(Received for publication, January 16, 1996; and in revised form, February 22, 1996)
From the
µ opiate receptors, the principal sites for opiate analgesia
and reward, can display compensatory responses to opiate agonist drug
administration. Agonist-induced K channel responses
mediated by these receptors desensitize when examined in Xenopus oocyte expression systems. Mechanisms underlying such processes
could include phosphorylation events similar to those reported to
desensitize other G-protein-linked receptors. We used C-terminally
directed anti-µ receptor antibodies to immunoprecipitate a
phosphoprotein with size appropriate for the µ receptor from stably
expressing Chinese hamster ovary cells. Phosphorylation of this µ
opiate receptor protein was enhanced approximately 5-fold by treatment
with the µ agonist morphine. The time course and dose-response
relationships between µ receptor phosphorylation and
agonist-induced desensitization display interesting parallels.
Phosphorylation of µ opiate receptor protein is also enhanced
5-fold by treatment with the protein kinase C activator phorbol
12-myristate 13-acetate. The protein kinase inhibitor staurosporine
blocked the effect of phorbol 12-myristate 13-acetate on µ receptor
phosphorylation. However, staurosporine failed to block
morphine-induced phosphorylation. These observations suggest that
several biochemical pathways can lead to µ receptor phosphorylation
events that may include mechanisms involved in µ receptor
desensitization.
Opioid receptors are G-protein-coupled, seven transmembrane domain receptors. These receptors mediate the actions of opiate drugs and endogenous opioid neuropeptides in producing euphoria, modulating pain perception, and altering other important functions in the central and peripheral nervous systems (Herz, 1993). Pharmacological characterization and cDNA cloning have focused attention on the µ opiate receptor subtype, the site at which opiate drugs such as morphine and its derivatives exert most of their analgesic and euphoric effects (Chen et al., 1993; Fukuda et al., 1993; Wang et al., 1993; Thompson et al., 1993). G-protein cascades activated by µ receptors can reduce adenylyl cyclase activity, alter inositol trisphosphate turnover, activate G-protein-linked, inward potassium channels, and close calcium channels (Childers, 1991; Smart et al., 1994; North et al., 1987).
A prominent characteristic of morphine-like drugs is their
ability to induce drug tolerance and dependence in humans. Another
feature of these drugs' actions is the rate-sensitivity. ()Rapidly acting opiates such as heroin induce much more
striking euphoria than more slowly acting opiates such as
methadone.
Potential µ opiate receptor mechanisms that
might contribute to opiate tolerance, dependence, and rate-sensitivity
have thus been of interest. Receptor desensitization is one of the
cellular mechanisms that could play possibly significant roles in these
neuroadaptive processes.
Phosphorylation is a post-translational modification used to regulate the functions of a wide variety of proteins, including G-protein-linked neurotransmitter receptors (Walaas and Greengard, 1991). Studies of adrenergic and other receptors demonstrate that agonist-dependent phosphorylation of the receptors contribute to mechanisms of receptor desensitization, although receptor internalization and altered rates of receptor gene expression can also play roles (e.g. Kobilka, 1992). Such studies also point to the possibilities that mechanisms of homologous desensitization occurring as a result of agonist occupancy of the receptor can be different from heterologous desensitization mechanisms produced by the consequences of occupation of other G-protein-linked receptors coexpressed on the same cell.
Xenopus oocytes
co-expressing cDNAs encoding the µ receptor and GIRK1, an inwardly
rectifying potassium channel that can be activated by µ
receptor-activated G-proteins, have allowed detection of
agonist-mediated desensitization of µ receptor-mediated
K channel responses in two laboratories (Chen and Yu,
1994; Kovoor et al., 1995). These two reports, however,
disagree on the effects of stimulators of protein kinase A and/or
protein kinase C (PKC) (
)in this desensitization.
The possibility that the µ opiate receptor might undergo phosphorylation was suggested by the presence of sequences with homology to consensus sites for protein kinase A and C phosphorylation in putative intracellular receptor domains when the receptor's sequence was elucidated by cDNA cloning (Wang et al., 1993). Antibodies recognizing epitope-tagged opiate receptors have immunoprecipitated apparent phosphorylated tagged µ receptor protein from expressing HEK293 cells (Arden et al., 1995). However, no current work has established the biochemical pathways for such phosphorylation or examined the parallels between biochemical evidence for µ receptor phosphorylation and the receptor desensitization noted electrophysiologically.
In order to approach this problem, we have studied agonist-dependent and protein kinase-mediated µ receptor phosphorylation events and sought parallels with patterns of desensitization of µ receptor-mediated potassium channel responses. We have documented agonist-induced and PKC-induced µ receptor phosphorylation and agonist- and PKC-mediated receptor desensitization. Observed patterns of phosphorylation, desensitization, and PKC-inhibitor effects provide evidence for at least two pathways for µ receptor phosphorylation events, some of which could be involved in receptor desensitization.
Oocytes were isolated from mature female Xenopus laevis (Xenopus I, Ann Arbor, MI), defolliculated by treatment
with 0.2% collagenase A, injected with 16-20 ng of RNAs encoding
the µ opiate receptor and GIRK1 in molar ratios of 3:1, and
incubated for 2-3 days at 19-20 °C in ND96 solution (96
mM NaCl, 2 mM KCl, 2.5 mM CaCl,
1.0 mM MgSO
, and 5 mM HEPES, pH 7.5)
supplemented with 2 mM sodium pyruvate, 10,000 units/liter
penicillin, 10 mg/liter streptomycin, and 0.5 mM theophylline.
Figure 1:
A, currents induced in Xenopus oocytes coexpressing µ opiate receptors and GIRK1 during
superfusion with ND96 (open bars), hK solution (cross-hatched bars), or 10 nM DAMGO in hK solution (solid bars). B, dose-response relationship for DAMGO
in inducing inward currents greater than those found in hK medium
alone. Peak currents during DAMGO/hK superfusion were corrected for
currents found during superfusion of hK alone and reported as mean
± S.E. of maximal currents determined for each of 5-7
individual expressing oocytes determined with 1 µM DAMGO.
The EC of DAMGO is 4.6
nM.
Agonist-induced desensitization was observed, manifested as a gradual decline in the amplitude of current responses to short test applications of 1 µM DAMGO applied in hK solution to oocytes previously exposed to 1 µM DAMGO superfused in ND96 medium for varying periods of time. hK-elicited currents were assessed during washout periods using hK medium alone (Fig. 2A). This paradigm reveals agonist-induced desensitization after as little as 3-min oocyte exposures to 1 µM DAMGO; the desensitization reaches maximal values by 20 min and is reversible. Short test doses of 1 µM DAMGO administered following as little as 10 min of washing with ND96 medium reveal recovery to near-normal function. The magnitude of observed desensitization depends on both the exposure time and concentration of agonist (Fig. 2, B and C). In experiments in which oocyte responses to test DAMGO administrations were recorded after 30 min of continuous DAMGO exposure, maximum desensitization was observed at 10 µM DAMGO (Fig. 1C).
Figure 2:
Currents induced in Xenopus oocytes coexpressing opiate receptors and GIRK1. A,
recordings of responses to superfusion with ND96 or test superfusions
with 1 µM DAMGO in hK (solid bars) followed by hK
solution superfusion for 2 min in oocytes previously exposed for 10 or
20 min to superfusion of 1 µM DAMGO in ND96. B,
time course of desensitization of µ opiate receptor/GIRK1 responses
in oocytes pretreated with opiate agonist for various periods. Peak
currents during test DAMGO/hK superfusions following 0-30-min
treatment with 1 µM DAMGO in ND96 were corrected for
currents found during superfusion of hK alone and reported as mean
± S.E. of maximal currents determined for DAMGO/hK responses
obtained before DAMGO pretreatments (times -20 to 0 min).
Recovery of peak currents obtained during test DAMGO/hK superfusions
performed during DAMGO washout (times 30-60 min). C,
dose-response relationship for DAMGO in inducing desensitization of
µ opiate receptor/GIRK1 responses. Oocytes were pretreated for 30
min with varying concentrations of DAMGO in ND96 and then tested by
DAMGO/hK superfusions. Values obtained during test DAMGO/hK
superfusions were corrected for currents found during superfusion of hK
alone and reported as mean ± S.E. of maximal currents determined
for DAMGO/hK responses obtained before DAMGO/ND96 pretreatments.
Maximal inhibition of control currents peaks at 70%; half of this
effect is reached at DAMGO concentrations
0.4 µM. D, recordings of responses to test superfusions with 10 nM DAMGO in hK (solid bars) followed by hK solution
superfusion for 2 min in oocytes pretreated for 40 min with ND96
control superfusions alone (left), 60 min with 100
nM4
-PMA (second from left), 40 min with 10
nM PMA (third from left), and 60 min with 10 nM PMA (right trace). E, time course of effects of
pretreatments with 100 nM PMA or 4
-PMA (solid
bar) on test responses to 10 nM DAMGO in hK medium. Peak
currents during test DAMGO/hK superfusions performed at various times
before or after 10-min superfusions of phorbol esters in ND96 are
reported as mean ± S.E. of maximal currents determined for
DAMGO/hK responses obtained before phorbol ester treatment. F,
dose-response relationships for the effects of 10-min PMA treatments in
inhibiting responses to 10 nM DAMGO obtained during test
DAMGO/hK superfusions performed 40 min later. Currents are corrected
for currents found during superfusion of hK alone and reported as mean
± S.E. of maximal currents determined for DAMGO/hK responses
obtained before PMA treatments. Maximal inhibition of control currents
peaks at
67%; half of this effect is reached at PMA concentrations
of 10.4 ± 3 nM (n =
5-7).
In
order to seek evidence for involvement of protein kinase C activation
on desensitization, we examined effects of bath-application
pretreatments with the active phorbol ester PMA and the inactive
control compound 4-PMA on opiate agonist-activated K
currents. Although these agents required DMSO for solubilization,
control experiments revealed that the 0.01% DMSO concentrations that
resulted in the superfusate produced no detectable effects on
DAMGO-activated currents (data not shown). To avoid concealing PMA
effects by agonist-induced desensitization manifested at greater
magnitude, experiments were conducted using 10 nM DAMGO
exposures for 1 min. These DAMGO concentration and exposure times did
not lead to desensitization (e.g.Fig. 2D).
Currents activated by 10 nM DAMGO were significantly
attenuated after application of 100 nM PMA for 10 min (Fig. 2E). This inhibition was maximal by 40 min after
PMA pretreatment. In initial experiments, the effect persisted for as
much as 2 h after the end of PMA treatment (data not shown). By
contrast, the amplitude of the current activated by 10 nM DAMGO in oocytes pretreated for 60 min with the inactive
4
-PMA was indistinguishable from that induced in oocytes not
pretreated (Fig. 2, D and E). The effect of
PMA was concentration-dependent, with an EC
of 10.4
± 3 nM (Fig. 2F). Maximal inhibition to
68 ± 6% of control values was reached at
300 nM PMA (p < 0.001, n = 5).
Figure 3:
A, autoradiogram of phosphoproteins
extracted from nonexpressing CHO cells (lanes 1 and 2) and hµCHO cells (lanes 3-9)
immunoprecipitated with anti-µ receptor antibodies and separated on
SDS-PAGE. Cells were pretreated for 20 min at 37 °C with 1
µM concentrations of morphine (lanes 2 and 4), morphine and naloxone (lane 5), morphine and 2
µM staurosporine (lane 6), PMA (lane 7),
PMA and 2 µM staurosporine (lane 8), and
4-PMA (lane 9). Autoradiogram was exposed for 2 days, and
size standards were derived from prestained markers electrophoresed in
adjacent lanes. B, effect of staurosporine on DAMGO-induced
desensitization. Expressing oocytes were pretreated with 2 µM staurosporine for 10 min (dotted bar) and then exposed to
desensitizing treatments with 1 µM DAMGO and 2 µM staurosporine in ND96 for various times (solid bar).
Responses to test DAMGO/hK superfusions performed at various times were
corrected for currents found during superfusion of hK alone and
reported as mean ± S.E. of maximal currents determined for
DAMGO/hK responses obtained during staurosporine pretreatments. C, the effects of staurosporine on PMA-induced
desensitization. Expressing oocytes were pretreated (solid
bar) with 2 µM staurosporine and 100 nM PMA
or with 100 nM PMA alone. Responses to test DAMGO/hK
superfusions performed at various times were corrected for currents
found during superfusion of hK alone and reported as mean ± S.E.
of maximal currents determined for DAMGO/hK responses obtained prior to
or during PMA or staurosporine and PMA
pretreatments.
The time course of receptor phosphorylation induced by morphine appeared to be rapid; it reached near-peak levels after 5 min of morphine exposure (Fig. 4A). This pattern differed slightly from that observed with PMA (Fig. 4C), which manifested stimulation of phosphorylation that began at least as early but was maintained perhaps more poorly with prolonged incubations. µ receptor phosphorylation was concentration-dependent for both receptor agonist and PMA (Fig. 4, B and D). Morphine-induced receptor phosphorylation was detectable after 10-min exposures to 10 nM morphine, and maximum phosphorylation was found after exposure to 1 µM morphine (Fig. 4B). Concentrations of PMA as low as 1 nM enhanced µ opiate receptor phosphorylation, whereas maximum effects were observed at 100 nM PMA (Fig. 4D).
Figure 4: Autoradiograms displaying the time course (A and C) and concentration dependence (B and D) of morphine (A and B) and PMA (C and D) effects on µ opiate receptor phosphoprotein densities in proteins extracted from hµCHO cells. A, µ opiate receptor phosphoproteins in hµCHO cells incubated with 1 µM morphine for 0 min (lane 1), 1 min (lane 2), 2 min (lane 3), 5 min (lane 4), 10 min (lane 5), and 20 min (lane 6). B, µ opiate receptor phosphoproteins in hµCHO cells incubated for 10 min with morphine at 0 nM (lane 1), 1 nM (lane 2), 10 nM (lane 3), 100 nM (lane 4), 1000 nM (lane 5), and 5000 nM (lane 6). C, µ opiate receptor phosphoproteins in hµCHO cells incubated with 1 µM PMA for 0 min (lane 1), 2 min (lane 2), 5 min (lane 3), 10 min (lane 4), 20 min (lane 5), and 40 min (lane 6). D, µ opiate receptor phosphoproteins in hµCHO cells incubated for 10 min with PMA at 0 nM (lane 1), 1 nM (lane 2), 10 nM (lane 3), 100 nM (lane 4), 1000 nM (lane 5), and 10,000 nM (lane 6).
Electrophysiological experiments also suggested that
staurosporine exerts differential effects on PMA and on opiate
agonist-induced alterations in inward K currents.
Staurosporine pretreatments virtually abolished PMA effects on inward
K
currents induced by nondesensitizing opiate agonist
treatments but failed to alter agonist-induced desensitization (Fig. 3, B and C).
The current results provide data that add to the identification of the µ opiate receptor as a potentially rapidly regulated phosphoprotein. It also provides evidence for parallels between the effects of time of opiate agonist exposure and agonist dose on the phosphorylation and desensitization of a major µ receptor function, opening inwardly rectifying potassium channels. Studies of the effects of agonists and PKC on µ receptor phosphorylation and function provide some of the first evidence that µ receptor phosphorylation is likely to be heterogeneous and that different phosphorylation patterns may contribute to µ receptor desensitization.
Several lines of biochemical evidence support the likelihood that the immunoprecipitated phosphoproteins examined in these studies include authentic phosphorylated µ opiate receptors. The immunoprecipitable phosphoproteins isolated from CHO cells expressing the µ opiate receptor include a 70-kDa band of the size found for the mature µ receptor purified from striatum but not found in protein extracted from CHO cells that do not express the µ receptor. The specificities of the anti-µ receptor C-terminal antibodies used here for immunoprecipitation have been well documented in Western blotting and immunohistochemical experiments (Surratt et al., 1994; Cheng et al., 1995). The morphine-specific patterns of regulation also support detection of authentic µ receptor in this protein band.
Our studies in Xenopus oocytes coexpressing µ receptor and potassium channel cDNAs
indicated initially brisk DAMGO-induced alteration of potassium
currents that desensitized to 50% of basal values with repeated
DAMGO applications and recovered with repeated washing over periods of
30 min. These desensitizing currents displayed several other
characteristics anticipated to µ receptor-mediated events. They
were also noted with repeated morphine administration, were blocked by
coadministration of the opiate antagonist naloxone, and were not seen
in oocytes that did not express the µ receptor. Interestingly, two
preliminary experiments each document that µ receptor-coupled
inhibition of adenylyl cyclase also displays desensitization. cAMP
levels in hµCHO cells decline 30-40% less following a second
challenge with DAMGO than after initial DAMGO application (data not
shown). Physiological desensitization of µ receptor responses can
thus occur in two distinct cellular systems and in two distinct
physiological responses.
Several observations suggest that protein
kinase C can induce µ receptor desensitization in the Xenopus expression system. The ability of the active phorbol PMA to
produce this effect, the failure of the inactive phorbol 4-PMA to
reproduce it, and the ability of staurosporine to inhibit it each
support its mediation through protein kinase C. These results are also
in accord with those reported by Yu and co-workers (Chen and Yu, 1994)
in a similar expression system, although they differ from those
obtained by Chavkin and colleagues (Kovoor et al., 1995).
These substantial effects of PKC stimulation contrast markedly with our
failure to detect any effects of the protein kinase A stimulator
forskolin on this desensitization (data not shown).
PMA and µ
receptor agonists both stimulate µ receptor phosphorylation by
5-fold. Since experiments assessing phosphorylation are performed
in the presence of a phosphatase inhibitor mixture, this magnitude of
phosphate accumulation may not provide an accurate estimate of the
magnitude of the effects of receptor or protein kinase C stimulation in
systems with normal balances between phosphatase and kinase activities.
Conceivably, differences in balances between phosphatase and kinase
activities in HEK293 cells could account for the stronger levels of
basal phosphorylation and weaker magnitudes of agonist-induced
phosphorylation recently reported in this expression system by Sadee
and co-workers (Arden et al., 1995). The magnitude of the
5-fold changes in phosphorylation found in our CHO cell expression
system is nevertheless equivalent to that found in studies of other
G-protein-coupled receptors e.g. (Pei et al., 1995;
Tobin and Nahorski, 1993). Our µ receptor phosphorylation results
could also represent underestimates of the extent of this receptor
phosphorylation if the antibody used to immunoprecipitate the receptor
recognized the phosphorylated µ receptor less avidly than the
nonphosphorylated receptor.
Comparisons between the time course and dose-response relationships for agonist-mediated µ receptor phosphorylation and agonist-mediated µ receptor desensitization reveal remarkable parallels. Each can be detected as early as 3 min following application of modest agonist concentrations. Each is reversible but persists for minutes following agonist washout. These observations are consistent with a role for phosphorylation in agonist-induced desensitization, although neither of these parallels by itself provides proof for causal relationships.
The differential
effects of staurosporine on agonist-induced and PKC-mediated responses,
however, provide striking evidence that these two means of receptor
modulation employ different biochemical pathways. While this PKC
inhibitor eliminated PMA influences on desensitization, as anticipated,
it failed to influence desensitizations induced by repeated
administration of agonist alone. These results point to the likelihood
that PKC activation may alter µ receptor phosphorylation in a
fashion different from that induced by agonists, even though agonist
activity at µ receptors can increase inositol trisphosphate
turnover in a fashion that may well stimulate PKC (Johnson et
al., 1994; Smart et al., 1994). Conceivably, these
results could also point toward utilization of different µ receptor
phosphorylation sites in different modes of receptor regulation.
Ongoing experiments with µ receptors mutated in these sites will
help to define such distinctions. They could argue as well for
participation of different members of the growing family of
G-protein-coupled receptor kinases in such distinct paths (Lefkowitz,
1993). Indeed, recent studies of agonist-dependent phosphorylation of
the opiate receptor suggest involvement of one or more
G-protein-coupled receptor kinases in this receptor's
desensitization (Pei et al., 1995).
Many brain neurons normally express several types of receptors. Effects of agonists at one receptor in desensitizing the same receptor are termed homologous desensitization, whereas effects of agonists at coexpressed receptors are termed heterologous desensitization. The presence of staurosporine-sensitive and staurosporine-insensitive pathways for stimulation of µ receptor phosphorylation makes more plausible the idea that µ receptor function could be modulated by prior exposure of cells to both agonists at the µ receptor and agonists at other coexpressed receptors. Conceivably, for example, heterologous desensitization could result from actions induced at coexpressed receptors by drugs often coadministered with opiates. Elucidating how dopamine/µ receptor costimulation as a result of psychostimulant/opiate coadministration or cannabanoid CB1/µ receptor costimulation with marijuana/opiate coadministration could alter the function of the µ receptor would help to understand the interactions between these commonly coadministered drug classes.
The expression systems used in current experiments rely on endogenous complements of G-proteins to interconnect µ receptors, cellular effectors, kinases, and G-protein-linked ion channels. Studies in normally receptor-expressing neurons may be required for accurate determination of the roles actually played by receptor phosphorylation events in vivo.
Agonist-induced desensitization, documented in functional assays, could be caused through a variety of mechanisms. Phosphoreceptor function could be altered while µ receptor was still expressed on cell surfaces, phosphoreceptor internalization could be speeded and/or phosphoreceptor degradation could be accelerated. Our data document recovery of receptor function within less than 3 min after beginning agonist washout in the Xenopus system. This time course is poorly compatible with that expected for reversal of receptor internalization or degradation but fits well with the rapid time course with which phosphorylation/dephosphorylation cycles can be observed for a number of cellular signaling proteins. Indeed, µ receptor phosphorylation could be noted at above background levels within less than 2 min of agonist or PMA treatment. Functional and biochemical analyses of µ receptor phosphorylation thus make this receptor's phosphorylation a plausible candidate to contribute to the mechanisms of the physiologically observed acute agonist-induced desensitization. However, other cellular proteins, including the GIRK1 and G-proteins that are necessary for the responses measured here, could also represent plausible phosphorylation targets and possible sites for agonist-induced desensitization. Studies of the magnitudes, time courses, and physiological effects of GIRK1, G-protein, and other important protein phosphorylation events will allow comparisons with the results presented here for the µ receptor and help to place their roles in physiological desensitization processes into the appropriate context.
It will be of interest to localize the sites of phosphorylation on the receptor, to determine which protein kinases are responsible for agonist-induced desensitization, and to understand how different receptor phosphorylation events could interact with each other. Each of these results should help to elucidate the nature of the regulatory mechanisms employed by this important brain receptor for pain and pleasure.