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Vol. 295, Issue 1, 274-283, October 2000
Department of Biochemical Pharmacology (J.F.M.V., K.J., J.E.L.), and Department of Functional Genomics (W.H.M.L.L.), Janssen Research Foundation, Belgium; and Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, The Netherlands (A.J.D.)
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Abstract |
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 Top
 Abstract
 Introduction
Experimental Procedures
Results
Discussion
References
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Human dopamine D2 and D3 receptors were expressed in Chinese hamster ovary (CHO) and Escherichia coli cells to compare their ligand binding properties in the presence or absence of G-proteins and to analyze their ability to interact with Gi/o-proteins. Binding affinities of agonists (dopamine, 7-OH-DPAT, PD128907, lisuride) and antagonists/inverse agonists (haloperidol, risperidone, domperidone, spiperone, raclopride, nemonapride), measured using [125I]iodosulpride and [3H]7-OH-DPAT, were similar for hD3 receptors in E. coli and CHO cell membranes. Both agonists and antagonists showed 2- to 25-fold lower binding affinities at hD2 receptors in E. coli versus CHO cell membranes (measured with [3H]spiperone), but the rank order of potencies remained similar. Purported inverse agonists did not display higher affinities for G-protein-free receptors. In CHO membranes, GppNHp decreased high affinity agonist ([3H]7-OH-DPAT) binding at hD2 receptors but not at hD3 receptors. Also, [3H]7-OH-DPAT (nanomolar concentration range) binding was undetectable at hD2 but clearly measurable at hD3 receptors in E. coli membranes. Addition of a Gi/o-protein mix to E. coli membranes increased high affinity [3H]7-OH-DPAT binding in a concentration-dependent manner at hD2 and hD3 receptors; this effect was reversed by addition of GppNHp. The potency of the Gi/o-protein mix to reconstitute high affinity binding was similar for hD2 and hD3 receptors. Thus, agonist binding to D3 receptors is only slightly affected by G-protein uncoupling, pointing to a rigid receptor structure. Furthermore, we propose that the generally reported lower signaling capacity of D3 receptors (versus D2 receptors) is not due to its lower affinity for G-proteins but attributed to its lower capacity to activate these G-proteins.
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Introduction |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Dopamine
receptors belong to the superfamily of G-protein-coupled receptors.
Five dopamine receptors have been found and classified into the D1-like
family (D1 and D5
receptors) and the D2-like family (D2,
D3, and D4 receptors)
(Missale et al., 1998
). Receptor genes of the D1-like family do not
contain introns, whereas members of the D2-like family possess multiple
introns. For instance, two splice variants of the
D2 receptor have been found; the
D2short (D2S) receptor,
lacking a 29-amino acid insert in the third cytoplasmic loop, and the
D2long (D2L) receptor
(Monsma et al., 1989). Differences in G-protein coupling of these
splice variants have been reported (Picetti et al., 1997), but the
physiological relevance of these splice variants remains unclear.
Human dopamine D2 and D3
receptors have an overall amino acid sequence similarity of 52%, which
increases to 78% if only transmembrane regions are considered (Giros
et al., 1990). Most ligands that bind to hD2
receptors also bind to hD3 receptors and their
rank order of potency is quite similar. In general, dopamine agonists have a higher affinity for hD3 receptors, whereas
antagonists have a slightly higher affinity for
hD2 receptors. The pharmacological properties of
the hD2 receptor splice variants are almost
identical (Liu et al., 1992; Leysen et al., 1993; Schotte et al.,
1996). Although both hD2 and
hD3 receptors are thought to couple to
Gi/o proteins, this is much less clear for the
hD3 than for the hD2 receptor. Guanine nucleotide modulation of agonist binding at hD2 receptors has been clearly demonstrated,
whereas conflicting results have been obtained for
hD3 receptors (Sokoloff et al., 1992; Freedman et
al., 1994; MacKenzie et al., 1994; Tang et al., 1994; Akunne et al.,
1995; McAllister et al., 1995). In addition, coupling of
hD2 receptors to various
pertussis-toxin-sensitive signaling pathways has been reported, whereas
a total lack or a weak coupling to these pathways has been described
for hD3 receptors (MacKenzie et al., 1994; Tang
et al., 1994; McAllister et al., 1995). Only recently, we succeeded in
demonstrating clear activation of hD3 receptors
at different levels of the signal transduction cascade when expressed
at high levels in Chinese hamster ovary (CHO) cells (Vanhauwe et al.,
1999).
According to the allosteric ternary complex model (Lefkowitz et al.,
1993; Samama et al., 1993), receptors, by themselves, can occur in an
active and an inactive state. In the absence of agonist, a fraction of
the receptor population can exist in an active state, leading to basal
or constitutive activity. Agonist binding increases the population of
active receptors. In contrast, binding of inverse agonists drives the
receptor to the inactive state, diminishing the basal activity. It is
not yet clear whether agonist binding recruits G-proteins to the
activated receptor or that G-protein coupling promotes subsequent
agonist binding. Probably, both processes play in concert (ternary
complex formation). However, it is generally found that agonists bind
with higher affinity to G-protein-coupled receptors than to uncoupled
receptors, whereas neutral antagonists do not distinguish between
G-protein-coupled and uncoupled receptors. Furthermore, it was
suggested that inverse agonists have a higher affinity for uncoupled
than for G-protein-coupled receptors (Costa et al., 1992). Inverse
agonists have been identified for D2 (Hall and
Strange, 1997; Kozell and Neve, 1997; Vanhauwe et al., 2000) and
D3 receptors (Griffon et al., 1996; Malmberg et
al., 1998), indicating that both receptors can have basal activity.
To demonstrate clearly the effect of G-protein receptor coupling on
ligand binding, one would need the receptor in a G-protein-free environment. Escherichia coli cells, which do not contain
G-proteins, provide a system for producing uncoupled receptors. Certain
G-protein-coupled receptors have been successfully expressed in
E. coli (Marullo et al., 1990; Freissmuth et al., 1991;
Bertin et al., 1992; Grisshammer et al., 1993; Munch et al., 1995).
In this study, we succeeded for the first time to express hD2S, hD2L, and hD3 receptors in E. coli. To scrutinize the difference in G-protein-coupling properties of hD2S, hD2L, and hD3 receptors, we compared the binding properties of agonists and antagonists for recombinant receptors expressed in E. coli versus CHO cells. Measurements on recombinant hD2 and hD3 receptors in E. coli membranes were carried out in the absence or presence of added Gi/o-proteins. Our study illustrates that agonist binding to hD3 receptors is poorly sensitive to G-protein coupling, whereas hD2 receptor high affinity agonist binding is highly dependent on G-proteins. Nevertheless, hD3 and hD2 receptors were found to have equal affinity for the Gi/o-protein mix. A constrained hD3 receptor structure that retains a conformation of high affinity agonist binding in the uncoupled and G-protein state is hypothesized, along with a conformation that binds well, but poorly activates G-proteins.
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Experimental Procedures |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Bacterial Expression and Membrane Preparation.
The cDNAs
of hD2S, hD2L, and
hD3 receptors were cloned, and the sequence was
verified by DNA sequencing (Schotte et al., 1996). For each receptor,
the cDNA was ligated in the expression vector pMal-p so that
it was in-frame with the maltose binding protein (MalE). All plasmid
constructs were confirmed by DNA sequencing. The resulting plasmids
coded for the MalE protein fused via a polylinker, containing a
protease factor Xa cleavage site, to the receptor at the C terminus.
Fusion and wild-type plasmids were transformed into competent E. coli TB1 cells [ara
(lac proAB) rpsL (
80 lacZM15) hsdR]
by electroporation and plated on 2xYT plates containing 100 µg/ml ampicillin. A single colony was inoculated into 2 ml of 2xYT
medium containing 100 µg/ml ampicillin. After 8 h of growth, the
2-ml starter culture was added to 500-ml 2xYT medium containing 100 µg/ml ampicillin and grown overnight in a shaking incubator (New
Brunswick Scientific, Edison, NJ) at 300 rpm and 37°C. The optical
density was determined, and the cells were harvested by centrifugation
(2000g for 10 min at 4°C) and resuspended to an optical
density of 1.5 in 2xYT, containing 100 µg/ml ampicillin and 0.5 mM
isopropyl-
-D-thiogalactopyranoside. Cells were
further incubated for 4 h at 25°C, harvested by centrifugation, and resuspended in ice-cold 50 mM Tris-HCl, pH 7.4, containing 20%
sucrose and 1 freshly dissolved tablet of protease inhibitor cocktail
(Complete) per 50 ml. In a first series of experiments on the
pharmacological characterization of hD3
receptors, E. coli cell suspensions were frozen as pellets
and thawed before use. In subsequent studies on the characterization of
D2 receptors and addition of G-proteins to
E. coli membranes, frozen droplets were prepared by freezing
drops of the cell suspension in liquid nitrogen followed by storage at
70°C until use. After thawing of the droplets, the cell suspension
was passed four times at 800 psi through a French press (Spectronic
Instruments, Cheshire, England). The suspension was diluted in 50 mM
Tris-HCl, pH 7.4, containing 10% glycerol. After a low spin
centrifugation (1400g for 5 min at 4°C) for removal of
intact cells, the supernatant was collected and centrifuged at
90,000g for 1 h at 4°C. The membrane pellet was
suspended in 50 mM Tris-HCl, pH 7.4, containing 10% glycerol and used
in radioligand binding experiments.
CHO Cell Culture and Membrane Preparation.
CHO cells
expressing the hD2S, hD2L,
or D3 receptors (Vanhauwe et al., 1999) were
grown in Dulbecco's modified Eagle's medium supplemented with 2 mM
L-glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% fetal calf serum in a humidified
atmosphere of 5% CO2 at 37°C. Cells were
subcultured at 80 to 90% confluence.
). The medium was removed, and the Petri dishes
were washed once with 5 ml of ice-cold PBS and stored at 70°C.
Cells on Petri dishes were thawed, and 5 ml of 10 mM Tris-HCl (pH 7.4),
1 mM EDTA, 1 mM 4-(2-aminoethyl)benzenesulfonylfluoride hydrochloride was added per dish. The cells were harvested and homogenized with a dual homogenizer (motor-driven Teflon pestle and
conical glass tube). The homogenate was centrifuged (10 min at
1000g at 4°C), and the resulting pellet was resuspended
and centrifuged again (10 min at 1300g at 4°C). Both
supernatants were pooled and centrifuged at 50,000g for
1 h at 4°C. The resulting pellet was resuspended in 50 mM
Tris-HCl (pH 7.4), containing 10% glycerol and stored in aliquots at
70°C. The protein concentration in membrane preparations was
measured with the Bradford protein assay using BSA as a calibration standard.
Radioligand Binding Assays. For radioligand binding experiments on hD3R-E. coli cells, the cell suspension was thawed and washed twice in the incubation buffer for the ligand. For radioligand binding experiments on E. coli or CHO cell membranes, the membranes were thawed and resuspended in the incubation buffer for the ligand.
[3H]7-OH-DPAT was used to measure agonist binding to hD3, hD2S, and hD2L receptors. The incubation with [3H]7-OH-DPAT was performed for 30 min at 25°C in a total volume of 0.5 ml, containing 50 mM Tris-HCl, pH 7.4, 10 mM MgCl2, and 1 mM EGTA (for hD2R-E. coli membranes, the total incubation volume was 1 ml). [125I]Iodosulpride was used to measure antagonist binding to hD3 receptors in E. coli cells and CHO cell membranes. The incubation with [125I]iodosulpride was performed for 30 min at 25°C in a total volume of 0.25 ml, containing 50 mM Tris-HCl, pH 7.4, 120 mM NaCl, and 0.1% BSA. [3H]Spiperone was used to measure antagonist binding to hD2S and hD2L receptors in E. coli membranes and CHO cell membranes. The incubation with [3H]spiperone was performed for 30 min at 37°C in a total volume of 0.5 ml, containing 50 mM Tris-HCl, pH 7.4, and 120 mM NaCl (for hD2R-E. coli membranes the total incubation volume was 1 ml). The amount of protein per incubation was 10 to 20 µg for E. coli membranes and cells and for hD2SR-CHO cell membranes. For hD3R-CHO and hD2LR-CHO cell membranes 5 to 10 µg of protein was used. Nonspecific binding was estimated in the presence of 10 µM haloperidol. The reaction was terminated by filtration through Whatman GF/B filters, presoaked in 0.1% polyethyleneimine. Filters were rinsed twice with 5 ml of ice-cold incubation buffer. The filter-bound radioactivity was measured in a liquid scintillation spectrometer (Tricarb, Packard, Meriden, CT), using 3 ml of scintillation fluid. Specific binding was calculated as the difference between total binding and nonspecific binding. For ligand concentration binding isotherms, [3H]7-OH-DPAT was used at 10 to 12 concentrations in the range of 0.1 to 20 nM for CHO cell membranes and 0.1 to 10 nM for E. coli cell membranes (high concentrations of [3H]7-OH-DPAT resulted in high nonspecific binding at E. coli membranes), and [125I]iodosulpride was used at 10 concentrations in the range of 0.1 to 3 nM and [3H]spiperone was used at 10 to 12 concentrations in the range of 0.01 to 1 nM. Ligand concentration binding isotherms were fitted to a rectangular hyperbola by nonlinear regression analysis in which the apparent equilibrium dissociation constant (Kd) and the maximum number of binding sites (Bmax) were free parameters. Computerized curve fitting was performed using the GraphPad Prism software. In competition binding experiments at hD3 receptors, serial dilutions of unlabeled compounds were incubated with [3H]7-OH-DPAT (2 nM) or [125I]iodosulpride (0.4 nM) and CHO or E. coli membranes. In the case of hD2S and hD2L receptors, serial dilutions of unlabeled compounds (10-12 concentrations, range 0.1 mM to 0.1 nM) were incubated with [3H]spiperone (0.5 nM) and CHO or E. coli membranes. Competition curves were fit to a sigmoid by nonlinear regression analysis according to algorithms described by Oestreicher and Pinto (1987), in which the
pIC50 and the Hill coefficient were free
parameters. Inhibition constants (Ki) were
calculated according to Cheng and Prusoff (1973). Assays were run in
duplicate in ligand concentration binding isotherms and in singlets in
competition binding experiments and repeated in independent experiments
(n numbers in tables and figures). Curves were calculated
for individual experiments, and the mean of the derived parameters was calculated.
Reconstitution of High Affinity [3H]7-OH-DPAT
Binding with G-Proteins.
A commercially available bovine
Gi/o-protein mix (G
i1,
Gi2, Gi3,
Go, G
purified from bovine brain) was
diluted in 50 mM Tris-HCl, pH 7.4, containing 10 mM
MgCl2, 1 mM EGTA, and 0.15% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS)
and then 5-fold diluted and mixed with E. coli membrane suspension by vortexing. At this concentration of CHAPS, 90% of the
binding is retained (results not shown). E. coli membranes (30-40 µg of protein/300 µl) were incubated in a total volume of
0.5 ml with serial dilutions of the G-protein mix and 0.75 nM or 2 nM
[3H]7-OH-DPAT for hD3 and
hD2S receptors, respectively. The assay buffer
contained 50 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 1 mM EGTA, and 0.03% CHAPS. After a 30-min incubation period at 25°C,
the reaction was terminated by filtration and the amount of bound radioactivity was determined as described above. The concentration of
the Gi/o-protein mix was calculated assuming an
average molecular mass of 87,000 Da.
Materials. [3H]Spiperone (3.5 TBq/mmol), [125I]iodosulpride (74.1 TBq/mmol), and [3H]7-OH-DPAT (5.5 TBq/mmol) were purchased from Amersham Pharmacia Biotech (Little Chalfont, UK). The E. coli strain (TB1) was from Stratagene (La Jolla, CA). The expression vector pMal-p was purchased from New England BioLabs (Leusden, Netherlands). The Gi/o-protein mix purified from bovine brain was obtained from Calbiochem (La Jolla, CA).
The 2xYT, Dulbecco's modified Eagle's medium, and fetal bovine serum were from Life Technologies (Gaithersburg, MD). The Bradford protein assay kit was from Bio-Rad (Richmond, CA). PD128907 and dopamine were purchased from Research Biochemicals International (Natick, MA). Raclopride and nemonapride were from Astra Arcus (Stockholm, Sweden) and Yamanouchi (Tokyo, Japan), respectively. TL99 was from ICN Pharmaceuticals (Costa Mesa, CA). Haloperidol, domperidone, risperidone, and spiperone are original products of Janssen Pharmaceutica (Beerse, Belgium). 7-OH-DPAT (racemic mixture) was synthesized in-house. Ampicillin, Complete protease inhibitor cocktail, CHAPS, isopropyl--D-thiogalactopyranoside, and
4-(2-aminoethyl)benzenesulfonylfluoride hydrochloride were purchased
from Boehringer Mannheim (Mannheim, Germany). All other reagents were
analytical grade from Merck (Haasrode, Belgium) or Sigma (St. Louis,
MO). GF/B glass-fiber filters were from Whatman (Kent, UK). The
scintillation fluid (Ultima Gold MV) was from Packard (Meriden, CT).
The GraphPad Prism program was from GraphPad Software, Inc. (San Diego,
CA). Dopamine, 7-OH-DPAT, and PD128907 were dissolved and diluted in
assay buffer. Lisuride and TL99 were dissolved and diluted in ethanol.
Haloperidol, spiperone, domperidone, risperidone, raclopride, and
nemonapride were dissolved and diluted in dimethyl sulfoxide (DMSO).
For compounds that were dissolved and diluted in DMSO or ethanol, a
last dilution step of 20-fold in assay buffer was performed just before
addition to the assay mixture in which the dilution was 10-fold. In
control assays, ethanol or DMSO was added to a final concentration of 0.5%.
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Results |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Ligand Concentration Binding Isotherms at hD3
Receptors.
Ligand concentration binding isotherms on
hD3 receptors were measured with the partial
agonist [3H]7-OH-DPAT and the antagonist
[125I]iodosulpride on E. coli cells
and CHO cell membranes expressing the recombinant receptor (Vanhauwe et
al., 1999). The ligand concentration binding curve of
[125I]iodosulpride on E. coli cells
is shown in Fig. 1A.
Bmax and apparent
Kd values obtained with
hD3R-E. coli cells and
hD3R-CHO cell membranes are shown in Table
1A. The affinity of
[125I]iodosulpride for
hD3 receptors expressed in E. coli or
CHO cells was similar (Kd = 1.2-1.4 nM).
Also, the agonist [3H]7-OH-DPAT bound with
comparable affinity to hD3R-E. coli
cells and hD3R-CHO cell membranes
(Kd = 1.5 nM). The affinity of
[3H]7-OH-DPAT for
hD3R-CHO cell membranes was not affected by
addition of GppNHp. No binding could be detected in vector-transformed E. coli cells and CHO cell membranes with either
radioligand.
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Ligand Concentration Binding Isotherms for hD2S and hD2L Receptors. Ligand concentration binding isotherms on hD2 receptors were measured with the partial agonist [3H]7-OH-DPAT and the antagonist [3H]spiperone. To improve the signal-to-noise ratio, membranes were prepared from E. coli cells expressing hD2S or hD2L receptors. The ligand concentration binding curves of [3H]spiperone on hD2SR- and hD2LR-E. coli membranes are shown in Fig. 1, B and C, respectively. Bmax and apparent Kd values are listed in Table 1B. The antagonist [3H]spiperone bound with a similar affinity to hD2S and hD2L receptors in E. coli than in CHO cell membranes (Student's t test, P > .05; Kd = 0.1-0.2 nM)). [3H]7-OH-DPAT binding was undetectable in hD2SR-E. coli or hD2LR-E. coli cell membranes within the tested concentration range (0.1-10 nM). In contrast, [3H]7-OH-DPAT bound to hD2SR- and hD2LR-CHO cell membranes; its affinity was similar for either splice variant (Kd = 2.3-2.9 nM). Addition of GppNHp significantly decreased by nearly 4-fold the affinity of [3H]7-OH-DPAT for hD2S (Student's t test, P < .005) and hD2L (Student's t test, P < .05) receptors in CHO cell membranes.
Inhibition Binding Experiments at hD3 Receptors.
The agonists dopamine, 7-OH-DPAT, PD128907, TL99, and lisuride and the
presumed antagonists haloperidol, spiperone, domperidone, risperidone,
raclopride, and nemonapride were tested for their potency to inhibit
[125I]iodosulpride and
[3H]7-OH-DPAT binding to
hD3 receptors. The pIC50
and Ki values are listed in Table
2. In general, the apparent binding
affinities of the antagonists for hD3 receptors
expressed in E. coli versus CHO cells were similar when
using [125I]iodosulpride or
[3H]7-OH-DPAT. The apparent affinities of
agonists were comparable for hD3 receptors
expressed in E. coli or CHO cells, when tested with
[125I]iodosulpride. When
[3H]7-OH-DPAT was used, dopamine and 7-OH-DPAT
were significantly (5-7-fold) less potent at E. coli than
at CHO cell membranes (paired Student's t test,
P < .05); no significant decrease in potency could be
found for PD128907, TL99, and lisuride in E. coli cells (paired Student's t test, P > .05). The
rank order of potencies of agonists and antagonists was quite similar
in hD3R-E. coli and
hD3R-CHO cell membranes. In
hD3R-E. coli cells and CHO membranes, raclopride and nemonapride showed lower apparent binding affinities with [3H]7-OH-DPAT than with
[125I]iodosulpride. It should be noted that
both compounds are benzamides like
[125I]iodosulpride; close structural
similarities between nonlabeled and labeled ligands often leads to more
effective inhibition.
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Inhibition Binding Experiments at hD2S and
hD2L Receptors.
The same series of agonists and
antagonists were tested for their potency to inhibit
[3H]spiperone binding;
pIC50 and Ki values
are listed in Table 3. The rank order of
antagonist potencies was similar for hD2S and hD2L receptors when expressed in E. coli or CHO cell membranes. The apparent binding affinities of
antagonists for hD2L receptors in E. coli membranes were two to five times lower than those in CHO cell
membranes. Some compounds showed higher differences with the
hD2S receptor. Dopamine and 7-OH-DPAT were about
10 to 20 times less potent in E. coli membranes than in CHO
cell membranes; PD128907 and lisuride were only three times less potent
at hD2-E. coli than at
hD2-CHO cell membranes. In CHO cell membranes,
the potencies of compounds at hD2S and
hD2L receptors were virtually similar. This was
also the case for these splice variants when expressed in E. coli (except for raclopride).
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Reconstitution of G-Protein Interaction with hD2S and
hD3 Receptors in E. coli.
In a first
series of experiments (see Fig. 2),
radioligand binding isotherms were determined with
[3H]7-OH-DPAT using a fixed concentration
(±2.3 nM) of the G-protein mix, which corresponds to a
G-protein/receptor ratio as indicated in Fig.
3. To compare the agonist binding
properties of hD2 and hD3
receptors, we have prepared membranes from E. coli cells and incubated them with the G-protein mix. The apparent affinity of [3H]7-OH-DPAT for hD3
receptors in prepared membranes was virtually equal to that in
hD3R-E. coli cells, indicating that
preparation of the membranes does not alter the binding properties of
the receptor. [3H]7-OH-DPAT had an apparent
binding affinity of 1.7 ± 0.2 nM (n = 4) for
untreated (Bmax = 2000 ± 300 fmol/mg
of protein) and 1.1 ± 0.2 nM (n = 4) for
G-protein-treated hD3R-E. coli
membranes (Bmax = 2200 ± 300 fmol/mg
of protein); addition of GppNHp along with the G-protein mix, resulted
in an apparent binding affinity of 2.0 ± 0.3 nM
(n = 4) (Bmax = 2200 ± 200 fmol/mg of protein). As mentioned before,
[3H]7-OH-DPAT binding at
hD2R-E. coli membranes could not be
determined in the absence of G-proteins. However, when the G-protein
mix was added to the membranes, [3H]7-OH-DPAT
binding was clearly detectable at hD2SR-E.
coli membranes (see Fig. 2) with an apparent binding affinity of
3.5 ± 0.1 nM (n = 2)
(Bmax = 160 ± 30 fmol/mg of protein);
addition of GppNHp thereupon abolished
[3H]7-OH-DPAT binding. At
hD2LR-E. coli membranes,
[3H]7-OH-DPAT binding became apparent on
addition of G-proteins, but the signal-to-noise ratio was too low for
further characterization.
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7 M in hD3R-E.
coli membranes and 9.4 × 108 M in
hD2SR-E. coli membranes (for details
of calculation see Fig. 3). Addition of the
Gi/o-protein mix to
hD2SR-E. coli or hD3R-E. coli membranes had no effect
on antagonist binding (results not shown). At the highest concentration
of the Gi/o-protein mix, we estimated a
G-protein/receptor ratio of 260 for hD3 and of 780 for hD2S receptors.
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Discussion |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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We have used an E. coli and CHO expression system to investigate the ligand binding properties of hD2S, hD2L, and hD3 receptors in the absence or presence of G-proteins, respectively. The goal was to obtain information on their receptor-G-protein interaction.
Successful Expression of hD2S, hD2L, and
hD3 Receptors in E. coli.
We have
demonstrated expression of hD2S,
hD2L, and hD3 receptors in
E. coli membranes. The antagonist
[125I]iodosulpride and partial agonist
[3H]7-OH-DPAT bound with similar affinity to
hD3 receptors expressed in E. coli and
CHO cells (Table 1). In general, the apparent binding affinities and
the rank order of potency of the antagonists and agonists tested were
similar in hD3R-E. coli cells and
hD3R-CHO cell membranes (Table 2).
[3H]Spiperone bound to
hD2SR-E. coli and
hD2LR-E. coli cell membranes with an
apparent affinity comparable with hD2SR-CHO and
hD2LR-CHO cell membranes (Table 1). The tested
antagonists revealed apparent affinities at
hD2SR-E. coli and
hD2LR-E. coli membranes that were lower than their apparent affinities in hD2SR-CHO
and hD2LR-CHO cell membranes, but the rank order
of potency was similar, suggesting successful expression (Table 3). The
lower apparent affinities of the ligands at hD2
receptors in E. coli membranes may be attributed to sticking
of the compounds and/or [3H]spiperone (see Fig.
1, B and C: high nonspecific binding of [3H]spiperone) to E. coli membranes.
Alternatively, it may be attributed to the different composition of
bacterial membranes; lower affinities of antagonists have also been
observed for dopamine D2 receptors expressed in
Saccharomyces cerevisiae (Sander et al., 1994). Also, the
different protein amount and assay volume in the binding assays for
hD2R-E. coli and -CHO membranes may
lead to the lower apparent affinities of the compounds in E. coli membranes versus CHO membranes. Inhibition binding constants
using [3H]7-OH-DPAT in membranes of
hD2SR- or hD2LR-expressing
E. coli could not be determined due to the absence of
radioligand binding at nanomolar concentration. In CHO and E. coli cell membranes, compounds had similar affinities for
hD2S and hD2L receptors, respectively. This confirms previous findings and expands the idea of
similar binding properties of these splice variants also to the
G-protein-free environment in E. coli membranes (Schotte et
al., 1996).
G-Protein Uncoupling Decreases High Affinity Agonist Binding at hD2 Receptors. GppNHp treatment significantly decreased the affinity of [3H]7-OH-DPAT at hD2SR-CHO and hD2LR-CHO cell membranes (Table 1). Moreover, the lack of [3H]7-OH-DPAT binding at nanomolar concentration in hD2SR-E. coli and hD2LR-E. coli membranes indicates that hD2 receptors have a low affinity for [3H]7-OH-DPAT in the total absence of G-proteins. Probably, G-proteins in CHO membranes cannot be fully dissociated from their receptor by GppNHp leading to an intermediate ("low") affinity for agonists. It was observed that [3H]7-OH-DPAT bound to hD2SR-E. coli membranes in the presence of a Gi/o-protein mix with an affinity similar to what was found in hD2SR-CHO cell membranes, indicating proper folding of the receptor and reconstitution of the high affinity state. This reconstitution was reversible, because GppNHp treatment abolished [3H]7-OH-DPAT binding. Furthermore, we found significantly lower potencies (10-20-fold lower) of dopamine and 7-OH-DPAT for inhibition of [3H]spiperone binding to hD2SR- and hD2LR-E. coli membranes (Table 3), indicating again that high affinity agonist binding to D2 receptors requires the presence of G-proteins.
G-Protein-Coupled and Uncoupled States of hD3 Receptors
Are Similar.
It is generally assumed that D3 receptors
poorly couple to G-proteins or that the investigated cell systems do
not possess the suitable G-proteins to explain a small (or no) shift in
high affinity agonist binding on GTPS treatment. The latter
assumption is further substantiated by studies reporting the inability
of D3 receptors to regulate effector systems
(Freedman et al., 1994; Tang et al., 1994). However, in recent
publications it was shown that D3 receptors do
activate G-proteins (Vanhauwe et al., 1999). However, it has not
been established whether the slight modulation of agonist binding by
guanine nucleotides is due to a particular interaction between the
D3 receptor and its target G-protein or that it
is due to specific properties of the receptor itself. Our results
confirm that GppNHp treatment does not decrease the affinity of an
agonist ([3H]7-OH-DPAT) for
hD3 receptors in CHO cell membranes. Most
interestingly, we found that [3H]7-OH-DPAT had
similar affinities for hD3 receptors in E. coli and CHO cell membranes (Table 1). This clearly shows that
high affinity agonist binding to hD3 receptor is
almost independent of G-proteins, which makes it unlikely that a tight
association between receptor and G-protein would prevent the effect of
guanine nucleotides.
Inverse Agonists Do Not Have Higher Affinities for Uncoupled
hD2 and hD3 Receptors.
It was hypothesized
that inverse agonists would have higher affinities for uncoupled than
for coupled receptors (Costa et al., 1992). Antagonists tested in this
study have been reported to be inverse agonists at
hD2S (haloperidol, domperidone, risperidone, spiperone, and nemonapride) and hD3 receptors
(haloperidol and raclopride) (Griffon et al., 1996; Hall and Strange,
1997; Kozell and Neve, 1997; Malmberg et al., 1998; Vanhauwe et al.,
2000). However, Malmberg et al. (1998) reported no shift to higher
affinities for inverse agonists at hD3 receptors
after pertussis toxin treatment of the membranes. Here, we demonstrate
that in E. coli membranes, which are devoid of G-proteins,
these presumed inverse agonists do not have higher affinities for
hD2S, hD2L, or
hD3 receptors than in CHO cell membranes.
Therefore, the theory of the allosteric ternary complex model with
regard to inverse agonists seems not to be generally applicable.
A Purified Bovine G-Protein Mix Has a Comparable Affinity for hD3 and hD2 Receptors. It is not known why D3 receptors activate G-proteins (much) less efficiently than D2 receptors. Several hypotheses have been put forward, such as inability to activate and/or couple to G-proteins, unavailability of suitable G-proteins, or a low affinity toward G-proteins. To address this issue, we have tried to estimate the affinity of a subset of pertussis toxin-sensitive G-proteins for the hD2S and hD3 receptors. Therefore, we have reconstituted high affinity [3H]7-OH-DPAT binding at hD2SR- and hD3R-E. coli membranes by addition of increasing concentrations of exogenous Gi/o-proteins, purified from bovine brain. As was found before, no specific [3H]7-OH-DPAT binding was detectable in the absence of G-proteins at hD2SR-E. coli membranes, in contrast to hD3R-E. coli membranes. However, the G-protein mix increased [3H]7-OH-DPAT binding in a dose-dependent manner at either receptor, whereas it had no effect on antagonist binding (latter results not shown).
Most surprisingly, the G-protein mix was at least as potent in increasing high affinity agonist binding at hD3 receptors than at hD2S receptors. The estimated Kd of 1.4 × 107 M for the
hD3R·Gi/o complex and the
Kd of 9.4 × 108 M for the
hD2SR·Gi/o complex
corresponded to reported affinities of G-proteins for G-protein-coupled
receptors. In addition, the G-protein/receptor ratio for
half-maximal effect was similar to previous findings for
A1-adenosine and 5HT1A
receptors in E. coli membranes (Bertin et al., 1992; Jockers
et al., 1994) and slightly higher for dopamine
D2S receptors in Sf9 membranes (Grünewald et al., 1996). The G-protein/receptor ratio is probably overestimated due to the presence of inactive, purified G-protein contaminations and/or nonoptimal insertion of G-proteins in E. coli
membranes. It cannot be concluded that more G-proteins per receptor
were required for reconstituting high affinity binding at
hD3 receptors (versus hD2S
receptors), because more receptors were expressed in the
hD3-E. coli membranes. Therefore, a
more efficient way was to calculate the Kd
values based on our results (see Fig. 3 and legend), which showed that
there was no difference in affinity between hD2S
and hD3 receptors for the
Gi/o-protein mix. Until recently, it was
generally believed that hD3 receptors couple poorly to G-proteins when compared with D2
receptors. Now, we found an equally strong interaction of
hD3 receptors compared with
hD2 receptors with a
Gi/o-protein mix. Robinson and Caron (1996)
suggested a weak interaction between hD3
receptors and G-proteins due to a constrained receptor structure. We
propose a strong interaction between hD3
receptors and G-proteins, but a rigid receptor structure that is little
influenced by G-protein coupling. The rigid hD3
receptor activates G-proteins less efficient than
D2 receptors. Hence, the receptor-G-protein
interaction can be described by two independent parameters, analogous
to the agonist-receptor interaction, i.e., affinity and intrinsic
activity. In this respect, hD3 receptors have a
high affinity and a low intrinsic activity at G-proteins, whereas
hD2 receptors have a high affinity and a high
intrinsic activity at G-proteins. In future studies, it will be
interesting to define the different regions of the receptor that
contribute to either G-protein coupling or activation.
In conclusion, we have expressed in E. coli
hD2S, hD2L, and
hD3 receptors, which retained their antagonist
binding properties. In E. coli, as well as in CHO cell
membranes, agonist binding to uncoupled receptors was severely impaired
at hD2S and hD2L receptors,
whereas only a small effect was observed at hD3
receptors. We found that the low and high affinity states of
hD3 receptors are similar, whereas for
hD2S and hD2L receptors
they are clearly distinguishable. Furthermore, our results indicate
that hD2S and hD3 receptors
have a similar affinity for an exogenously added Gi/o-protein mix. Thus, the reported low
signaling capacity of hD3 receptors, as compared
with hD2 receptors, is not due to a low affinity
for G-proteins but may be attributed to a constrained structure of the receptor.
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Acknowledgments |
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We greatly appreciate the technical assistance of Monique Berben and the late Walter Gommeren.
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Footnotes |
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Accepted for publication July 3, 2000.
Received for publication April 12, 2000.
1 This work was supported by a grant (project IWT 940232) from the IWT (Vlaams Instituut voor de Bevordering van het Wetenschappelijk-Technologisch Onderzoek in de Industrie).
2 Present address: the laboratory of Dr. Heidi Hamm, Northwestern University, Institute of Neuroscience, Chicago, IL.
Send reprint requests to: Josée E. Leysen, Janssen Pharmaceutica, Turnhoutseweg 30, B-2340 Beerse, Belgium. E-mail: jleysen2{at}janbe.jnj.com
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Abbreviations |
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CHO, Chinese hamster ovary;
7-OH-DPAT, (+)-7-(dipropylamino)-5,6,7,8-tetrahydro-2-naphthalenol;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate;
DMSO, dimethyl sulfoxide;
FCS, fetal calf serum;
G-protein, heterotrimeric
guanine nucleotide binding protein;
GppNHp, guanylyl imidodiphosphate;
hD2, human dopamine D2;
hD3, human dopamine D3;
pIC50, log IC50
(concentration of the compound producing 50% inhibition of the
specific binding of the radioactive ligand);
pEC50, log EC50
(concentration of the compound producing 50% effect).
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References |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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