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Vol. 58, Issue 4, 677-683, October 2000
Department of Pharmacology (K.D.K., N.K., R.L.), Neuroscience Graduate Program (R.L.), Penn State College of Medicine, Milton S. Hershey Medical Center, Hershey, Pennsylvania
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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We have generated a stable cell line expressing FLAG epitope-tagged D3 dopamine receptors and used this cell line to study D3 receptor-protein interactions. To analyze protein interactions, we separately introduced into the stable cell line either D3 receptors carrying an hemagglutinin (HA) epitope tag, or an HA-tagged version of the D3 receptor splice variant D3nf. A combination of confocal laser microscopy and coimmunoprecipitation was used to assay the formation and expression pattern of D3-D3 homodimers or D3-D3nf heterodimers. When coexpressed in HEK 293 cells, FLAG- and HA-tagged D3 receptors exhibited a similar plasma membrane distribution. Using an HA epitope tag-specific antibody, we coimmunoprecipitated HA- and FLAG-tagged D3 receptors, suggesting that D3 receptors are capable of forming homodimers. Epitope-tagged D3nf polypeptides exhibited a markedly different cellular distribution than D3 receptors. When expressed in HEK 293 cells, either alone or in combination with FLAG-tagged D3 receptors, D3nf exhibited a punctate perinuclear distribution. When D3nf was introduced into the stable D3-expressing cell line, D3 receptors were no longer visualized at the plasma membrane. Instead, D3 and D3nf showed a similar, predominantly cytosolic, localization. Using the HA-specific antibody, we were able to coimmunoprecipitate D3 and D3nf polypeptides from transfected cells. These data suggest the existence of physical interaction between D3 and D3nf. This interaction appears to result in the mislocalization of D3 receptors from the plasma membrane to an intracellular compartment, a finding that could be of significance in the etiology of schizophrenia.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Dopamine
neurotransmission in mammalian brain is mediated by a cohort of
receptors that are members of the superfamily of G protein-coupled
receptors (GPCRs). In humans, five dopamine receptor subtypes (D1-D5)
have been identified by molecular cloning (reviewed in Missale et al.,
1998
). The five dopamine receptors have been grouped into two
subfamilies based upon sequence homologies and pharmacologic profiles.
The D1 class of dopamine receptors is comprised of the D1 and D5
receptor subtypes. These receptors are expressed at high levels in
cerebral cortex and are coupled to stimulatory subsets of
heterotrimeric G proteins. The D2 class of dopamine receptors,
consisting of the D2, D3, and D4 subtypes, is coupled to inhibitory
subsets of G proteins and is a major target of antipsychotic drugs.
Among the D2 class of dopamine receptors, the D3 receptor is
distinctive in that it is distributed preferentially in limbic areas of
the brain (nucleus accumbens, olfactory tubercle, islands of Calleja,
and hippocampus) thought to control cognitive and emotional aspects of
behavior (Bouthenet et al., 1991). The D3 receptor has also been shown
to bind most antipsychotic drugs, both typical and atypical, with high
affinity (Levant, 1997). Because of its anatomic distribution and
interaction with antipsychotic drugs, the D3 dopamine receptor has been
suggested to play a role in the etiology of schizophrenia. Post-mortem
examination of patients with chronic schizophrenia has shown a
selective loss of D3 mRNA sequences in the parietal and motor cortex
(Schmauss et al., 1993). A novel D3 receptor splice variant, termed
D3nf, has recently been identified and shown to be present in regions
of schizophrenic brains lacking D3 mRNA transcripts (Schmauss et al.,
1993). D3nf mRNA encodes a polypeptide with a carboxyl terminus
distinct from that of the original human D3 receptor (Schmauss et al.,
1993). Immunohistochemical analysis using antibodies raised against the unique carboxyl terminus of D3nf has revealed expression of D3nf polypeptides in rat and monkey brain (Nimchinsky et al., 1997). It is
not clear, however, whether the D3nf polypeptide is correctly targeted
to, or inserted into, the plasma membrane. The functional significance
of D3nf in either normal or aberrant dopaminergic neurotransmission is
also an issue that has not been clearly elucidated.
Signaling through the D3 receptor plays an important role in regulating
a variety of neural processes including brain development (Fishburn et
al., 1996; Levant, 1997), modulation of locomotor activity (Accili et
al., 1996), and motivational aspects of behavior (Caine and Koob,
1993). However, the mechanisms that control signaling through the D3
and other neurotransmitter receptors are complex and, at present, not
well understood. Recent work has suggested protein-protein interactions
may play a crucial role in receptor-mediated signaling events. For
example, specialized proteins such as rapsyn and gephyrin, as well as a
large class of proteins containing PDZ domains, have been shown to
cluster and localize signaling molecules at neuronal synapses (Craven
and Bredt, 1998). To begin to investigate potential mechanisms of D3
receptor regulation, a stable cell line expressing an epitope-tagged D3
receptor was generated and used to analyze D3 receptor-protein
interactions. In this study, we provide evidence for the existence of
D3-D3 and D3-D3nf interactions. When coexpressed in transfected cells, D3nf appears to function in a dominant-negative fashion to prevent D3
receptors from localizing to the plasma membrane. These observations are an important first step in understanding the functional
significance of D3nf in D3 receptor-mediated neurotransmission.
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Materials and Methods |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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DNA Constructs and Transfections.
FLAG or hemagglutinin (HA)
epitope tags were inserted at the amino termini of the D3 and D3nf
polypeptides by polymerase chain reaction mutagenesis as
described by Nelson and Long (1989). Each tagged polypeptide was
verified by DNA sequencing and then subcloned into the eukaryotic
expression vector pCB6 (Brewer and Roth, 1991). Human embryonic kidney
(HEK) 293 cells were used as recipients for transfection. Cells were
grown in Dulbecco's modified Eagle's medium supplemented with 10%
fetal calf serum. Transfections were performed by the calcium phosphate
coprecipitation method as previously described (Canfield et al., 1996).
For stable cell lines, cells were transfected with a FLAG-tagged D3
receptor cDNA construct and maintained under selection using 800 mg/ml
G418 (LifeTechnologies Inc., Rockville, MD). Individual clones were
selected approximately 3 weeks following transfection and expanded into
cell lines that were maintained in medium containing 800 mg/ml G418.
For transient transfections, HEK 293 cells were plated either in 100-mm
tissue culture dishes or on glass coverslips and transfected under
conditions described above. Neuro-2a cells were obtained from American
Type Culture Collection (Manassas, VA) and grown in minimum essential Eagle's medium supplemented with 10% fetal calf serum. Transfection of Neuro-2a cells was performed using the LipofectAMINE 2000 transfection reagent (Life Technologies Inc.) according to instructions
supplied by the manufacturer.
Immunofluorescence and Confocal Microscopy. Transiently transfected HEK 293 cells grown on glass coverslips were examined 72 h after transfection. Cells were fixed in 1:1 methanol/acetone (v/v) solution, blocked with PBS containing 2% bovine serum albumin and 10% goat serum at room temperature for 1 h, and then incubated in the same medium with a 1:1000 dilution of the HA-specific monoclonal antibody (mAb) 16B12 (Babco, Berkeley, CA) or a 1:4000 dilution of the anti-FLAG mAb M2 (Kodak, Rochester, NY). Secondary antibody (Cy-3-conjugated goat anti-mouse IgG; Jackson ImmunoResearch, West Grove, PA) was diluted 1:800 and applied in the same buffer. For double labeling, cells were coincubated with a 1:200 dilution of a polyclonal HA antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and a 1:4000 dilution of the FLAG-specific M2 mAb antibody. Labeling was detected by incubation with rhodamine red-conjugated goat anti-mouse (diluted 1:200) and fluorescein isothiocyanate (FITC)-conjugated donkey anti-rabbit (diluted 1:200) secondary antibodies (Jackson ImmunoResearch). Immunofluorescence was visualized by confocal laser scanning microscopy using a Zeiss LSM 210 confocal microscope.
Membrane Preparation, Immunoblotting, and
Immunoprecipitation.
Crude membrane fractions from either stably
or transiently transfected HEK 293 cells were prepared as described
previously (Shyjan and Levenson, 1989), and protein concentrations were
determined by the method of Bradford (1976). Solubilized membrane
fractions were separated on an SDS-containing 12% polyacrylamide gel
(70 µg of protein/lane) and transferred to a polyvinylidene fluoride membrane (ICN Biomedicals, Aurora, OH) as described (Karpa et al.,
1999). The filter was blocked for 2 h in PBS containing 10% dry
milk and 5% goat serum, and then incubated with a 1:1000 dilution of
the anti-FLAG M2 mAb. The filter was rinsed with PBS and then incubated
with either horseradish peroxidase-conjugated goat anti-rabbit or goat
anti-mouse secondary antibodies (Jackson ImmunoResearch) for 1 h.
Immunoreactivity was detected by enhanced chemiluminescence using an
ECL Plus kit (Amersham, Piscataway, NJ).
). Samples were digested for 1 h at 37°C.
For immunoprecipitation, cell lysates were prepared from transfected
cells using protocols described in the ImmunoCatcher immunoprecipitation kit (CytoSignal, Irvine, CA). Cell lysates were
incubated with a 1:100 dilution of the anti-HA mAb 12CA5 (Boehringer
Mannheim) for 2 h at room temperature. Protein A/G resin
(Cytosignal) was used to capture antigen, and bound proteins were
eluted with SDS-polyacrylamide gel electrophoresis gel loading buffer.
Proteins were analyzed by electrophoretic separation on an
SDS-containing 12% polyacrylamide gel.
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Results |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Generation and Characterization of D3 Dopamine Receptor Expressing
Cell Lines.
To study D3 dopamine receptor-protein interactions, we
generated an HEK 293 cell line stably expressing FLAG epitope-tagged D3
dopamine receptors. Confocal laser microscopy was used to analyze the
cellular distribution of the epitope-tagged D3 receptors. As shown in
Fig. 1A, cells transfected with the
FLAG-tagged D3 receptor were reactive with the FLAG-specific M2 mAb.
Strong staining was visualized at cell margins, indicating a
predominantly plasma membrane localization of the D3 receptor
polypeptides. Untransfected cells, or cells treated with secondary
antibody alone, produced no visible staining (data not shown).
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lane) shows that the FLAG-specific mAb reacted most
intensely with a broad band ~40 to ~45 kDa in size. A series of
less intense bands ~58, ~60, and ~80 kDa in size were also
reactive with the M2 mAb. Each of these immunoreactive bands appear to
represent differentially glycosylated forms of the D3 receptor.
Treatment of the microsomes with N-glycosidase F, an enzyme
that removes N-linked sugars, produced a predominant immunoreactive band ~40 kDa in size (Fig. 1B, + lane). This band corresponds well with the size of the D3 dopamine receptor predicted from cDNA cloning (Sokoloff et al., 1990). These results demonstrate that transfected HEK 293 cells stably express epitope-tagged D3 receptors. These receptors are post-translationally modified by the
addition of N-linked sugars and appear to be properly
targeted to the plasma membrane.
Cellular localization of epitope-tagged D3 and D3nf polypeptides was
examined in transiently transfected HEK 293 cells. Cells transiently
transfected with either FLAG- (Fig. 2B)
or HA-tagged (Fig. 2D) D3 constructs showed predominantly plasma
membrane localization of D3 receptors. Untransfected cells produced no
visible staining with anti-FLAG (Fig. 2A) or anti-HA (Fig. 2C)
antibodies. These results indicate that transiently expressed D3
receptors exhibit the same plasma membrane distribution as stably
expressed D3 receptors. Transient transfection of epitope-tagged D3nf
constructs produced a strikingly different staining pattern. Cells
transiently transfected with either FLAG- (Fig.
3B) or HA-tagged (Fig. 3D) D3nf
constructs showed a predominantly punctate, cytosolic staining pattern.
Little, if any, staining was observed at the margins of cells
transiently transfected with D3nf constructs. Untransfected cells
showed virtually no antibody reactivity with either the anti-FLAG (Fig.
3A) or anti-HA (Fig. 3C) antibodies. These results provide the first evidence that the D3 splice variant, D3nf, does not appear to traffic
to the plasma membrane. Instead, D3nf polypeptides appear to be
retained within some intracellular compartment in transfected HEK 293 cells.
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D3-D3nf and D3-D3 Interactions.
The distinct intracellular
distributions exhibited by D3 and D3nf prompted us to examine the
effect of coexpression of D3 and D3nf constructs on the subsequent
localization of the two polypeptides. To address this issue, we used
confocal laser microscopy to localize epitope-tagged D3 and D3nf
polypeptides after cells stably expressing FLAG-tagged D3 receptors
were transiently transfected with an HA-tagged D3nf construct. As shown
in Fig. 4A, cells reactive with anti-HA
antibodies showed a punctate staining pattern (arrow), suggesting
cytosolic localization of D3nf polypeptides. To localize D3 receptors,
the same field of cells was stained with anti-FLAG antibodies (Fig.
4B). In cells not expressing D3nf, anti-FLAG D3 receptor staining was
observed at cell margins (arrowhead). In cells expressing D3nf (arrow),
anti-FLAG antibodies gave a predominantly cytosolic staining pattern.
Superimposition of anti-HA and anti-FLAG images (Fig. 4C) showed
overlap in the cytosolic distribution of anti-FLAG and anti-HA
immunoreactivity in cells (arrow) exhibiting coexpression of D3 and
D3nf polypeptides. These results suggest that when D3 and D3nf
polypeptides are coexpressed in the same cell, D3 receptors no longer
traffic to the plasma membrane.
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In this study, we describe several novel features of D3 dopamine receptor structure and function. The ability to coimmunoprecipitate HA- and FLAG-tagged D3 receptors from doubly transfected cells provides biochemical evidence that D3 receptors can form homodimers. Immunoprecipitation analysis indicates that physical interaction between D3 and D3nf polypeptides can occur as well. As a result of this interaction, D3 receptors no longer appear to be capable of trafficking to the plasma membrane. D3nf, whose normal cellular function is unknown, may act in transfected cells as a dominant-negative regulator of D3 receptor activity.
A number of GPCRs have been shown to be capable of forming homodimeric
(Hebert et al., 1996; Fukushima et al., 1997; Xie et al., 1999) or
heterodimeric (Jordan and Devi, 1999; Marshall et al., 1999; Xie et
al., 1999) structures. It has recently been suggested that the dopamine
D3 receptor may also form higher order (dimeric and tetrameric)
structures (Nimchinsky et al., 1997). However, this conclusion is based
primarily on the identification of D3 antibody-reactive polypeptides
that migrate on gels at the positions expected for multimers of the D3
receptor core protein (Nimchinsky et al., 1997). Here we provide direct
biochemical data supporting the view that the D3 receptor is capable of
forming homodimers. This conclusion is based on coexpression of D3
receptors containing HA or FLAG epitope tags in transfected HEK 293 cells. The ability of an antibody directed against one of the epitope tags to immunoprecipitate D3 receptors carrying the heterologous tag
provides very strong evidence for D3-D3 receptor interaction. It is
possible that D3 receptor polypeptides may interact directly with one
another, as has been described for muscarinic (Maggio et al., 1996) and
GABAB (White et al., 1998) receptors.
Alternatively, D3 receptors could interact indirectly via interaction
with receptor accessory proteins, as has been described for
GABAA receptors (Essrich et al., 1998; Wang et
al., 1999). At present, the functional significance of D3 receptor
homodimerization remains unknown. It will clearly be of interest to
define the domains through which D3 receptors interact, and to
ascertain whether dimerized D3 receptors exhibit different
pharmacological properties than do monomeric D3 receptors. Recently,
dopamine D5 receptors have been shown to interact directly with
GABAA receptors (Liu et al., 2000). Our
expression system could therefore be used to study interaction between
D3 and other dopamine receptor subtypes, as well as additional GPCR
family members.
The dopamine D3 receptor splice variant D3nf was originally identified
in post-mortem brains from patients with schizophrenia (Schmauss et
al., 1993). The polypeptide encoded by D3nf mRNA lacks the sixth and
seventh transmembrane segments found in the full-length D3 receptor.
Although D3nf polypeptides have been detected in human, monkey, and
rodent brain (Liu et al., 1994; Nimchinsky et al., 1997), it is not
clear whether D3nf functions as an authentic GPCR (Schmauss et al.,
1993). Here we show that in transiently transfected HEK 293 and
Neuro-2a cells, epitope-tagged D3nf polypeptides do not traffic to the
plasma membrane. Instead, D3nf polypeptides localize to an as yet
undefined intracellular compartment. It is possible that transient D3nf
overexpression could lead to the cytoplasmic accumulation of D3nf
polypeptides. We view this possibility as unlikely, however, since
transiently expressed wild-type D3 receptors are correctly targeted to
the plasma membrane in both HEK 293 and Neuro-2a cells. Our data thus lends support to the idea that D3nf is not likely to function as a
typical GPCR.
In cells stably expressing FLAG-tagged D3 receptors, anti-FLAG staining
was detected almost exclusively at the plasma membrane. Introduction of
D3nf into these cells produced a dramatic shift in anti-FLAG staining
from the plasma membrane to the cytoplasm. In contrast, the plasma
membrane distribution of anti-FLAG staining was not affected by
transient expression of HA-tagged D3 receptors in stably transfected
HEK 293 cells. These results strongly imply that D3nf causes
mislocalization of D3 receptors in doubly transfected cells and that
mislocalization of D3 receptors is not an artifact of the transient
expression system. The fact that D3 and D3nf can be
coimmunoprecipitated from doubly transfected cells suggests that the
physical interaction of D3 and D3nf polypeptides is the mechanism that
underlies the failure of D3 receptors to traffic to the plasma
membrane. A number of truncated receptor variants have recently been
described, including truncated forms of the V2 vasopressin (Zhu and
Wess, 1998), gonadotropin-releasing hormone (Grosse et al., 1997), and
chemokine 5 (Benkirane et al., 1997) receptors. In each case,
coexpression of truncated and wild-type receptors results in diminished
cell surface expression of the full-length receptors. The
immunolocalization studies reported here provide the first evidence
that D3nf may play an inhibitory role in dopaminergic signaling through
D3 receptors.
The physiological significance of D3-D3nf protein interaction is
not yet understood. A truncated version of the D3 receptor has recently
been generated in transgenic mice (Accili et al., 1996). Binding of the
dopamine antagonist iodosulpride was greatly reduced in mice
heterozygous for the D3 receptor mutation, suggesting that the
truncated D3 receptor may act in a dominant-negative fashion to inhibit
antagonist binding to D3 receptors produced from the wild-type allele
(Accili et al., 1996). It will therefore be of considerable interest to
determine whether in coexpression studies, D3nf acts in a
dominant-negative fashion to alter the pharmacological profile of the
D3 receptor for various ligands, or whether D3-D3nf interaction affects
activation of appropriate subsets of G proteins in transfected cells.
For D3-D3nf interactions to have physiological relevance in vivo, it is
necessary that the two polypeptides be coexpressed in the same neurons.
Immunostaining with D3- and D3nf-specific antibodies has revealed
overlap in the distribution of D3 and D3nf polypeptides within cortical
pyramidal neurons of rat brain (Nimchinsky et al., 1997). Experiments
designed to determine the subcellular localization of D3 and D3nf
polypeptides within specific neuronal cell types could help clarify the
role D3nf plays in the intracellular trafficking of D3 receptors.
Finally, D3-D3nf interaction may be of relevance for understanding the
etiology of schizophrenia. D3nf has been detected in brains of normal
human subjects as well as brains from patients with chronic
schizophrenic (Schmauss et al., 1993). In schizophrena, the ratio of D3
to D3nf mRNA sequences is altered in several brain regions including
the parietal and motor cortices. It is conceivable that within these
brain regions, alterations in the ratio of D3 to D3nf polypeptides
could profoundly affect cell surface expression of D3 receptors and
thus contribute to the manifestation of schizophrenia. Clearly,
investigation of the role of D3 receptor function is just beginning.
Further study is required to verify whether alterations in the homo- or
heterodimeric structure of this receptor contribute to abnormalities in
dopaminergic neurotransmission in various pathological conditions.
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Footnotes |
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Received March 24, 2000; Accepted June 21, 2000
This work was supported by National Institute of Mental Health Grant P50-MH44866.
Send reprint requests to: Dr. Robert Levenson, Department of Pharmacology, H078, Penn State University College of Medicine, Milton S. Hershey Medical Center, P.O. Box 850, Hershey, PA 17033. E-mail: rlevenson{at}hmc.psu.edu
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Abbreviations |
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GPCRs, G protein-coupled receptors;
HA, hemagglutinin;
HEK, human embryonic kidney;
mAb, monoclonal antibody;
FITC, fluorescein isothiocyanate;
GABA, 
-aminobutyric acid.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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M. A. Uberti, R. A. Hall, and K. P. Minneman Subtype-Specific Dimerization of {alpha}1-Adrenoceptors: Effects on Receptor Expression and Pharmacological Properties Mol. Pharmacol., December 1, 2003; 64(6): 1379 - 1390. [Abstract] [Full Text] [PDF]
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J. J. Carrillo, J. Pediani, and G. Milligan Dimers of Class A G Protein-coupled Receptors Function via Agonist-mediated Trans-activation of Associated G Proteins J. Biol. Chem., October 24, 2003; 278(43): 42578 - 42587. [Abstract] [Full Text] [PDF]
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 A. Leanos-Miranda, A. Ulloa-Aguirre, T. H. Ji, J. A. Janovick, and P. M. Conn Dominant-Negative Action of Disease-Causing Gonadotropin-Releasing Hormone Receptor (GnRHR) Mutants: A Trait That Potentially Coevolved with Decreased Plasma Membrane Expression of GnRHR in Humans J. Clin. Endocrinol. Metab., July 1, 2003; 88(7): 3360 - 3367. [Abstract] [Full Text] [PDF]
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T. Seck, R. Baron, and W. C. Horne The Alternatively Spliced {Delta}e13 Transcript of the Rabbit Calcitonin Receptor Dimerizes with the C1a Isoform and Inhibits Its Surface Expression J. Biol. Chem., June 13, 2003; 278(25): 23085 - 23093. [Abstract] [Full Text] [PDF]
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 N. Kabbani, L. Negyessy, R. Lin, P. Goldman-Rakic, and R. Levenson Interaction with Neuronal Calcium Sensor NCS-1 Mediates Desensitization of the D2 Dopamine Receptor J. Neurosci., October 1, 2002; 22(19): 8476 - 8486. [Abstract] [Full Text] [PDF]
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A. V. Binda, N. Kabbani, R. Lin, and R. Levenson D2 and D3 Dopamine Receptor Cell Surface Localization Mediated by Interaction with Protein 4.1N Mol. Pharmacol., September 1, 2002; 62(3): 507 - 513. [Abstract] [Full Text] [PDF]
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M. A. Ayoub, C. Couturier, E. Lucas-Meunier, S. Angers, P. Fossier, M. Bouvier, and R. Jockers Monitoring of Ligand-independent Dimerization and Ligand-induced Conformational Changes of Melatonin Receptors in Living Cells by Bioluminescence Resonance Energy Transfer J. Biol. Chem., June 7, 2002; 277(24): 21522 - 21528. [Abstract] [Full Text] [PDF]
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 A. Christopoulos and T. Kenakin G Protein-Coupled Receptor Allosterism and Complexing Pharmacol. Rev., June 1, 2002; 54(2): 323 - 374. [Abstract] [Full Text] [PDF]
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 R. Lin, K. Karpa, N. Kabbani, P. Goldman-Rakic, and R. Levenson Dopamine D2 and D3 receptors are linked to the actin cytoskeleton via interaction with filamin A PNAS, April 24, 2001; 98(9): 5258 - 5263. [Abstract] [Full Text] [PDF]
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M. McVey, D. Ramsay, E. Kellett, S. Rees, S. Wilson, A. J. Pope, and G. Milligan Monitoring Receptor Oligomerization Using Time-resolved Fluorescence Resonance Energy Transfer and Bioluminescence Resonance Energy Transfer. THE HUMAN delta -OPIOID RECEPTOR DISPLAYS CONSTITUTIVE OLIGOMERIZATION AT THE CELL SURFACE, WHICH IS NOT REGULATED BY RECEPTOR OCCUPANCY J. Biol. Chem., April 20, 2001; 276(17): 14092 - 14099. [Abstract] [Full Text] [PDF]
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M. Scarselli, F. Novi, E. Schallmach, R. Lin, A. Baragli, A. Colzi, N. Griffon, G. U. Corsini, P. Sokoloff, R. Levenson, et al. D2/D3 Dopamine Receptor Heterodimers Exhibit Unique Functional Properties J. Biol. Chem., August 3, 2001; 276(32): 30308 - 30314. [Abstract] [Full Text] [PDF]
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