From the Ottawa Health Research Institute, Ottawa Hospital (Civic Campus), and Departments of Medicine/Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario K1Y 4K9, Canada
Received for publication, August 7, 2002, and in revised form, December 17, 2002
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ABSTRACT |
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A chimeric D1A dopaminergic receptor harboring
the cytoplasmic tail (CT) of the D1B subtype (D1A-CTB) has been used
previously to show that CT imparts high dopamine (DA) affinity
and constitutive activity to the D1B receptors. However, the D1A-CTB
chimera, unlike the D1B subtype, exhibits a significantly lower DA
potency for stimulating adenylyl cyclase and a drastically lower
maximal binding capacity (Bmax). Here, using a functional
complementation of chimeric D1-like receptors, we have
identified the human D1B receptor regions regulating the
intramolecular relationships that lead to an increased DA potency and
contribute to Bmax. We demonstrate that the addition of
variant residues of the third extracellular loop (EL3) of the human D1B
receptor into D1A-CTB chimera leads to a constitutively active mutant
receptor displaying an increased DA affinity, potency, and
Bmax. These results strongly suggest that constitutively
active D1-like receptors can adopt multiple active conformations,
notably one that confers increased DA affinity with decreased DA
potency and Bmax and another that imparts increased DA
affinity with a strikingly increased DA potency and Bmax.
Overall, we show that a novel molecular interplay between EL3
and CT regulates multiple active conformations of D1-like receptors and
may have potential implications for other G protein-coupled receptor classes.
Dopamine (DA)1 elicits
its physiological and endocrine effects through the interaction with
D1-like and D2-like G protein-coupled receptor (GPCR) subfamilies (1).
D1-like receptors couple to the activation of adenylyl cyclase (AC) and
in mammals are further divided into D1A (or D1) and D1B (or D5)
subtypes. The D1B receptor distinguishes itself from the D1A subtype by
a higher constitutive activity, increased affinity and potency for
agonists, decreased affinity for antagonists, and a lower
agonist-mediated maximal activation (2). Recently, a study highlighted
the potential physiological relevance of the D1B receptor constitutive
activity (3). Indeed, the extent of the D1B receptor constitutive
activity controls the estrogen-induced mRNA expression of atrial
natriuretic factor in primary hypothalamic neurons, a neuroendocrine
process that potentially plays a role in D1B receptor-mediated
facilitation of female sexual behavior (3). Moreover, the detection of
D1-like subtype mRNA expression and/or activity in human breast and
neuroendocrine gastrointestinal tumor cells (4, 5) underscores the
importance of these receptors as well as, potentially, their levels of
constitutive activation in regulating DA function in pathophysiological
conditions, as shown previously with other constitutively active mutant
GPCRs (6). Thus, D1A and/or D1B subtype-specific ligands may help in
the treatment of pathologies for which compromised D1-like receptor
responsiveness is purported (7-9). However, the structure-function relationships shaping the functional properties of the D1-like receptors remain unclear.
Previously, we have shown that a structural domain (referred to as the
terminal receptor locus (TRL)) encompassing the sixth and seventh
transmembrane regions (TM6 and TM7), third extracellular loop (EL3) and
cytoplasmic tail (CT) plays an important role in the phenotypic
expression of ligand binding and G protein coupling properties of
D1-like receptors (10). To narrow down the structural determinants
within TRL involved in the subtype-specific ligand binding and G
protein activation properties of the D1-like receptors, additional
mutant receptors were constructed in which either the EL3 (10) or CT
sequences (11) were swapped between the D1A and D1B receptors. Chimeric
receptors harboring EL3 of their respective wild-type counterparts
exhibited a complete reversal of agonist-mediated maximal activation of
AC, whereas DA affinity and constitutive activity of the chimeras were
only partially modulated by the exchange (10). In contrast, chimeric
receptors harboring the CT of their respective wild-type counterparts
displayed a full switch in DA affinity and constitutive activity,
whereas DA potency decreased and agonist-mediated maximal activation of
AC increased for both chimeras (11). The decrease in DA potency is
particularly intriguing for the constitutively active D1A-CTB chimera
in light of its ability to bind DA with high affinity, reminiscent of
the DA affinity for wild-type D1B receptors (11). Moreover, mutagenesis studies revealed a previously unappreciated role of these receptor regions in determining the maximal binding capacity (Bmax)
of D1-like subtypes (10-13), a facet of the D1-like receptor function that may underlie the cognitive and working memory deficits observed in
schizophrenia (14, 15). Our previous findings hint at the complexity of
intramolecular relationships within the TRL and indicate that discrete
receptor regions responsible for regulating specific GPCR activation
properties can be dissociated by mutations (10-13). Furthermore, these
studies suggest the existence of multiple active conformations of
constitutively active D1-like receptors, notably an active conformation
imparting increased constitutive activity, DA affinity, and potency
with unchanged Bmax (e.g. wild-type D1B
versus wild-type D1A receptors) or another conferring
increased constitutive activity and DA affinity but decreased DA
potency and Bmax (e.g. D1A-CTB chimera
versus wild-type D1-like receptors).
In the present study, we tested whether EL3 and CT regions control the
phenotypic expression of D1-like subtype-specific ligand binding and G
protein coupling properties. To do so, we used a mutagenesis approach
and functional complementation of different chimeric D1-like receptors
expressed in human embryonic kidney 293 (HEK 293) cells to probe the
potential molecular interplay between EL3 and CT regions. Here we
report that a functional complementation of the constitutively active
D1A-CTB chimera with the EL3 region of the D1B receptor leads to slight
increase in DA affinity while causing a drastic increase in DA potency
and Bmax. Thus, these results indicate the existence of
distinct molecular interplay between the CT and EL3 involved in the
regulation of discrete aspects of D1-like receptor signaling
(e.g. multiple active conformations and
Bmax).
Drugs--
N-[Methyl-3H]SCH23390
(82 Ci/mmol), [3H]adenine (27 Ci/mmol), and
[14C]cAMP (275 mCi/mmol) were from Amersham
Biosciences. DA, cis-flupentixol, and
1-methyl-3-isobutylxanthine were from Sigma.
Construction of Chimeric Human D1A and D1B Receptors--
To
construct chimeric receptors harboring only the EL3 and CT regions of
their wild-type counterparts, we took advantage of existing chimeras in
which the EL3 region was swapped between the D1A and D1B subtypes (10).
We have utilized these chimeras (D1A-EL3B and D1B-EL3A) and wild-type
receptors as templates to engineer two additional chimeric receptors
(D1A-EL3CTB and D1B-EL3CTA) in which the CT regions were exchanged by
gene splicing using a PCR-based overlap extension approach. The
receptor sequences were swapped at the junction between the TM7 and CT
regions. The junction corresponds to amino acid 334 and 362 in the D1A
and D1B receptor, respectively. The D1A-CTB and D1B-CTA chimeras have been described previously (11).
In the case of the D1A-EL3CTB chimera, the first round of PCR generated
two fragments: the AB1 fragment encoding the third intracellullar loop,
TM6, EL3, and TM7 of D1A-EL3B receptors; and the B2 fragment coding for
the CT of D1B receptors. The AB1 fragment was amplified using
primers 5'-CACCACAGGTAATGGAAA-3' (forward) and 5'-ATTAAACGCGTAAAT-3'
(reverse). The B2 fragment was generated using primers
5'-ATTTACGCGTTTAATGCCGACTTTCAGAAGGTGTTTG-3' (forward) and
5'-TGCAACTTAATTTTATTA-3' (reverse). Likewise, in the case of the
D1B-EL3CTA chimera, the first round of PCR generated two fragments: the
BA1 fragment coding for the N-terminal region of the D1B-EL3A receptor
up to TM7 and the A2 fragment encoding the CT of D1A receptor. The BA1
fragment was amplified using primers 5'-TACGGTGGGAGG-3' (forward) and
5'-GTTGAAGGCATAGAT-3' (reverse). The A2 fragment was amplified using
primers 5'-ATCTATGCCTTCAACGCTGATTTTCGGAAAGCTTTTTCAACCCTC-3' (forward)
and 5'-TGCAACTTAATTTTATTA-3' (reverse). To facilitate the construction
and identification of the chimeric receptors, a silent mutation
was introduced in each construct to create a unique restriction
endonuclease site. For the D1A-EL3CTB chimera, a MluI site
was introduced at a nucleotide sequence corresponding to amino acids
335 and 336 (5'-TATGCC-3' Cell Culture and Transfection--
HEK 293 cells (American Type
Culture Collection, Manassas, VA) were cultured at 37 °C and 5%
CO2 in minimum essential medium supplemented with 10%
heat-inactivated fetal bovine serum and gentamicin (10 µg/ml)
(Invitrogen). Cells were seeded into 100-mm dishes (2.5 × 106 cells/dish) and transiently transfected with 5 µg of
DNA/dish using a modified calcium phosphate precipitation procedure as described (16). When less than 5 µg of DNA was used in transfections, empty pCMV5 vector was added to normalize the total amount of DNA. All
experiments were performed with cells from 38 to 52 passages.
Membrane Preparation--
Following an overnight incubation with
the DNA-calcium phosphate precipitate, HEK 293 cells were washed with
phosphate-buffered saline, trypsinized, reseeded in 150-mm dishes, and
grown for an additional 48 h. Transfected HEK 293 cells were then
washed with cold phosphate-buffered saline, scraped from the dish in ice-cold lysis buffer (10 mM Tris-HCl, pH 7.4, 5 mM EDTA), and centrifuged at 40,000 × g
for 20 min at 4 °C. The crude membrane pellet was resuspended in
lysis buffer using a Brinkmann Polytron (17,000 rpm for 15 s) and
centrifuged at 40,000 × g for 20 min at 4 °C. The
final pellet was resuspended in lysis buffer, and membranes were either
used immediately (saturation studies) or frozen in liquid nitrogen and
stored at Radioligand Binding Assays--
Fresh or frozen membranes were
diluted in binding buffer (final in binding assays: 50 mM
Tris-HCl, pH 7.4, 120 mM NaCl, 5 mM KCl, 4 mM MgCl2, 1.5 mM CaCl2,
1 mM EDTA) and mixed briefly using a Brinkmann Polytron.
Binding assays were performed with 100 µl of membranes in a total
volume of 500 µl using
N-[methyl-3H]SCH23390 as
radioligand. Saturation studies were done using fresh membranes and
concentrations of
N-[methyl-3H]SCH23390 ranging from
0.01 to 6 nM. Nonspecific binding was assessed in the
presence of a final concentration of 10 µM
cis-flupentixol (dissolved in milli-Q-water). For competition studies,
frozen membranes were thawed on ice and incubated with a constant
concentration of
N-[methyl-3H]SCH23390 (~0.6
nM) and increasing concentrations of DA in the presence of
0.1 mM ascorbic acid (dissolved in milli-Q-water). Binding
assays were incubated for 90 min at room temperature and terminated
using rapid filtration through glass fiber filters (GF/C, Whatman). The
filters were washed four times with 5 ml of cold washing buffer (50 mM Tris-HCl, pH 7.4, 120 mM NaCl), and bound
radioactivity was determined by liquid scintillation counting (Beckman,
LS 6500). Protein concentrations were measured using the Bio-Rad assay
kit with bovine serum albumin as standard. To determine the equilibrium
dissociation constant (Kd) of the ligands and the
maximal binding capacity (Bmax) of
N-[methyl-3H]SCH23390, binding
isotherms were analyzed using the nonlinear curve-fitting program,
LIGAND (17).
Whole Cell cAMP Assay--
Regulation of AC activity by
wild-type and chimeric receptors was assessed using a whole cell cAMP
assay as described previously (10, 11). Following overnight incubation
with the DNA-calcium phosphate precipitate, HEK 293 cells were reseeded
in 6- or 12-well dishes. The next day, cells were labeled with
[3H]adenine (2 µCi/ml) in fresh minimum essential
medium containing 5% (v/v) fetal bovine serum and gentamicin (10 µg/ml) for 18 h at 37 °C and 5% CO2. The
labeling medium was then removed, and HEK 293 cells were incubated in
20 mM HEPES-buffered medium containing 1 mM
1-methyl-3-isobutylxanthine in the presence or absence of DA for 30 min
at 37 °C. At the end of the incubation period, the medium was
aspirated, 1 ml of lysis solution (2.5% (v/v) perchloric acid, 1 mM cAMP, and [14C]cAMP (2.5-5 nCi,
~5,000-10,000 cpm)) was added to each well, and cells were lysed for
30 min at 4 °C. The lysates were then transferred to tubes
containing 0.1 ml of 4.2 M KOH (neutralizing solution), and
precipitates were sedimented by a low-speed centrifugation (1,500 rpm)
at 4 °C. The amount of intracellular [3H]cAMP was
determined from supernatants purified by sequential chromatography
using Dowex (AG 50W-X4) and alumina columns as described previously
(18). The amount of [3H]cAMP (CA) over the total amount
of [3H]adenine uptake (TU) was calculated to determine
the relative intracellular cAMP levels (CA/TU × 1000).
Dose-response curves to DA were analyzed by a four-parameter logistic
equation using ALLFIT (19). The Bmax values of wild-type and
chimeric receptors were determined using a saturating concentration
(~6 nM) of
N-[methyl-3H]SCH23390.
Statistics--
Equilibrium dissociation constants
(Kd) are expressed using the geometric mean ± S.E. as described previously (20). All other data are reported as
arithmetic means ± S.E. unless stated otherwise. All statistical
tests used in this study have been described elsewhere (21).
Homoscedasticity of variances was assessed using Bartlett or
Fmax tests prior to statistical analyses. A
one-sample t test, Student's t test, and
one-way analysis of variance (with Newman-Keuls multiple comparison
test) were performed using GraphPad Prism, version 3.0 for Windows
(GraphPad Software, San Diego; www.graphpad.com). The level of
significance was established at p < 0.05.
EL3 and CT Regions Coordinate the D1-like Subtype-specific DA
Affinity--
Table I shows the
Kd values for
N-[methyl-3H]SCH23390 binding to
wild-type and chimeric human D1-like receptors obtained with saturation
studies. As indicated by the Kd values, the chimeric
receptors maintained their ability to bind this D1-like antagonist with
high affinity, suggesting that the protein folding required for ligand
binding is functional. Specifically, D1A-EL3CTB chimera displayed an
increase in binding affinity for
N-[methyl-3H]SCH23390 compared with
the wild-type D1A receptor. These results are consistent with studies
showing that benzazepine-like compounds can behave as partial agonists
in cells expressing D1-like receptors (2, 22, 23). Meanwhile, the
binding affinity of D1B-EL3CTA chimera was unchanged in comparison with
the wild-type D1B receptor.
Furthermore, competition studies were performed to examine the changes
in DA affinity for wild-type and chimeric receptors. As shown in Table
I and in agreement with previous studies (10-13), DA exhibited a
higher affinity for the D1B receptor compared with the D1A receptor.
Furthermore, swapping the EL3 region between the D1A and D1B subtypes
resulted in partial modulation of DA affinity, whereas swapping the CT
resulted in a nearly complete switch in DA affinity to that of cognate
wild-type counterparts. Exchange of both EL3 and CT regions resulted in
a further pronounced switch in the DA affinity for chimeric receptors.
Namely, the D1A-EL3CTB chimera exhibited an increased DA affinity
extending beyond that of chimeric D1A-CTB receptors. Meanwhile, the
chimeric D1B-EL3CTA receptor displayed a decreased DA affinity, falling below that of the D1B-CTA chimera. Importantly, the DA affinity values
for EL3CT chimeras are essentially indistinguishable from their
respective cognate wild-type receptors (Table I). However, based upon
the EL3B- and CTB-induced effects individually exerted on the D1A
subtype for DA affinity, it would be expected that a chimeric D1A
receptor harboring the EL3B and CTB regions would display an even
higher DA affinity than that of either the wild-type D1B subtype or
D1A-EL3CTB chimera. To address this issue, we have used the standard
free energy ( Subtype-specific Regulation of the Maximal Binding Capacity by EL3
and CT Regions of Human D1A and D1B Dopaminergic Receptors
Expressed in HEK 293 Cells--
One interesting finding stemming from
our saturation studies is the potential role of the distinct molecular
interplay between the EL3 and CT regions of the D1A and D1B receptors
in regulating their Bmax values. Indeed, our saturation
studies reveal stark differences in Bmax values among the
various chimeric receptors (Fig. 2). As
reported previously (11), there was a significant reduction in the
Bmax of D1A-CTB chimera (Fig 2A). Conversely, there was a large increase in the Bmax value of D1A-EL3B
chimera (Fig. 2A). Importantly, inserting the EL3 of the D1B
subtype into the D1A receptor harboring the D1B tail (D1A-CTB) led to a
complete rescue of the Bmax value of D1A-CTB chimera. In
fact, D1A-EL3CTB chimera displayed a Bmax that is
indistinguishable from the wild-type D1A receptor (Fig. 2A).
The reverse was partially true for the chimeric D1B receptors, where
insertion of EL3 of the D1A subtype decreased the Bmax value
as we reported previously (10). Meanwhile, insertion of CT of the D1A
subtype significantly increased the Bmax value of D1B-CTA
chimera compared with the wild-type D1B receptor (Fig 2B).
In a previous study, a similar trend was detected but not established
as statistically significant (11). Interestingly, however, a chimeric
D1B receptor harboring the EL3 and CT regions of the D1A subtype
(D1B-EL3CTA) displayed a Bmax that is significantly increased from those measured with wild-type and chimeric D1B receptors
(Fig. 2B).
To assess whether the molecular interplay between the EL3 and CT
regions exerts additive, synergistic, or interfering effects on the
spatial relationships controlling the maximal binding capacity, the
Bmax values of chimeric D1A-like (EL3B, CTB, and EL3CTB
chimeras) and D1B-like receptors (EL3A, CTA, and EL3CTA) were expressed as net changes relative to their respective wild-type counterparts (Fig. 2, C and D). Data depicted in Fig. 2C
indicate that the net change observed upon insertion of both EL3B and
CTB (black bar) is significantly lower than the sum of net
changes induced individually by EL3B and CTB (white bar),
suggesting a nonadditive effect or interference. In contrast, the sum
of the net change obtained by introducing both EL3A and CTA
(black bar) is significantly higher than the sum of net
changes produced separately by EL3A and CTA (Fig. 2D, white
bar), indicative of a synergism. These results indicate
that EL3 and CT regions exert in the overall receptor conformation not
only unique but also opposing and synergistic effects on the
intramolecular interactions regulating the Bmax value of D1A
and D1B receptors. These results suggest a potential role for these two
regions in regulating the receptor turnover (synthesis, degradation)
and/or protein stability. In a series of preliminary experiments, we
have tested the potential role of EL3 in regulating the thermal
stability of D1-like receptor proteins using a membrane assay as
described previously (24). Membrane preparations from HEK 293 cells
expressing D1A-EL3B or D1B-EL3A chimeras were incubated at 37 °C for
24 h and tested for
N-[methyl-3H]SCH23390 functional
binding to assess the role of EL3 region in controlling the thermal
stability of D1-like receptor proteins. In agreement with a potential
role of EL3 in the regulation of thermal stability of proteins,
membranes expressing D1A-EL3B or D1B-EL3A chimeras, when compared with
wild-type receptors, displayed a lower and higher reduction of
Bmax, respectively, following a 24-hour incubation at
37 °C (data not shown).
Opposing EL3- and CT-induced Intramolecular Interactions Confer
D1-like Receptor-specific G Protein Coupling Properties--
To
determine the combined effect of EL3 and CT on agonist-independent AC
activation, whole cell cAMP assays were performed in HEK 293 cells
expressing wild-type or chimeric D1-like receptors (Fig.
3A). Reminiscent of
constitutively active mutant GPCRs, the D1B receptor displayed higher
agonist-independent activity compared with the D1A subtype. In
agreement with previously published results (10), the exchange of the
EL3 region caused a partial modulation in agonist-independent activity
(data not shown). Furthermore, the swap of the CT further substantiated
the functional role of this cytoplasmic region in subtype-specific
agonist-independent activity of the D1A and D1B receptors (data not
shown), which was likewise supported by our previous findings (11). The
exchange of both the EL3 and CT resulted in chimeras displaying a full switch in agonist-independent activation of AC as compared with parent
receptors (Fig. 3A). Indeed, the constitutive activation of
D1A-EL3CTB chimera was not statistically different from the wild-type
D1B receptor. In this particular series of experiments, the slightly
lower degree of constitutive activation of D1A-EL3CTB displayed in HEK
293 cells when compared with wild-type D1B receptors (~24%) is in
agreement with the smaller Bmax value of D1A-EL3CTB (~20%). Thus, our results strongly suggest that the EL3B and CTB regions in the D1A-EL3CTB chimera partake in the full switch of agonist-independent activity of wild-type D1A receptors. Similarly, an
exchange of both the EL3 and CT regions of D1B receptors with those of
the D1A subtype resulted in a chimeric D1B receptor displaying a
statistically significant diminution of its constitutive activity to a
level akin to that of wild-type D1A receptors (Fig. 3A).
Some of the molecular determinants underlying D1-like subtype-specific
agonist-mediated G protein coupling properties have been identified
previously (2, 10, 11). Nonetheless, the molecular interplay between
the extracellular and intracellular domains that may impart the D1-like
subtype-specific G protein coupling properties remains unclear. As
shown in Fig. 3B, the DA-mediated maximal activation of AC
activity (an indicator of DA efficacy) was essentially unchanged in
cells expressing D1A-EL3CTB and D1B-EL3CTA chimeras compared with their
parent receptors; i.e. wild-type and chimeric D1A receptors
display a higher DA-mediated maximal activation of AC as compared with
their respective counterparts. To examine further the role of EL3 and
CT in shaping the intramolecular interactions governing
subtype-specific G protein coupling properties, dose-response curves
were performed in HEK 293 cells expressing wild-type or chimeric
receptors at similar Bmax values (~1 pmol/mg protein).
Under these experimental conditions, we observed that the
subtype-specific DA potency and efficacy in HEK 293 cells was modulated
differentially by EL3 and CT regions (Fig.
4). As depicted in Fig. 4, DA potency (as
indexed by EC50 values) is ~10-fold higher in HEK 293 cells expressing the wild-type D1B receptor in comparison with the D1A
subtype. In agreement with binding results, the EL3 region exerts a
partial modulation of DA potency in cells expressing D1A-EL3B or
D1B-EL3A chimeras (Fig. 4). In striking contrast to what would be
expected from constitutively active GPCRs, cells expressing D1A-CTB
chimera displayed a decreased DA potency in comparison with cells
expressing wild-type D1B receptors and also a significantly lower
potency value than wild-type D1A receptors (Fig. 4, B and
C). Interestingly, insertion of the EL3B region into the
D1A-CTB chimera rescued the DA potency for the chimeric receptor,
i.e. the EC50 value of D1A-EL3CTB chimera is closer to that of wild-type D1B receptor (Fig. 4, B and
C). Indeed, the D1A-EL3CTB chimera displayed an ~6-fold
increase in DA potency compared with wild-type D1A receptor. As
reported previously (11), D1B-CTA chimera displayed a statistically
significant decrease in DA potency compared with wild-type D1B
receptors (Fig. 4, E and F). These results are
consistent with a role of the CTA region in imparting a constrained
conformation to heptahelical D1-like receptors. This idea is supported
further by our results showing that HEK 293 cells expressing wild-type
D1A receptors display higher DA potency in comparison with those
transfected with the D1B-CTA chimera (Fig. 4, E and
F). Importantly, the CTA-induced constrained conformation
was attenuated by insertion of the EL3A into D1B-CTA chimera, which
brought the EC50 to a value indistinguishable from that in
cells expressing wild-type D1A receptors (Fig. 4, E
and F). Overall, these results suggest that interfering
intramolecular interactions between the EL3 and CT regions also control
DA potency in HEK 293 cells. A similar assertion can also be made about
the DA efficacy (Fig. 4, A and D).
In the present study, we used a functional complementation of
chimeric D1-like receptors to address the molecular interplay between
an extracellular region, EL3, and an intracellular region, CT, in
coordinating the functional properties (ligand binding, G protein
coupling, and Bmax) of GPCRs and potentially their multiple active conformations. By constructing chimeric receptors in which EL3
and/or CT were exchanged between the D1A and D1B receptors, we were
able to establish that interfering, synergistic, and additive intramolecular interactions put forth by these regions coordinate D1-like subtype-specific functional properties and multiple active GPCR
conformations. It is also worth mentioning that the results obtained in
the present study are not attributed to differences in the transfection
efficiency of the different wild-type and chimeric receptor constructs.
Indeed, we have obtained evidence that HEK 293 transfected with
different epitope- or GFP (green fluorescent protein)-tagged versions
of wild-type and chimeric dopamine receptor constructs show similar
transfection efficiencies as assessed by immunofluorescence microscopy
and fluorescence-activated cell sorter (data not shown).
The prime objective of our study was to explore the underlying
molecular basis for constitutively active wild-type and chimeric forms
of D1-like receptors displaying similar increased agonist affinity but
unexpectedly divergent agonist potency. Such dissociation of DA potency
and affinity deviates from one of the established paradigms for
constitutively active mutant GPCRs, i.e. increased agonist
affinity translates into increased agonist potency (2, 25, 26). An
interesting finding from our studies is the potential role EL3 plays in
controlling the CT-mediated regulation of D1-like subtype-specific DA
potency in HEK 293 cells. Evidence pertaining to the role of EL3 in
regulating the GPCR coupling function remains minimal (27).
Interestingly, a study using EL3 chimeras made between the hamster
Meanwhile, the CT region has also been shown to regulate
agonist-induced phosphorylation, desensitization, and internalization of several GPCR types including D1A receptors (29-34). Therefore, the
observed loss of agonist potency of D1A-CTB chimera in HEK 293 cells
could be explained potentially by a constitutive desensitization of the
receptor. Indeed, there is evidence that constitutively active mutant
GPCRs are subjected to desensitization in the absence of agonist
stimulation (35-37). If constitutive desensitization plays a
significant role in regulating DA potency in HEK 293 cells expressing
D1A-CTB, this process is attenuated by the insertion of EL3 region of
the D1B receptor into the D1A-CTB chimera as indexed by DA potency. At
the present time, the issue of constitutive desensitization remains to
be resolved fully. However, we believe that the loss of DA potency
exhibited by the constitutively active D1A-CTB chimera is likely to be
explained for the most part by CT-specific intramolecular interactions
that underlie the agonist-induced receptor conformation for G protein
coupling rather than constitutive or tonic desensitization. This idea
is supported by our current and previous (11) findings showing that
DA-induced maximal stimulation of AC in HEK 293 cells expressing
D1A-CTB is significantly increased instead of being decreased when
compared with wild-type D1-like receptors or D1A-EL3CTB.
In fact, the results presented herein pertaining to agonist-mediated
maximal activation of AC address an important issue raised by our
previous study showing that EL3 controls the extent of DA-mediated
maximal activation of AC displayed by D1A and D1B receptors in HEK 293 cells (10). In striking contrast, the extent of DA-mediated maximal
activation of AC (higher for D1A and lower for D1B) was not
significantly changed by using chimeric receptors in which
sequences composed of the TM6, TM7, EL3, and CT (TRL region) were
switched between the D1A and D1B subtypes (10). In the present study,
we demonstrate that in a similar fashion to TRL chimeras (10), the
constitutive activity was switched, whereas the extent of
agonist-mediated maximal activation of AC was not affected by the
exchange of both EL3 and CT regions. Thus, other structural
determinants within the TRL of D1A and D1B receptors must prevent EL3
from regulating DA-mediated maximal activation of AC. Based on the
results obtained with EL3CT chimeras, we propose that these structural
determinants are located within the CT. Importantly, our data suggest
that TM6 and TM7 residues do not prevent EL3 from exerting its effects
on agonist-mediated maximal activation (10). In fact, our studies using
D1A and D1B receptors harboring single-point mutations of their variant
TM6 and TM7 residues support this assertion. Indeed, TM6 and TM7
single-point mutant receptors do not exhibit any major changes in their
agonist-independent and -dependent activation
properties.2 Thus, EL3 and CT
appear to impose on the overall receptor conformation interfering intramolecular interactions in controlling the extent of
DA-mediated maximal activation of AC in HEK 293 cells.
Furthermore, we have observed that the EL3 and CT regions play an
important role in controlling the Bmax of D1A and D1B
receptors in HEK 293 cells. For instance, the chimeric D1A-CTB receptor exhibited a drastically reduced Bmax, possibly reflective of
its high constitutive activity profile, although the wild-type D1B receptor did not show a lower Bmax value in
comparison with HEK 293 cells expressing the wild-type D1A subtype.
Meanwhile, marked structural instability has been reported as a result
of constitutive activation of mutant GPCRs (24, 38, 39). Thus, one
likely possibility is that swapping of the CT region of D1A subtype
with that of the D1B receptor imparts a decreased structural stability to the D1A-CTB chimera, which is functionally rescued by an
insertion of the EL3B region (D1A-EL3CTB chimera). It is also worth
mentioning that the Bmax values of D1-like chimeras measured
in transfected HEK 293 cells may also depend on the rates of both
receptor synthesis and degradation (40, 41). Potentially, the EL3
and/or CT regions may play an important role in controlling the rate of
D1-like chimera degradation through GPCR internalization-independent
and -dependent processes (29, 42, 43).
Taken together, the findings of our study suggest that the EL3 and CT
regions regulate D1-like subtype-specific ligand binding and G protein
coupling properties. However, the conformational determinants may also
require residues located outside of the TRL, notably for
agonist-mediated maximal activation of AC. Furthermore, the distinct
molecular interplay between EL3 and CT may also underlie the multiple
active conformations adopted by constitutively active D1-like subtypes.
These subtypes can be grouped into receptors displaying
increased DA affinity with decreased DA potency and Bmax
(D1A-CTB chimera), increased DA affinity with increased DA potency and
Bmax (D1A-EL3B chimera), or increased DA affinity with
increased DA potency and unchanged Bmax (wild-type D1B
receptors and D1A-EL3CTB chimera). The topological locations of EL3 and CT suggest that they may serve as conformational switches in
In conclusion, our study has clearly demonstrated that a set of
molecular interactions between EL3 and CT of the D1A and D1B receptors controls the underlying spatial relationships of D1-like subtype-specific active conformations. Whether acting together or
against each other, these interactions define the spatial relationships underlying DA binding, as well as the agonist-dependent and
-independent G protein coupling properties of D1-like receptors.
Further studies are under way in our laboratory to identify specific
residues of the CT and EL3 that participate in the formation of these
intramolecular bonds. Finally, our results may be of potential
physiological relevance for the treatment of pathological conditions
caused by a compromised D1-like receptor function (9). This
issue is further underscored by the identification of missense
and nonsense mutations in the EL3 region of the D1B receptor, which may
play a role in the phenotypic expression of neuropsychiatric disorders (55, 56).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
5'-TACGCG-3')
near the 3' end of the D1A receptor TM7 region, immediately upstream of
the D1B receptor CT sequence (altered nucleotides are underlined). For
D1B-EL3CTA chimera, a HindIII restriction site was
introduced at a nucleotide sequence encompassing the residues 363 and 364 (5'-AAGGCA-3'
5'-AAAGCT-3'),
located 3' of the junction between the D1B receptor TM7 region
and D1A receptor CT sequence. Amplified fragments were separated on a
1% agarose gel, and appropriate bands were excised and purified by
QIAEX II gel extraction method (Qiagen, Valencia, CA). Diluted
aliquots of the paired fragments were combined (AB1+B2 or BA1+A2) and
subjected to overlap PCR using appropriate 5' and 3' flanking primers.
The resulting PCR products were cut with BclI and
XbaI and subcloned into pBluescript II SK+
(Stratagene) containing the HindIII/XbaI fragment
of the wild-type D1A receptor (for PCR product AB1/B2) or the
full-length coding sequence of D1B receptor (for PCR product BA1/A2).
The full-length expression constructs for wild-type and chimeric
receptors were ultimately engineered into the pCMV5 expression vector.
The D1A-EL3CTB chimera was subcloned into pCMV5 together with the
N-terminal portion (EcoRI/HindIII fragment) of
wild-type D1A receptor in a three-piece ligation reaction. The
EcoRI/HindIII D1A fragment was ligated to the
HindIII fragment of D1A-EL3CTB and linearized pCMV5
(EcoRI/HindIII). The D1B-EL3CTA chimera was
subcloned into empty pCMV5 linearized with SalI and
XbaI. The nucleotide sequence of PCR products (encompassed
by BclI and XbaI) and cloning sites was confirmed
by dideoxy sequencing using Sequenase version 2.0 from Amersham Biosciences.
80 °C until required (competition studies).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Equilibrium dissociation constants (Kd) for wild-type and
chimeric D1-like receptors
, p < 0.05 when compared with D1B; #, p < 0.05 when compared
with D1A-EL3CTB;
, p < 0.05 when compared with
D1B-EL3CTA.
Go) values derived from DA
affinity constants (
Go =
RTln(1/Kd)) to calculate the sum of net
changes induced separately by EL3 and CT regions (EL3+CT) and have
compared that with the net change caused by the addition of both
regions (Fig. 1). The net change
calculated for the D1B-EL3CTA chimera is not statistically different
from zero, suggesting that a full switch has been implemented (Fig. 1,
right panel). Interestingly, similar results were obtained
using the sum of net changes produced individually by EL3A and CTA
regions, which was not statistically different from zero, or changes
observed with the D1B-EL3CTA chimera. These results suggest an additive
effect of EL3 and CT in regulating the DA affinity of D1B-EL3CTA
chimera. Likewise, the net change measured for the D1A-EL3CTB was not
statistically different from zero. However, in stark contrast, the sum
of the net changes elicited independently by the EL3B and CTB regions
was statistically different from zero and D1A-EL3CTB value, indicative
of an interfering effect of these regions in the D1A-EL3CTB chimera
(Fig. 1, left panel).
View larger version (17K):
[in a new window]
Fig. 1.
Net changes in standard free
energy values for DA affinity of EL3CT chimeras relative to
wild-type receptors. Standard free energy values
( Go) of DA affinities for wild-type and
chimeric D1-like receptors were computed as
RTln(1/Kd). Variations in standard
free energy (
Go) values between wild-type
D1A and D1B receptors
(
GoD1A
D1B),
wild-type and chimeric D1A receptors
(
GoD1A
D1A
chimera), wild-type D1B and D1A receptors
(
GoD1B
D1A), and
wild-type and chimeric D1B receptors
(
GoD1B
D1B chimera) were
also calculated. The sum of individual
Go
values of EL3A and CTA or EL3B and CTB was used to obtain the
Go values for EL3A+CTA
(
GoD1B
EL3A+CTA) and EL3B+CTB
(
GoD1A
EL3B+CTB),
respectively. Net changes were computed using
Go values to determine whether the
differences between D1A-EL3CTB and D1A-EL3B+D1A-CTB (left
panel) or D1B-EL3CTA and D1B-EL3A+D1B-CTA (right panel)
were statistically significant. *, p < 0.05 when compared with a value of zero; #, p < 0.05 when
compared with EL3CTB.
View larger version (34K):
[in a new window]
Fig. 2.
Maximal binding capacity
(Bmax) of
N-[methyl-3H]SCH23390
for wild-type and chimeric D1-like receptors expressed in HEK 293 cells. Values are expressed as the arithmetic means ± S.E.
of five experiments done in duplicate determinations of wild-type and
chimeric D1A receptors (A) and wild-type and chimeric D1B
receptors (B) using saturation studies. Net changes of
Bmax of chimeric D1A receptors relative to the wild-type D1A
receptor (C) and Bmax of chimeric D1B receptors
relative to the wild-type D1B receptor (D) are shown.
A, *, p < 0.05 when compared with D1A; #,
p < 0.05 when compared with D1A-EL3B. B,
, p < 0.05 when compared with D1B;
,
p < 0.05 when compared with D1B-EL3A.
C, *, p < 0.05 when compared with a value
of zero; #, p < 0.05 when compared with EL3B+CTB.
D,
, p < 0.05 when compared with a value
of zero;
, p < 0.05 when compared with
EL3A+CTA.
View larger version (16K):
[in a new window]
Fig. 3.
Constitutive activity and dopamine-mediated
maximal stimulation of adenylyl cyclase by wild-type and chimeric
D1-like receptors expressed in HEK 293 cells. HEK 293 cells were
transfected with wild-type or chimeric D1-like subtypes using 5 µg of
DNA/dish of receptor expression constructs. Basal and maximal levels of
AC activity in transfected HEK 293 cells were determined in single
wells of a 6-well dish using whole cell cAMP assays in the absence or
presence of 10 µM DA. Constitutive activation and
DA-mediated maximal stimulation of AC are expressed as the geometric
and arithmetic means ± S.E., respectively, of eight experiments
done in triplicate determinations. The Bmax values of
N-[methyl-3H]SCH23390 in pmol/mg of
membrane proteins (expressed as the arithmetic mean ± S.E.) were
16.2 ± 1.8 (D1A), 14.4 ± 1.0 (D1B), 11.6 ± 0.8 (D1A-EL3CTB), and 24.6 ± 3.0 (D1B-EL3CTA). A, basal
activity of wild-type D1B and chimeric D1-like receptors relative to
the D1A subtype. B, DA-mediated maximal stimulation of AC
activity of wild-type and chimeric D1-like receptors. *,
p < 0.05 when compared with D1A; #, p < 0.05 when compared with D1A-EL3CTB.
View larger version (31K):
[in a new window]
Fig. 4.
Dose-response curves of DA for AC stimulation
by wild-type and chimeric D1-like receptors expressed in HEK 293 cells. HEK 293 cells were transfected with wild-type or chimeric
D1-like receptors using the following amounts (in µg) of DNA per
dish: 0.02 D1A, 0.02 D1B, 0.02 D1A-EL3B, 5.0 D1A-CTB, 0.04 D1A-EL3CTB,
0.1 D1B-EL3A, 0.02 D1B-CTA, or 0.02 D1B-EL3CTA. Under these
experimental conditions, the Bmax values were similar. The
Bmax values in pmol/mg membrane protein (expressed as the
arithmetic mean ± S.E.) were 1.6 ± 0.3 (D1A,
n = 8), 1.4 ± 0.3 (D1B, n = 8),
0.8 ± 0.3 (D1A-EL3B, n = 5), 0.8 ± 0.3 (D1B-EL3A, n = 5), 1.4 ± 0.3 (D1A-CTB,
n = 5), 0.7 ± 0.2 (D1B-CTA, n = 5), 1.1 ± 0.2 (D1A-EL3CTB, n = 8), and 0.9 ± 0.2 (D1B-EL3CTA, n = 8). Intracellular cAMP levels
were measured in single wells of a 12-well dish in the absence or
presence of increasing concentrations of DA as described under
"Experimental Procedures" and plotted as a function of log of DA
concentrations. Each point is the arithmetic mean ± S.E. of five
to eight experiments done in triplicate determinations and expressed as
[3H]cAMP (CA) over the total amount of
[3H]adenine uptake (TU) × 1000 (shown in
A and D) or the percentage of maximal activation
obtained with the respective wild-type or chimeric receptor after
subtracting the basal value (shown in B and E).
Curves were analyzed by simultaneous curve fitting using ALLFIT.
Statistical significance was determined using unconstrained and
constrained simultaneous curve fitting. For the graphical
representation, a representative example of dose-response curves of
wild-type and chimeric D1A and D1B receptors using raw data are shown
in A and D, respectively. The best-fitted values
for DA-mediated maximal activation of adenylyl cyclase ((CA/TU × 1000) ± approximate S.E. as obtained with ALLFIT) are as follows:
14.5 ± 0.4 (D1A), 7.1 ± 0.3 (D1B), 10.6 ± 0.4 (D1A-EL3B), 32.2 ± 0.5 (D1A-CTB), 16.1 ± 0.3 (D1A-EL3CTB),
9.1 ± 0.4 (D1B-EL3A), 8.9 ± 0.5 (D1B-CTA), and 7.6 ± 0.4 (D1B-EL3CTA). Normalized and averaged dose-response curves are
shown in B and D, respectively. The
EC50 values (in nM ± approximate S.E. as
obtained with ALLFIT) are as follows: 19.9 ± 3.0 (D1A), 1.6 ± 0.2 (D1B), 7.3 ± 1.1 (D1A-EL3B), 38.6 ± 6.0 (D1A-CTB),
3.5 ± 0.5 (D1A-EL3CTB), 3.4 ± 0.5 (D1B-EL3A), 127.1 ± 19.3 (D1B-CTA), and 30.7 ± 4.7 (D1B-EL3CTA). The EC50
ratios of the wild-type D1B and chimeric receptors relative to the D1A
subtype are shown as values ± approximate S.E. (as obtained with
ALLFIT) in C and F. The values for
EC50 ratios are as follows: 0.08 ± 0.02 (D1B),
0.37 ± 0.08 (D1A-EL3B), 0.17 ± 0.04 (D1B-EL3A), 1.94 ± 0.42 (D1A-CTB), 6.4 ± 1.4 (D1B-CTA), 0.18 ± 0.04 (D1A-EL3CTB), and 1.5 ± 0.3 (D1B-EL3CTA). *, p < 0.05 when compared with D1A; #, p < 0.05 when compared
with D1B.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-adrenergic receptor (Gs-coupled GPCR) and rat
1A-adrenergic receptor (Gq-coupled GPCR)
has serendipitously found that EL3 can modulate
2-adrenergic receptor/G protein affinity (28). In
agreement with the latter study, we show that the partial modulation of
DA potency induced by the different EL3 regions of D1-like receptors
(increase for D1A-EL3B and decrease for D1B-EL3A) is consistent with
the partial modulation of DA affinity observed herein and previously
(10). Thus, we believe that the EL3 primary structure plays a key role
in shaping the CT-induced intramolecular interactions regulating
D1-like subtype-specific G protein coupling properties. Most
importantly, this issue is best highlighted by the insertion of EL3 of
the D1B subtype into the D1A-CTB chimera, which imparts to the
constitutively active D1A-EL3CTB chimera an increased DA affinity and potency.
-helical packing and/or movement/tilting of the TM6 and TM7 of D1A
and D1B receptors, a biophysical process involved in rhodopsin and
2-adrenergic receptor activation (27, 44-48).
Importantly, regulation of TM6 and TM7 intramolecular bonds have also
been implicated in agonist-independent and -dependent
activation of GPCRs (27, 44-54).
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ACKNOWLEDGEMENTS |
---|
We thank Marie-France Nantel and Amy Slater for expert technical assistance with the cell culture. We thank members of our laboratory for critical reading of the manuscript.
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FOOTNOTES |
---|
* This work was supported by Operating Grant 203694 from the Natural Sciences and Engineering Research Council of Canada (to M. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Holder of an Ontario Graduate Scholarship in Science and
Technology from the Government of Ontario and Ottawa Health Research Institute.
§ Recipient of the K. M. Hunter doctoral research award from the Canadian Institutes of Health Research.
¶ To whom correspondence should be addressed: Ottawa Health Research Institute, Moses and Rose Loeb Research Centre, 725 Parkdale Ave., Ottawa, Ontario K1Y 4K9, Canada. Tel.: 613-798-5555 (ext. 18749); Fax: 613-761-5365; E-mail: mtiberi@ohri.ca.
Published, JBC Papers in Press, December 30, 2002, DOI 10.1074/jbc.M208059200
2 R. M. Iwasiow and M. Tiberi, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: DA, dopamine; GPCR, G protein-coupled receptor; AC, adenylyl cyclase; TRL, terminal receptor locus; EL3, third extracellular loop; HEK 293, human embryonic kidney 293 cells; TM, transmembrane; Bmax, maximal binding capacity.
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