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Departments of Pharmacology (G.V., B.F.O., S.R.G.), Psychiatry (G.Y.K.N., P.S.), and Medicine (H.T.C., S.R.G.), University of Toronto; and the Addiction Research Foundation (B.F.O., S.R.G.), and the Eye Research Institute (J.T.), Toronto Western Hospital, Toronto, Ontario, Canada
Address all correspondence and requests for reprints to: Dr. Susan R. George, Department of Pharmacology, University of Toronto, Medical Sciences Building, Room 4358, 8 Taddle Creek Road, Toronto, Ontario, Canada M5S 1A8. E-mail: s.george{at}utoronto.ca
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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1 pmol receptor/mg
protein in Sf9 insect cells. Treatment of D2L
receptor-expressing cells with dopamine for up to 3 h resulted in
no detectable change in the ligand-binding properties of the receptor
and a 120-fold reduction in the potency, but not the efficacy, of
D2L receptors to mediate dopamine inhibition of
forskolin-stimulated adenylyl cyclase activity. This resistance of the
D2L receptor to agonist-induced desensitization was
accompanied by a 28% translocation of intracellular D2L
receptors to the cell surface, as quantified by cellular fractionation
and radioligand binding and visualized by whole cell immunocytochemical
staining and confocal microscopy. Immunoblot analysis of the P2
membrane fraction revealed that surface D2L receptors
comprised monomers and dimers. Treatment of D2L
receptor-expressing cells with the protein synthesis inhibitor
cycloheximide significantly reduced the basal expression level of
receptors, but did not block the agonist-induced up-regulation of
receptors. Longer periods of dopamine exposure for 24 h brought
about a small increase in surface receptor density. However, when these
studies were conducted in the presence of cycloheximide, receptor
density was marginally reduced, suggesting that receptor synthesis
accounts for the maintenance of cellular receptor density under these
conditions. We conclude that the resistance of the D2L
receptor-coupled adenylyl cyclase system to agonist-induced
desensitization is attributed to the up-regulation of surface receptors
after the translocation of existing intracellular receptors and
de novo receptor synthesis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies have shown that desensitization of the D2L receptor-inhibited adenylyl cyclase system is slow to occur and results only after very prolonged agonist exposure for 24 h (9, 10, 11). Yet, other studies have reported the absence of desensitization (12, 13). Similarly, the effects of agonist on D2L receptor density are controversial. Agonist exposure has been shown to not affect receptor density (9), sequester receptors (14), or up-regulate receptors on cultured cells (10, 11, 12, 13). A clear explanation has been lacking, and it is unlikely to be attributed to differences in receptor expression level, because most of these studies used stably transfected or endogenously D2 receptor-expressing cells generating about 1 pmol receptor/mg protein (10, 11, 12, 13, 14). Agonist-induced up-regulation of D2L receptors was also observed in cells expressing approximately 50 fmol of the receptor (12). The likely explanation may be that these studies reported on different aspects of the biology of the D2L receptor.
The recombinant baculovirus/Sf9 insect cell system has been shown to be highly suitable for the study of the structure and function of a wide range of heterologous genes. We have shown that Sf9 cells can express D2L receptors to a specific activity of 1–2 pmol receptor/mg protein, and that expressed D2L receptors are pharmacologically similar to their neuronal counterparts (15). Further, we showed that D2L receptors are appropriately posttranslationally modified in this cell line (15), and others have shown that D2L receptors expressed in the Sf9 cells are coupled to endogenous G proteins and mediate inhibition of adenylyl cyclase activity (16). Hence, the baculovirus/Sf9 model system can be highly suitable for studies of a fundamental nature. The high target signal to low background is an advantage of the Sf9 cells that adds only to the confidence of the experimental results. Hence, the baculovirus/Sf9 model system was selected to clarify the mechanisms that may explain the differential regulation of the D2L receptor after agonist exposure. The experiments described herein show that resistance of the D2L receptor-coupled adenylyl cyclase to desensitization accompanies the up-regulation of surface receptors. This newly discovered replenishment mechanism is subject to at least two distinct mechanisms, involving, in the short term after agonist exposure, the redistribution of existing pools of receptors and, in the long term after agonist exposure, receptor synthesis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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5). All cells were harvested at the 48 h postinfection point.
To minimize variations in recombinant baculovirus infection
efficiencies among batches of cultures, the same recombinant virus
stock was used in all experiments for this study.
Dopamine and cycloheximide treatments
D2L receptor-expressing Sf9 cells were cultured in
serum-free Grace’s medium when examining the effects of dopamine.
Dopamine treatments were performed for 3–4 h, similar to the
t1/2 reported for the up-regulation of D2L
receptors expressed in HEK-293 cells (17) and CHO cells (10), and were
as long as 24 h, which was a common time point used in previous
studies as well (10, 12, 13). Dopamine was used at a
10-µM final concentration in the presence of 0.01
mM ascorbic acid.
To confirm that agonist-induced up-regulation of D2L receptors was not due to receptor synthesis, dopamine treatments were conducted in the presence of the protein synthesis inhibitor, cycloheximide. Cycloheximide was used at a final concentration of 10 µg/ml. Cycloheximide at a concentration of 5 µg/ml has been used to block protein synthesis in D2L receptor-expressing CHO (10) and C6 glioma (11) cells. Further, these studies used a Sf9 cell line adapted for permanent growth in serum-free Sf900II medium so as to eliminate serum catecholamine effects. Typically for all studies, cells were 90% viable or greater as assessed by exclusion of trypan blue stain. Incubations were terminated by pelleting cells at 100 x g for 10 min and washing the cells extensively with ice-cold PBS.
Statistical significance between control and treatment conditions was determined by Student’s paired t test.
Preparation of membrane fractions
Cells were pelleted by centrifugation at 100 x
g for 7 min, washed with PBS, and resuspended in 10 ml
buffer A (5 mM Tris-HCl and 2 mM EDTA buffer,
pH 7.4, containing the protease inhibitors: 10 µg/ml benzamidine, 5
µg/ml leupeptin, and 5 µg/ml soybean trypsin inhibitor). The cell
suspension was homogenized and centrifuged at 100 x g
for 7 min to pellet unbroken cells and nuclei. The S1 supernatant was
collected and centrifuged at high speed (27,000 x g
for 20 min) washed once with buffer A, and centrifuged again at high
speed to prepare the P2 membranes. P2 membranes were resuspended in
buffer A and stored at -80 C or resuspended in buffer B (75
mM Tris-HCl, 12.5 mM MgCl2, and 2
mM EDTA, pH 7.4) and assayed immediately for adenylyl
cyclase activity. Alternatively, the S1 supernatant was used to prepare
a cell surface heavy plasma membrane fraction and an intracellular
light vesicular membrane fraction that exhibits a lower density than
that of the heavy plasma membrane (18, 19). This was performed by
layering the S1 supernatant on top of a 35% sucrose cushion, which was
then subjected to centrifugation at 150,000 x g for 90
min at 4 C. As reported by Lohse et al. (20), the 0–35%
interface contains the light vesicular membrane fraction believed to
contain sequestered receptors, whereas the heavy plasma membrane
fraction containing surface receptors sediments at the bottom of the
sucrose cushion. Heavy plasma and light intracellular membrane
fractions were resuspended in buffer A and centrifuged at 200,000
x g for 60 min. Pelleted membranes were resuspended in
buffer A and stored at -70 C or resuspended in the appropriate buffers
for immediate use in various assays. Membrane protein content was
determined using a Bradford assay kit (Bio-Rad, Richmond, CA).
Radioligand binding
The binding of [3H]spiperone and
[3H]nemonapride (New England Nuclear, Boston, MA) to P2
membranes prepared from D2L receptor-expressing Sf9 cells
has been described previously (15, 21). Binding constants
[Kd, binding capacity (Bmax), and
Ki values] were determined by best-fit analysis using the
plotting program GraphPad InPlot Version 4.03 (San Diego, CA).
Adenylyl cyclase assay
Freshly prepared P2 membranes from D2L
receptor-expressing cells were used for adenylyl cyclase
determinations. The assay mix contained 0.02 ml membrane suspension
(25 µg protein), 0.012 mM ATP, 0.1 mM
cAMP, 0.053 mM GTP, 2.7 mM phosphoenolpyruvate,
0.2 U pyruvate kinase, 1 U myokinase, and 0.13 µCi
[32P]ATP in a final volume of 0.05 ml. Enzyme activities
were determined in triplicate assay tubes containing
10-3–10-9 M dopamine with 10
µM forskolin and incubated at 27 C for 20 min. Reactions
were stopped by the addition of 1 ml of an ice-cold solution containing
0.4 mM ATP, 0.3 mM cAMP, and
[3H]cAMP (25,000 cpm). cAMP was isolated by sequential
column chromatography using Dowex cation exchange resin and aluminum
oxide. Data were analyzed by computer-fitted nonlinear least squares
regression (GraphPad InPlot version 4.03).
In situ immunofluorescence labeling
Cells expressing the receptor subtype of interest cultured in
suspension (1.5 x 106/ml) were aliquoted into
Eppendorf tubes for indirect immunocytofluorescent staining. For the
visualization of receptors, cells were fixed with freshly prepared 4%
paraformaldehyde in PBS for 15 min, washed in PBS, pelleted, and
permeabilized with methanol at -70 C for 3 min. Immunocytochemical
staining was performed at room temperature. To reduce nonspecific
staining, cells were incubated for 1 h in a blocking solution of
1% BSA and 5% goat serum. The cells were then incubated with 9E10
primary antibody for the c-myc of c-myc D1
receptor or with the AL-26 polyclonal antibody for the D2L
receptor (a gift from Dr. Mark R. Brann, University of Vermont,
Burlington, VT) followed by incubation with a fluorescein
isothiocyanate-conjugated secondary antibody each for 1 h. Cells
were washed with PBS for 15 min and pelleted. This was repeated twice
before the cells were resuspended in the mounting medium Mowiol-88
(Hoechst, Montreal, Canada) to which 1,4-diazabicyclo[2.2.2]octane
(Sigma Chemical Co., St. Louis, MO) had been added to reduce
photobleaching. The visualization of whole cells by confocal microscopy
and the protocol used for the classification of cell staining patterns
have been reported in detail previously (8).
SDS-PAGE and immunoblot analysis
Tissues were solubilized in sample buffer consisting of 50
mM Tris-HCl (pH 6.5), 10% SDS, 10% glycerol, 0.003%
bromophenol blue, and 10% 2-mercaptoethanol and electrophoresed on
precast 12% mini-Tris-glycine gels (Novex, San Diego, CA) under
reducing and denaturing conditions. Samples were electroblotted onto
nitrocellulose membrane and incubated with a D2 receptor-specific
rabbit (AL-26) polyclonal antibody as reported previously (15). Primary
antibody-bound receptor was detected with a goat antirabbit IgG
alkaline phosphatase conjugate (Bio-Rad). Blots were developed with
5-bromo-4-chloro-3-indoyl-phosphate/4-nitro blue tetrazolium chloride
substrate (Bio-Rad) according to the manufacturer’s instructions.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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shows the saturation
isotherms for [3H]spiperone binding to P2 membranes
prepared from vehicle-treated D2L receptor-expressing cells
and D2L receptor-expressing cells treated with 10
µM dopamine for 30 min and 3 h.
[3H]Spiperone binding increased significantly after
agonist treatment, rising from a Bmax of 1.2 ± 0.4
pmol/mg protein (n = 3) to a Bmax of 1.9 ± 0.2
pmol/mg protein (n = 3; P < 0.05) after 3 h.
However, no significant changes in Kd values [153 ±
11 pM (n = 3) and 138 ± 25 pM
(n = 3), respectively] were observed in these dopamine-treated
tissues, indicating that increased receptor density was not due to an
increased affinity of the receptor for ligand. Sf9 cells infected with
wild-type baculovirus did not show any specific
[3H]spiperone binding (data not shown). These data
indicated that the D2L receptor system responds to
sustained agonist exposure at least in part by increasing receptor
density on the surface of cells.
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shows dopamine competition of specific
[3H]spiperone binding to P2 membranes prepared from
D2L receptor-expressing cells treated with vehicle or
dopamine for up to 3 h. Surprisingly, no significant differences
were observed for dopamine-detected high and low affinity receptor
states between vehicle-treated and dopamine-treated tissues.
Ki values and proportions for dopamine-detected high and
low affinity states in vehicle-treated tissues were 32 ± 4
nM (23%) and 31 ± 4 µM (77%) (n
= 4). Ki values and proportions for dopamine-detected high
and low affinity states for tissues treated with dopamine for 30 min
were 30 ± 7 nM (27%) and 41 ± 10
µM (73%) (n = 3), and those for 3 h were
26 ± 12 nM (24%) and 24 ± 5 µM
(76%) (n = 3). These data indicated that in comparison to
dopamine D1 receptors, agonist-induced uncoupling of D2L
receptors from G proteins is slow to occur.
Expressed D2L receptors mediated dopamine inhibition
(21.3 ± 1.9%; n = 4) of forskolin-stimulated adenylyl
cyclase activity with an IC50 of 22 ± 6
nM (n = 4; Fig. 1C). After exposure of D2L
receptor-expressing cells to dopamine for 30 min, the efficacy and
potency of receptors to mediate dopamine inhibition of
forskolin-stimulated adenylyl cyclase activity was essentially
unchanged (IC50 of 31 ± 11 nM; n =
3; Fig. 1C). However, longer periods of dopamine exposure (3 h)
resulted in a marked loss of potency (IC50 of 3.0 ±
0.5 µM; n = 3) with little alteration in the
efficacy of receptors to inhibit adenylyl cyclase activity (Fig. 1C).
All experiments were performed on cells expressing 2 pmol
receptor/mg membrane protein (48 h postinfection), as assessed by
[3H]spiperone binding, and were 85–90% viable, as
assessed by trypan blue staining. No dopamine-sensitive adenylyl
cyclase was detected in Sf9 cells infected with the wild-type
baculovirus (data not shown). These data indicate that functional
uncoupling of the D2L receptor is slow to occur after
agonist exposure.
Agonist-mediated translocation of D2Lreceptors from a light membrane fraction to a heavy membrane
fraction
As several studies have reported no association between D2
receptor up-regulation and messenger RNA (mRNA) levels (11, 12, 13), we
tested the hypothesis that the agonist-promoted up-regulation of
receptors involved the redistribution of existing pools of receptors.
The number of receptors present in the heavy cell surface plasma
membrane and in the light intracellular vesicular membrane fraction
were assessed directly by subcellular fractionation and radioligand
binding experiments. Agonist exposure of D2L
receptor-expressing cells resulted in a slowly evolving translocation
of D2L receptors from the light membrane fraction to the
heavy plasma membrane fraction (Fig. 2).
Receptor translocation was defined as an increase in the number of
receptors in the cell surface heavy plasma membrane accompanied by a
proportional decrease in the number of receptors in the light vesicular
membrane. This D2L receptor response to agonist stimulation
began to plateau (18 ± 4%; n = 3; P <
0.05) after 1 h and appeared to be near maximal (28 ± 6%;
n = 3; P < 0.05) after 6 h. Total receptor
densities in vehicle- and dopamine-treated cells over this time period
were not different. Total cellular receptor was defined as the total
receptor density present in the heavy plasma membrane fraction added to
the receptor density in the light intracellular vesicular membrane
fraction. These data provide the scientific basis to suggest that the
agonist-induced up-regulation of receptors on the cell surface was
attributed to the redistribution of existing intracellular receptor
pools.
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).
Receptor labeling was visualized by confocal immunofluorescent
microscopy, which optically sections a cell such that each section or
slice could be viewed individually. Cells were permeablized so that
both cell surface and internally located receptors could be detected;
hence, the technique provides a valuable spatial description of the
effects of agonist on the cellular distribution of the receptor. All
comparative analysis was performed under identical illuminating and
viewing conditions.
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). Dopamine exposure (10
µM dopamine for 4 h) mediated a redistribution of
internally located receptors to the cell surface, as evidenced by the
loss of label in the cytoplasm and an accompanying increase in the ring
of fluorescence on the cell surface (compare A to B).
For comparison, the effects of agonist exposure on the cellular
distribution of a c-myc epitope-tagged D1 receptor was
assessed in the identical Sf9 cell line. Figure 3D shows that at steady
state, c-myc D1 receptors are diffuse over the cell surface,
appearing as a bright and continuous ring. Internally located receptors
appear as clusters of label, which may represent localization to
intracellular compartments or vesicles. In cells pretreated with 10
µM dopamine for 1 h, cell surface receptor labeling
changed from a bright continuous ring to a broken and less intense ring
with increased intracellular pockets of label (compare D to E). These
data suggest that dopamine mediated a redistribution and reduction of
the number of surface D1 receptors, data we have described in detail
previously (8). Control cells, expressing no receptors, are barely
visible, reflecting the absence of specific immunocytochemical labeling
(Fig. 3, C and F). The results of these experiments demonstrated for
the first time distinct regulation of D1 and D2L receptors
when expressed in the same foster cell line. Moreover, the differences
in receptor redistribution observed for D1 and D2L
receptors after agonist treatment in the identical Sf9 cell line
provide evidence that these are receptor subtype-specific responses and
not an artifact of the cell line.
Agonist-promoted up-regulation of surface
D2L receptor monomers and dimers
Immunoblot analysis of the P2 membrane fraction prepared from
D2L receptor-expressing cells showed that D2L
receptors were composed of an approximately 44-kDa species representing
a receptor monomer and, at approximately twice the molecular mass, an
approximately 98-kDa form, representing a receptor dimer (Fig. 4, lane 1). The biophysicochemical
properties of the D2L receptor dimer have been recently
reported (21). Immunoblot of a P2 membrane fraction prepared from
D2L receptor-expressing cells treated with dopamine showed
an increase in both receptor monomer and dimer (Fig. 4, compare lanes 1
and 2), consistent with radioligand binding data. Taken together, these
results raise the speculation of a functional role for D2 dimers in the
response to agonist.
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).
D2L receptors were expressed to 706 ± 143 fmol
receptor/mg protein (n = 3) in serum-free adapted Sf9 cells, which
was significantly reduced to 460 ± 37 fmol receptor/mg protein
(n = 3) after treatment with cycloheximide for 4 h
(P < 0.05). Figure 5
shows that treatment of D2L receptor-expressing cells with
cycloheximide did not block the dopamine-induced up-regulation of
D2L receptor density after 4 h of agonist exposure.
These findings support the radioligand binding and whole cell
immunocytochemical studies showing that the agonist-promoted
up-regulation of surface D2L receptors involved existing
pools of receptors.
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). Taken
together, these studies suggest the need for de novo
receptor synthesis in the maintenance of cell surface receptor density
after prolonged 24-h dopamine treatment. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Agonist treatment of D2L receptor-expressing cells for 3 h resulted in no detectable change in ligand-binding properties, but a loss of potency (but not efficacy) of the D2L receptor to inhibit adenylyl cyclase activity was observed, indicating that sustained agonist exposure initiates the desensitization process. These findings are in good agreement with the reported resistance (and/or slow onset) of the D2L receptor-coupled adenylyl cyclase to negative regulation by agonist in other mammalian cell systems (9, 10, 11, 12, 13). However, no clear mechanism has yet been linked to this receptor/cellular adaptation. It has been suggested that agonist binding to vitamin D receptors may render the receptor conformationally more stable (23), a mechanism that may account for the differential response of the D2L receptor-coupled adenylyl cyclase system to sustained agonist exposure. Our data show that the resistance of the D2L receptor to desensitization occurred with an agonist-promoted up-regulation of surface receptors that comprised receptor monomers and dimers. D2L dimers may represent a newly discovered functional intermediate in the response to agonist exposure; this work is underway. In fact, recent evidence showed possible biological activity for the ß2-adrenergic receptor dimer (22). Our subcellular fractionation and radioligand binding and whole cell confocal microscopy studies suggested that the agonist-promoted receptor up-regulation involved existing pools of receptors. Indeed, we showed that the agonist-promoted up-regulation of surface D2L receptors was not blocked by the protein synthesis inhibitor cycloheximide, which supports this conclusion. Further, the GnRH receptor has been reported to up-regulate when exposed to GnRH and does not appear to involve regulation of the synthesis or stability of receptor mRNA (24). It has been postulated that intracellular receptors could serve as reserve pools of receptors that may be induced by agonist to translocate to the cell surface or into functional microdomains (25, 26). We provide evidence to support this claim. Interestingly, agonist treatment has been shown to result in the up-regulation of D2-like dopamine D3 receptors on cultured C6 cells as well (27), suggesting that this may constitute a more general adaptive response in this receptor subfamily.
In contrast to the majority of D2L receptor regulation studies, a single report has recently shown the agonist-promoted sequestration of the D2L receptor based on the loss of hydrophilic [3H]sulpiride binding, with total hydrophobic [3H]spiperone binding remaining unchanged (14). Differences in receptor expression among the studies probably does not account for the conflicting results, as most of the studies used stably transfected or endogenously D2L receptor-expressing cells or, as shown herein, D2L receptor expressing Sf9 cells generating approximately 1 pmol receptor/mg protein (10, 11, 12, 13, 14). It could be explained that agonist exposure triggers a conformational change in surface D2L receptors, possibly rendering receptors refractory or inaccessible to hydrophilic ligands, as suggested previously for a model of ß2-adrenergic receptor sequestration (28). Indeed, it was recently reported that agonist treatment of D2L receptor-expressing HEK-293 cells led to a decreased proportion of surface receptors in the high affinity, agonist-preferring state as assessed by [125I]7-hydroxy-2-[N-n-propyl-N-(3'-iodo-2'-propenyl)-amino]tetralin binding (17). In the same experiments, agonist treatment led to an increase in surface D2L receptor density, as measured by antagonist [125I]NCQ 298 binding (17). Hence, the loss of hydrophilic [3H]sulpiride binding, with total hydrophobic [3H]spiperone binding remaining unchanged, may indeed result from an agonist-induced conformational change in the receptor. Depending on the method of analysis, past D2L receptor regulation studies (10, 11, 12, 13, 14) may have simply monitored different aspects of D2L receptor biology. That total hydrophobic spiperone binding remained unchanged in the D2L receptor sequestration study is consistent with our data, which support the conclusion that the up-regulation of surface D2L receptors after shorter term agonist exposure (3–4 h) involved existing pools of receptors.
Different mechanisms appear to be involved in regulating cellular D2L receptor density after prolonged (24-h) dopamine treatment. We observed no significant increase in D2L receptor density after 24-h agonist exposure. When these studies were conducted in the presence of cycloheximide, dopamine exposure led to a marginal decrease in surface D2L receptor density. This suggests that prolonged dopamine exposure may lead to the down-regulation of receptors and that de novo protein synthesis plays an important role in the agonist-promoted up-regulation of D2L receptor density under these conditions. In agreement with this idea, sustained agonist exposure has been associated with an increase in the steady state level of D2L mRNA in D2L receptor-expressing CHO cells (10). Agonist-induced up-regulation of 5-HT2 receptors on cultured cerebellar neurons and of ß2-adrenergic and ß3-adrenergic receptors in cultured cells (29, 30, 31) has also been attributed to alterations in transcriptional or translational activity (biosynthesis rate and/or altered stability and levels of receptor mRNA). Taken together, these results suggest that surface D2L receptor density is regulated by protein synthesis over the long term (24 h) after agonist exposure.
Several observations provide interesting speculations about possible
mechanisms underlying D2L receptor trafficking. First,
G
i3 proteins, to which D2 receptors have been reported
to couple (32), have been implicated in the activation of noncoated
vesicle-mediated exocytosis (33). Second, Gß
dimers
have been shown to regulate not only receptor kinase-mediated
phosphorylation and desensitization of receptors, but also
intracellular vesicular trafficking (25, 34). These findings suggest
that G proteins may play a role in D2 receptor trafficking.
Interestingly, treatment of D2L/CHO cells and D2L/HEK 293
cells with pertussis toxin blocked dopamine-induced receptor
up-regulation (10, 16), although a single study showed no effect
(11).
D2 receptor-mediated mechanisms are important for autoreceptor function
in dopamine neurons and in tonic inhibitory functions, such as the
inhibition of PRL or MSH secretion. The sensitivity of D2 receptor
agonists during long term control of PRL-secreting adenomas is
exquisite, but the mechanisms remain unknown. Hence, the relative
insensitivity of the D2 receptor system to desensitization by agonist
reported herein appears to be functionally important. It should also be
noted that D2 receptor up-regulation and behavioral supersensitivity to
D2 agonist have been observed in animals treated with indirect dopamine
agonists (35, 36, 37, 38), for which we now provide a mechanistic basis. In
turn, this observation substantiates the high likelihood that our
results are physiologically relevant. We propose that the newly
discovered recruitment of existing intracellular D2L
receptors to the cell surface, resulting in the up-regulation of
D2L receptor monomers and dimers, and the longer term
de novo receptor synthesis after agonist exposure contribute
to the sustained pattern of agonist-mediated activation of
D2L receptor-linked intracellular events.
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Footnotes |
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2 Current address: Department of Biochemistry and Molecular Biology,
Merck Frosst Canada, Inc., P.O. Box 1005, Pointe-Claire-Dorval, Quebec,
Canada H9R 4P8.
Received April 17, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T3-1 cells is associated with unchanged receptor
messenger RNA (mRNA) levels and altered mRNA activity. Mol Endocrinol 7:1625–1633
-dimers in transmembrane signalling. Nature 365:403–406[CrossRef][Medline]
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 R. G. Vickery and M. von Zastrow Distinct Dynamin-dependent and -independent Mechanisms Target Structurally Homologous Dopamine Receptors to Different Endocytic Membranes J. Cell Biol., January 11, 1999; 144(1): 31 - 43. [Abstract] [Full Text] [PDF]
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 B. Adams and B. Moghaddam Corticolimbic Dopamine Neurotransmission Is Temporally Dissociated from the Cognitive and Locomotor Effects of Phencyclidine J. Neurosci., July 15, 1998; 18(14): 5545 - 5554. [Abstract] [Full Text] [PDF]
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) and from cells treated with dopamine for 30
min (•) or 3 h (
). Radioligand binding data were best fitted
statistically using computer analysis (InPlot 4, GraphPad). Nonspecific
binding for all radioligand binding experiments was defined by 1
µM (+)-butaclamol. Results shown are from single
experiments, each representative of three to five determinations. C,
Membranes were prepared from cells expressing D2L receptors in the
basal state (
