclase. Cells were treated with various
doses of DADLE, and the cAMP was determined by radioimmunoassay as
described under ``Experimental Procedures.'' The cAMP in
control cells that was not treated with DADLE is taken as 100%. The
data represent the average ± S.E. of triplicate values from
three separate determinations. The data for cells expressing 1-2
10
receptors/cell are presented; similar
dose-response curves were observed with additional clonal cultures
expressing different numbers of receptors.
Figure 3:
Time-dependent decrease in
[H]diprenorphine binding during chronic DADLE
treatment. Cells expressing the WT (
), F-WT (
),
C15
(
),
C37 (
), S344G (
), or T353A (
) were
treated with 100 nM DADLE for various periods of time. After
extensive washing with buffer, [
H]diprenorphine
binding to the cells was measured as described under
``Experimental Procedures.''
[
H]diprenorphine binding to untreated cells
(treated 1-3 min with 100 nM DADLE prior to extensive
washing) is taken as ``control'' (100%). The data represent
the average ± S.E. of triplicate determinations. The data for
cells expressing 1-2
10
receptors/cell is
presented. For each construct, a similar time course of down-regulation
was observed with at least two additional clonal cultures expressing
different numbers of receptors.
We chose CHO cells as a suitable host cell line to express
high levels of delta opiate receptor, since CHO cells have been useful
for stable expression of a number of neuropeptide
receptors(10, 11, 12, 13, 14) .
We stably transformed the CHO cells with full-length or mutated
opiate receptor; a schematic of the primary structure of full-length
and mutated
opiate receptor is presented in Fig. 1. We
selected 24-48 clones expressing varying numbers of receptors and
analyzed two or three clones in each case. The cell lines expressing
wild type or mutated
opiate receptor exhibited high affinity for
diprenorphine (Table 1). The affinity is similar to the reported
affinity of NG108-15 for diprenorphine(2) . It should be
pointed out that although the receptor number varied 100-fold between
cell lines, there was no substantial difference in the affinity for
diprenorphine between the various clones. The modest 2-3-fold
variation in affinity is in agreement with the 2-3-fold variation
in the K
reported for CHO cells expressing varying
levels of the full-length receptor(10) .
DADLE, an opiate
receptor agonist, was used to determine the inhibition of adenylate
cyclase, as a measure of functional coupling, in these cell lines.
Dose-response curves of the inhibition of cAMP accumulation by 20-min
DADLE treatment show that the cAMP levels decrease with increasing
amounts of DADLE i.e. the percentage of inhibition of cAMP
increases with increasing amounts of DADLE (Fig. 2). The
IC value for the DADLE-induced inhibition of cAMP
accumulation by cells expressing the full-length receptor is identical
to the IC
value reported for CHO cells expressing
full-length receptor (10) and about 10-fold lower than the
IC
value reported for NG108-15 cells(2) .
Interestingly, the dose-response curve for the DADLE-induced inhibition
of cAMP accumulation in receptor lacking the COOH-terminal 37 amino
acids is comparable with the dose-response curves for the wild type
receptors (Fig. 2). The fact that the removal of the
COOH-terminal 37 amino acids does not affect the efficiency of
functional coupling suggests that the COOH-terminal tail does not play
a role in signal transduction by the
opiate receptor. This is in
contrast to other G-protein coupled receptors where the COOH-terminal
tail is thought to play an important role in functional coupling to
G-proteins(15) . Studies with deletion and other mutational
analyses in
adrenergic and other G-protein-coupled
receptors have shown that in these receptors the COOH-terminal tail is
an integral part of signal transduction(16, 17) .
To examine the role of the COOH-terminal tail in receptor
down-regulation, cells expressing the wild type or the mutant receptors
were treated with 100 nM DADLE for various time periods, and
the amount of receptors was determined by the binding of
[H]diprenorphine to intact cells. Cells
expressing the full-length receptors (untagged or flag-epitope-tagged)
exhibit a time-dependent decrease in diprenorphine binding with a
maximal decrease of about 60-70% by about 16 h (Fig. 3).
Comparable results were obtained with two other transfected cultures
expressing different numbers of receptors (data not shown). The cells
expressing
C15 receptor (lacking the COOH-terminal 15 amino acids)
exhibit a time-dependent decrease in diprenorphine binding similar to
the pattern of down-regulation seen with the wild-type receptor,
although the maximal decrease is only about 40% even after 48 h of
treatment (Fig. 3). In contrast, the cells expressing
C37
receptor (lacking the COOH-terminal 37 amino acids) show no decrease in
diprenorphine binding even upon 48 h of treatment with DADLE. The cell
surface diprenorphine binding actually increases after 48 h as compared
with the control (untreated) cells (Fig. 3). The finding that
the
C15 receptor shows down-regulation and
C37 receptor does
not suggests that the region located between 15 and 37 residues from
the COOH terminus plays an important role in
opiate receptor
down-regulation.
In order to identify the residues involved in this
down-regulation process, we generated receptors with mutations of three
phosphorylatable residues within the region between 15 and 37 amino
acids from the COOH terminus, namely, Ser,
Thr
, and Thr
. The cells expressing the
Thr
Ala receptor do not exhibit a decrease in
diprenorphine binding even after 48 h of treatment ( Fig. 3and Fig. 4). In contrast, the cells expressing Ser
Gly receptor exhibit a time course of decrease in
diprenorphine binding identical to the time course seen for the wild
type receptor ( Fig. 3and Fig. 4). This change in
diprenorphine binding in cells treated with DADLE represents a change
in the receptor number and not a change in affinity as examined by
Scatchard analyses (Fig. 4). It should be pointed out that we
have been unable to isolate cell lines expressing significant levels of
the Thr
Ala receptors among 96 clones tested.
Taken together, these results suggest that Thr
is
critical for the agonist-induced down-regulation of the
opiate
receptor.
Figure 4:
Down-regulation of the opiate
receptor in cell lines expressing wild-type and mutant receptors. The
cell lines were treated with 100 nM DADLE for 1-5 min (filled bars) or for 24 h (striped bars), and the
diprenorphine binding was carried out as described under
``Experimental Procedures.'' K
and B
values of diprenorphine binding
were determined by whole cell binding studies, using at least five
concentrations of diprenorphine, and the data were analyzed using the
Ligand program. The receptor number in cells treated for 1-3 min
with DADLE is taken as ``control'' (100%). Standard error of
the mean is included for each value of the B
.
There is growing evidence that in many of the
G-protein-coupled receptors the COOH-terminal tail plays a role in
receptor down-regulation. Studies involving the prototypical
adrenergic receptor have demonstrated the importance
of this receptor's carboxyl terminus in mediating desensitization
through both second messenger-dependent and independent kinases, which
phosphorylate multiple serines and threonines in this
region(8) . The COOH-terminal tail of the opiate receptor
contains putative phosphorylation sites(6, 7) ;
phosphorylation could play a role in opiate receptor down-regulation.
Studies in cell lines and in Xenopus laevis oocytes have
implicated the involvement of phosphorylation in opiate receptor
function(18, 19, 20, 21) .
Phosphorylation by protein kinase C has been implicated in the
down-regulation of other G-protein-coupled
receptors(22, 23, 24) . It is possible that
protein kinase C plays a role in opiate receptor desensitization
and/or down-regulation since the COOH-terminal tail of the
opiate
receptor contains residues that fit the consensus for phosphorylation
by protein kinase C. Studies examining the desensitization of
opiate receptors have shown that a
-ARK-related kinase, and not
protein kinase C, plays an important role in the receptor
desensitization(19) . In contrast, studies with the
down-regulation of the endogenous opiate receptors in NG108-15 cells
have shown that modulators of protein kinase C affect receptor
down-regulation, suggesting an involvement of protein kinase C in this
process(25) . In this study we find that mutation of a protein
kinase C consensus site (Ser
) does not affect
down-regulation, suggesting that phosphorylation by protein kinase C at
this site may not be involved in
opiate receptor down-regulation.
This is further supported by studies with modulators of protein kinase
C on receptor down-regulation. Pretreatment of the cells expressing
wild type receptor with 30 nM phorbol ester for 3 h (to
activate protein kinase C) or with 1 µM phorbol ester for
24 h (to deplete protein kinase C) does not affect the DADLE-mediated
down-regulation (data not shown). Furthermore, treatment of the cells
for 24 h with 0.5 µM calphostin C has no effect on
down-regulation (not shown). Taken together, these results suggest that
protein kinase C does not play a major role in the down-regulation of
the
opiate receptor expressed in CHO cells.
The possibility
that the Thr may play a role other than being
phosphorylated cannot be ruled out. It is possible that the Thr
identified in this study specifically recognizes some cellular
factor(s) involved in the process of receptor down-regulation. Such a
possibility is supported by the studies with the avian
adrenergic receptor that does not exhibit agonist-mediated
down-regulation(26, 27) . Deletion of a certain
portion of the COOH-terminal tail of this receptor results in
down-regulation(26) , and addition of this COOH-terminal
portion to the mammalian
adrenergic receptor results
in a dramatic decrease in the down-regulation of the latter
receptor(27) . These results suggest that COOH-terminal domains
could interact with certain cellular protein(s) that mediate receptor
down-regulation.
In summary, our study has shown that removal of the
COOH-terminal tail does not affect functional coupling but completely
abolishes down-regulation of the opiate receptor. In addition,
these studies demonstrate that Thr
is essential for
opiate receptor down-regulation.