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J. Biol. Chem., Vol. 277, Issue 18, 15482-15485, May 3, 2002
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,From the Neuroscience Research Centre, Merck Sharp & Dohme Research Laboratories, Harlow, Essex CM20 2QR, United Kingdom
Received for publication, February 14, 2002, and in revised form, February 15, 2002
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Recent studies have shown that
G-protein-coupled receptors (GPCRs) can assemble as high molecular
weight homo- and hetero-oligomeric complexes. This can result in
altered receptor-ligand binding, signaling, or intracellular
trafficking. We have co-transfected HEK-293 cells with differentially
epitope-tagged GPCRs from different subfamilies and determined whether
oligomeric complexes were formed by co-immunoprecipitation and
immunoblot analysis. This gave the surprising result that the
5HT1A receptor was capable of forming hetero-oligomers with all GPCRs tested including the 5HT1B,
5HT1D, EDG1, EDG3,
GPR26, and GABAB2 receptors. The testing of
other GPCR combinations showed similar results with hetero-oligomer formation occurring for the 5HT1D with the
5HT1B and EDG1 receptor. Control studies showed
that these complexes were present in co-transfected cells before the
time of lysis and that the hetero-oligomers were comprised of GPCRs at
discrete stoichiometries. These findings suggest that GPCRs have a
natural tendency to form oligomers when co-transfected into cells.
Future studies should therefore investigate the presence and
physiological role of GPCR hetero-oligomers in cells in which they are
endogenously expressed.
Recent studies have shown that G-protein-coupled receptors
(GPCRs)1 may form dimers or
higher order oligomers (1-7). This has led to some re-evaluation of
the mechanisms thought to be involved in GPCR function.
Co-expression studies with chimeric Materials--
Restriction endonucleases and other DNA-modifying
enzymes were from New England Biolabs (Beverly, MA) or Amersham
Biosciences. Dulbecco's modified Eagle's medium and fetal calf
serum were from Invitrogen. All other biochemicals were obtained
from Sigma unless specified otherwise.
Construction of Epitope-tagged GPCRs and Transient
Transfections--
The c-Myc epitope (EQKLISEEDL) was inserted
at the amino termini of 5HT1A and 5HT1D
receptors by PCR mutagenesis using a Stratagene Robocycler with Hot-Top
assembly (Amsterdam, Holland) as described by Nelson and Long (15). The
FLAG epitope (DYKDDDDK) was inserted by a similar procedure into the
amino termini of 5HT1A, 5HT1B, 5HT1D, EDG1, and EDG2 receptors.
FLAG-tagged EDG3, GPR26, and GABAB2
receptors were a gift from Dr. Kevin Lynch (University of Virginia,
Charlottesville, VA). PCR products were cloned into the eucaryotic
expression vector pCDNA3.1+ (Invitrogen) using standard
techniques (16). Inserts were sequenced with an ABI Prism dye
terminator cycle sequencing kit and analyzed on an Applied Biosystems
373A stretch DNA sequencer. Transient transfection of HEK-293 cells
with the epitope-tagged constructs was performed by calcium
phosphate-mediated gene transfer as described previously (17).
Immunoprecipitation--
Forty-eight hours after transfection
cells were washed three times in phosphate-buffered saline, harvested
by scraping, and centrifuged for 5 min at 500 × g. The
pelleted cells were homogenized at 4 °C by drawing up and down
through a 20-gauge syringe needle in 1 ml of cell lysis buffer (Sigma).
The homogenates were centrifuged for 20 min at 14,000 × g at 4 °C, and the supernatants were combined with 12.5 µl (packed gel) of either anti-c-Myc or anti-FLAG M2 affinity agarose
(Sigma) and mixed overnight at 4 °C. The immunoadsorbents were
recovered by centrifugation for 5 min at 700 × g and
washed three times by resuspension and centrifugation (5 min at
700 × g) in cell lysis buffer and two times in 50 mM Tris (pH 7.5) containing 0.1% (w/v) SDS and 150 mM NaCl. The samples were eluted into 60 µl of SDS
loading buffer (Sigma).
Immunoblot Analysis--
Eluted samples were heated for 3 min at
100 °C and subjected to SDS-PAGE on slab gels (180 × 160 × 1.5 mm) polymerized from 10% (w/v) acrylamide and 0.1%
N,N'-methylenebisacrylamide in Tris/glycine/SDS buffer (Novex, San Diego, CA) using the discontinuous buffer system of
Laemmli (18). The electrophoresed proteins were subjected to semi-dry
electrophoretic transfer onto polyvinylidene difluoride membranes as
described previously (19). The membranes were incubated with either
mouse c-Myc antibodies (1:1000) or mouse FLAG antibodies (1:1000) and
then with peroxidase-conjugated sheep anti-mouse serum (1:1000) for
detection of immunoreactive bands by enhanced chemiluminescence
(Amersham Biosciences). Molecular size calibration was achieved with
the MultiMark standards (Novex).
Homo-oligomerization of 5HT1A, 5HT1B, and
5HT1D Receptors--
HEK-293 cells transfected with either
FLAG-tagged 5HT1A, 5HT1B, or 5HT1D
receptor constructs were immunoprecipitated with anti-FLAG-agarose, and
immunoblot analysis of the precipitates revealed the presence of 46, 44, and 42-kDa FLAG-immunoreactive bands, respectively (Fig.
1A). Higher molecular weight
immunoreactive forms were also present, which might represent
oligomeric forms of the 5HT1 receptors. An immunoreactive
band that migrated at a size predicted for a FLAG-tagged
5HT1A receptor dimer (92 kDa) showed a strong increase in
immunostaining with the reciprocal decrease of the 46-kDa monomer band
when the disulfide bond-reducing agent dithiothreitol was omitted from
the sample loading buffer (Fig. 1B). An immunoreactive band
that migrated at a size predicted for a FLAG-tagged 5HT1B
receptor dimer (88 kDa) showed increased immunostaining with the loss
of the 44-kDa monomer band when the samples were electrophoresed in the
absence of dithiothreitol (Fig. 1B). Similarly, a possible
FLAG-tagged 5HT1D receptor dimer (82 kDa) showed an
increase in immunostaining on non-reducing gels with the reciprocal
disappearance of the 41-kDa monomer band (Fig. 1B). Similar
results have been shown previously for the 5HT1B and
5HT1D receptors, which led to the suggestion that they form
homodimers (14). The mechanism of 5HT1B receptor
homodimerization appeared to differ from that of the 5HT1A
and 5HT1D receptors because a high proportion
5HT1B receptor homodimer was observed even when gels were
run under disulfide bond-reducing conditions (Fig. 1A).
Hetero-oligomerization of 5HT1A and 5HT1D
Receptors with Other GPCRs--
To directly test for
hetero-oligomerization of different GPCR combinations we used
sequential co-immunoprecipitation and immunoblot analyses of cells
co-transfected with differentially epitope-tagged receptors. This
showed that the c-Myc-tagged 5HT1A receptor co-precipitated with FLAG-tagged versions of all receptors tested including the 5HT1A, 5HT1B, 5HT1D,
EDG1, EDG3, GPR26, and
GABAB2 receptors (Fig. 2,
A and B). The same finding was observed for the
c-Myc-tagged 5HT1D receptor, which was co-precipitated with
FLAG-tagged versions of the 5HT1B and EDG1
receptors (Fig. 2, A and B). Previous studies have also shown that the 5HT1B and 5HT1D
receptors form heterodimers (14). The finding that the FLAG- and
c-Myc-tagged versions of the 5HT1A receptor were
co-precipitated provides direct evidence that this GPCR forms a
homo-oligomer. This was also found for the 5HT1D receptor
using the c-Myc- and FLAG-tagged versions of this GPCR (data not
shown). Co-precipitation of high molecular weight FLAG-immunoreactive
5HT1B, 5HT1D, EDG1, and
EDG3 species with the c-Myc-5HT1A receptor
(Fig. 2A) suggests that these GPCRs might be present as
homodimers and trimers in higher order hetero-oligomeric complexes
comprised of at least one c-Myc-5HT1A receptor and two or
more of the FLAG-tagged constructs.
Sequential Immunoprecipitation/Immunoblot Analysis of Mixed Singly
Transfected HEK-293 Cells--
To rule out the possibility that the
observed hetero-oligomerizations were caused by nonspecific aggregation
of receptors, extracts from cells separately expressing the
c-Myc-tagged 5HT1A and FLAG-tagged 5HT1B
receptors were mixed, immunoprecipitated with anti-c-Myc agarose, and
subjected to immunoblot analysis with FLAG antibodies. In this case,
the FLAG-5HT1B receptor was not detected in the
precipitated material (Fig.
3A). Similarly, immunoprecipitation of the mixed extract with anti-FLAG agarose followed by immunoblot analysis with c-Myc antibodies did not detect
the c-Myc-5HT1A receptor (Fig. 3B). This
confirmed that successful immunoprecipitation of
5HT1A/5HT1B hetero-oligomers requires
co-expression within the same cells before the time of lysis. Thus, the
oligomerization of the c-Myc-tagged 5HT1A and FLAG-tagged
5HT1B receptors in the present study is not likely to be
caused by nonspecific aggregation. The same results were found for all
of the other GPCR/GPCR hetero-oligomers tested in this study (data not
shown).
Sequential Immunoprecipitation/Immunoblot Analysis of HEK-293 Cells
Transfected with Varying Amounts of 5HT1A and
EDG1 Receptors--
To determine whether the
hetero-oligomers observed in this study are comprised of GPCRs at
discrete protein/protein ratios, HEK-293 cells were co-transfected with
a fixed amount (25 µg) of the c-Myc-tagged 5HT1A receptor
and varying amounts (25, 5, 1, and 0.2 µg) of the FLAG-tagged
EDG1 receptor. The relative amounts of the
c-Myc-5HT1A receptor that were associated with the
decreasing levels of the FLAG-EDG1 receptor were determined by sequential immunoprecipitation and immunoblot analysis.
Immunoprecipitation of the FLAG-EDG1 receptor with
anti-FLAG-agarose resulted in co-precipitation of similar decreasing
levels of c-Myc-5HT1A receptor (Fig.
4B) as shown by immunoblot
analysis with c-Myc antibodies. Similarly, immunoprecipitation of the
c-Myc-5HT1A receptor resulted in co-precipitation of
decreasing levels of the FLAG-EDG1 receptor (Fig.
4D). Sequential immunoprecipitation/immunoblot analysis of
the lysates with c-Myc/c-Myc or FLAG/FLAG confirmed that the
c-Myc-5HT1A receptor was actually expressed in the cells at
relatively constant levels and that expression of the
FLAG-EDG1 receptor decreased accordingly with the
amounts of cDNA transfected into the cells (Fig. 4, A
and C). Similar results were obtained for all of the other
receptors under study (data not shown).
We have used a co-immunoprecipitation approach to provide direct
evidence that GPCRs from different subfamilies are capable of forming
hetero-oligomers when co-expressed in HEK-293 cells. Cells were
co-transfected with GPCR constructs containing different epitope tags
followed by immunoprecipitation of one receptor and detection of
co-precipitated receptors by immunoblot analysis. The 5HT1A
receptor formed a homo-oligomer when expressed alone and formed
hetero-oligomers when co-expressed with the closely related
5HT1B and 5HT1D receptors (~36% amino acid
homology) and also with the more distantly related EDG1,
EDG3, GPR26, and GABAB2 receptors
(4-16% amino acid homology). Interestingly, hetero-oligomer formation was shown for all other GPCR combinations tested including the 5HT1D receptor with the 5HT1B and
EDG1 receptors. The heterodimerization of 5HT1B
and 5HT1D receptors has been shown previously in insect SF9
cells using a viral expression system followed by Western blot analysis
(14).
The results of this study suggest that the receptor complexes were
pre-existing in cells at the time of lysis and were not caused by
nonspecific aggregation. Oligomerization was observed only in lysates
from co-transfected cells and not in mixed lysates from singly
transfected cells. Also, the observed receptor/receptor interactions
appeared to occur at defined stoichiometries in co-transfected cells
because decreased expression of one receptor resulted in a similar
incremental decrease in the amount of the second receptor immunoprecipitated. The appearance of high molecular weight
immunoreactive bands corresponding in size to 5HT1A and
5HT1D receptor homo-oligomers appeared to require intact
disulfide bonds because these were only detected when gels were run
under non-reducing conditions. Little or no 5HT1A and
5HT1D receptor homo-oligomers could be detected when gels
were run under reducing conditions. In contrast, a significant
proportion of the 5HT1B receptor was detected as high
molecular weight oligomers when gels were run under reducing conditions
with increased levels of the oligomer and reciprocal loss of the
monomer under non-reducing conditions. This indicates that the
mechanism of 5HT1B receptor homo-oligomer formation differs from that of the 5HT1A and 5HT1D receptors.
Also, the observed immuno-isolation of the putative 5HT1B
receptor dimer by immunoprecipitation of the 5HT1A receptor
indicates that these receptors may be present as higher order
hetero-oligomers such as trimers or tetramers.
A major prerequisite for the physiological assembly of
hetero-oligomeric GPCRs is co-expression in the same cells. To
demonstrate this would require detailed immunological or specific
binding experiments that localize the receptors under study to the same cells combined with co-precipitation studies using receptor-specific antibodies or ligands. The GPCRs tested in this study show a wide range
of tissue distributions in the brain. Within the 5HT receptor subfamily
5HT1A, 5HT1B, and 5HT1D receptors
all show pre- and postsynaptic localizations and can be found together
in some of the same brain regions including the cortex and dorsal raphe
nucleus (20-22). The 5HT1B receptor shows co-expression
with the 5HT1A receptor in the hippocampus and with the
5HT1D receptor in the olfactory tubercle although the
5HT1B receptor is expressed separately in the anterior
caudate putamen, hypothalamus, and thalamus, and the 5HT1D
receptor is localized separately in the trigeminal nucleus and in parts
of the cerebellum (21). Hetero-oligomerization of the 5HT1A
with the GABAB2 and GPR26 receptors may be of interest because all of these receptors are widely distributed throughout the
cortex and may therefore share some regions of overlap (22-24). The
physiological relevance of the hetero-oligomerization of
5HT1A receptor with the EDG1 and
EDG3 receptors is not clear because no detailed studies
have been carried out on the distribution of the latter receptors
within the brain. However, the interactions observed between the
5HT1A and EDG3 receptors are not likely to occur in vivo because the relative expression levels of the
latter are relatively low in the brain with the highest levels observed in peripheral tissues such as lung and heart (25, 26).
Supporting evidence for oligomerization of endogenously expressed GPCRs
comes from Western blot studies of brain tissues revealing the presence
of high molecular weight immunoreactive forms of the dopamine D2
receptor (27) and the A1 adenosine receptor (28). More recently an
immunoelectron microscopy study showed that CCR5 and CXCR4 receptors
are clustered and closely apposed on microvilli and in trans-Golgi
vesicles of human macrophages and T cells (29). The strongest evidence
comes from studies showing that GABABR1 and
GABABR2 receptors could be co-immunoprecipitated from rat cortex (9).
This is the first study in which the phenomenon of GPCR oligomerization
has been investigated using a large number of diverse receptor
subtypes. Interestingly, all of the receptors tested were capable of
forming hetero-oligomers when co-expressed in tissue culture cells
suggesting that this might be a general characteristic of these
receptors. These findings highlight the importance for further studies
to investigate the occurrence of GPCR hetero-oligomerization in tissues
and cells where they are endogenously expressed. Such studies will help
to establish the physiological role of such complexes in GPCR function.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2C-adrenergic/M3 muscarinic receptor constructs have shown that intermolecular interactions can occur between different GPCR subtypes (1, 2). More
direct studies have shown that the GABABR1 and
GABABR2 receptors are not functional as separate units and
can only form a functional receptor complex with the correct
pharmacological properties and plasma membrane expression when they are
co-expressed (8-12). The finding that co-transfection of the 
and
opioid receptors results in the formation of heterodimers with
ligand binding and functional properties that are distinct from singly transfected receptors suggests that heterodimerization may be involved
in modulation of GPCR function (13). Also, Western blot studies have
shown that the 5HT1D and 5HT1B receptors
form homodimers when expressed alone although they preferentially form heterodimers when co-expressed (14). In the present study, we have
directly examined whether 5HT1 receptors are capable of
forming oligomeric complexes with a variety of GPCRs by specific
immunoprecipitation of each receptor followed by identification of the
co-precipitated proteins using immunoblot analysis. The main objective
was to determine the specificity of the interactions across different GPCR subfamilies to gain further insight into the mechanism of GPCR
hetero-oligomerization.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (36K):
[in a new window]
Fig. 1.
Immunoblot analysis of transfected HEK-293
cells showing homo-oligomerization of 5HT1A,
5HT1B, and 5HT1D receptors. A,
HEK-293 cells (5 × 107) expressing the FLAG-tagged
5HT1A (lane 1), 5HT1B (lane
2), or 5HT1D receptor (lane 3) were
immunoprecipitated and subjected to immunoblot analysis using FLAG
antibodies as described under "Experimental Procedures."
B, the same immunoprecipitated samples were subjected to
immunoblot analysis under non-reducing (minus dithiothreitol)
conditions. The migration of monomers is indicated by open
arrows (approximate molecular sizes are: 5HT1A, 46 kDa; 5HT1B, 44 kDa; 5HT1D, 42 kDa), the
putative dimers are indicated by solid arrows, and
immunoglobulin heavy chains are indicated by an arrowhead.
The molecular size markers are shown in kDa. The immunoblots shown are
representative of at least three independent experiments.
View larger version (82K):
[in a new window]
Fig. 2.
Sequential immunoprecipitation/immunoblot
analysis of transfected HEK-293 cells showing oligomerization of
5HT1A and 5HT1D receptors with other
GPCRs. HEK-293 cells (5 × 107) either
co-expressing the c-Myc-tagged 5HT1A receptor with
FLAG-tagged versions of the 5HT1A (lane 1),
5HT1B (lane 2), 5HT1D (lane
3), EDG1 (lane 4), EDG3
(lane 5), GPR26 (lane 6), and
GABAB2 (lane 7) receptors or co-expressing the
c-Myc-tagged 5HT1D receptor with FLAG-tagged versions of
the 5HT1B (lane 8) and EDG1
(lane 9) receptors were immunoprecipitated using either
anti-c-Myc (A) or anti-FLAG-agarose (B), and the
precipitates were subjected to immunoblot analysis using FLAG
(A) or c-Myc (B) antibodies as described under
"Experimental Procedures." The molecular size markers are shown in
kDa (approximate molecular sizes are: 5HT1A, 46 kDa;
5HT1B, 44 kDa; 5HT1D, 42 kDa; EDG1,
43 kDa; EDG3, 42 kDa; GPR26, 38 kDa;
GABAB2, 106 kDa). The immunoblots shown are representative
of four independent experiments. IP,
immunoprecipitate.
View larger version (26K):
[in a new window]
Fig. 3.
Sequential immunoprecipitation/immunoblot
analysis of mixed singly transfected HEK-293 cells. HEK-293 cells
(5 × 107) expressing either the c-Myc-tagged
5HT1A receptor (lane 1), the FLAG-tagged
5HT1B receptor (lane 2), or a mixture of both
membranes (lane 3) were immunoprecipitated (IP)
with either anti-c-Myc agarose (A and D) or
anti-FLAG agarose (B and C), and the precipitates
were subjected to immunoblot (Blot) analysis with either
c-Myc (A and B) or FLAG (C and
D) antibodies as described under "Experimental
Procedures." The immunoblots shown are representative of at least
three independent experiments.
View larger version (34K):
[in a new window]
Fig. 4.
Sequential immunoprecipitation/immunoblot
analysis of HEK-293 cells transfected with varying amounts of
5HT1A and EDG1 receptors.
HEK-293 cells (5 × 107) were co-transfected with 25 µg of the c-Myc-tagged 5HT1A receptor and either 25 (lane 1), 5 (lane 2), 1 (lane 3), or
0.2 (lane 4) µg of the FLAG-tagged EDG1
receptor followed by immunoprecipitation (IP) using either
anti-c-Myc agarose (A and D) or anti-FLAG agarose
(B and C), and the precipitates were subjected to
immunoblot (Blot) analysis using c-Myc (A and
B) or FLAG (C and D) antibodies as
described under "Experimental Procedures." The molecular sizes of
the c-Myc-5HT1A (45 kDa) and FLAG-EDG1 (42 kDa)
receptor constructs are shown. The immunoblots shown are representative
of three independent experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
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* 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.
To whom correspondence should be addressed: Dept. of Biochemistry
and Molecular Biology, Merck Sharp & Dohme, Terlings Park, Harlow,
Essex CM20 2QR, UK; Tel.: 01279-440-495; Fax: 01279-440-390; E-mail:
kamran_salim@merck.com.
Published, JBC Papers in Press, February 19, 2002, DOI 10.1074/jbc.M201539200
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ABBREVIATIONS |
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The abbreviation used is: GPCR, G-protein-coupled receptor.
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REFERENCES |
|---|
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
| 1. |
Maggio, R.,
Vogel, Z.,
and Wess, J.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
3103-3107 |
| 2. |
Maggio, R.,
Vogel, Z.,
and Wess, J.
(1993)
FEBS Lett.
319,
195-200[CrossRef][Medline]
[Order article via Infotrieve] |
| 3. |
Hebert, T. E.,
Moffett, S.,
Morello, J. P.,
Loisel, T. P.,
Bichet, D. G.,
Barret, C.,
and Bouvier, M.
(1996)
J. Biol. Chem.
271,
16384-16392 |
| 4. |
Romano, C.,
Yang, W. L.,
and O'Malley, K. L.
(1996)
J. Biol. Chem.
271,
28612-28616 |
| 5. |
Fukushima, Y.,
Asano, T.,
Saitoh, T.,
Anai, M.,
Funaki, M.,
Ogihara, T.,
Katagiri, H.,
Matsuhashi, N.,
Yazaki, Y.,
and Sugano, K.
(1997)
FEBS Lett.
409,
283-286[CrossRef][Medline]
[Order article via Infotrieve] |
| 6. |
Cvejic, S.,
and Devi, L. A.
(1997)
J. Biol. Chem.
272,
26959-26964 |
| 7. |
Nimchinsky, E. A.,
Hof, P. R.,
Janssen, W. G. M.,
Morrison, J. H.,
and Schmauss, C.
(1997)
J. Biol. Chem.
272,
29229-29237 |
| 8. |
Jones, K. A.,
Borowsky, B.,
Tamm, J. A.,
Craig, D. A.,
Durkin, M. M.,
Dai, M.,
Yao, W. J.,
Johnson, M.,
Gunwaldsen, C.,
Huang, L. Y.,
Tang, C.,
Shen, Q.,
Salon, J. A.,
Morse, K.,
Laz, T.,
Smith, K. E.,
Nagarathnam, D.,
Noble, S. A.,
Branchek, T. A.,
and Gerald, C.
(1998)
Nature
396,
674-679[CrossRef][Medline]
[Order article via Infotrieve] |
| 9. |
Kaupmann, K.,
Malitschek, B.,
Schuler, V.,
Heid, J.,
Froestl, W.,
Beck, P.,
Mosbacher, J.,
Bischoff, S.,
Kulik, A.,
Shigemoto, R.,
Karschin, A.,
and Bettler, B.
(1998)
Nature
396,
683-687[CrossRef][Medline]
[Order article via Infotrieve] |
| 10. |
White, J. H.,
Wise, A.,
Main, M. J.,
Green, A.,
Fraser, N. J.,
Disney, G. H.,
Barnes, A. A.,
Emson, P.,
Foord, S.,
and Marshall, F. H.
(1998)
Nature
396,
679-682[CrossRef][Medline]
[Order article via Infotrieve] |
| 11. |
Ng, G. Y.,
Clark, J.,
Coulombe, N.,
Ethier, N.,
Hebert, T. E.,
Sullivan, R.,
Kargman, S.,
Chateauneuf, A.,
Tsukamoto, N.,
McDonald, T.,
Whiting, P.,
Mezey, E.,
Johnson, M. P.,
Liu, Q.,
Kolakowski, L. F., Jr.,
Evans, J. F.,
Bonner, T. I.,
and O'Neill, G. P.
(1999)
J. Biol. Chem.
274,
7607-7610 |
| 12. |
Kuner, R.,
Kohr, G.,
Grunewald, S.,
Eisenhardt, G.,
Bach, A.,
and Kornau, H. C.
(1999)
Science
283,
74-77 |
| 13. |
Jordan, B. A.,
and Devi, L. A.
(1999)
Nature
399,
697-700[CrossRef][Medline]
[Order article via Infotrieve] |
| 14. |
Xie, Z.,
Lee, S. P.,
O'Dowd, B. F.,
and George, S. R.
(1999)
FEBS Lett.
456,
63-67[CrossRef][Medline]
[Order article via Infotrieve] |
| 15. |
Nelson, R. M.,
and Long, G. L.
(1989)
Anal. Biochem.
180,
147-151[CrossRef][Medline]
[Order article via Infotrieve] |
| 16. | Davis, L. G., Kuehl, W. M., and Battey, J. F. (1994) Basic Methods in Molecular Biology , Appleton & Lange, Norwalk, CT |
| 17. |
Chen, C. A.,
and Okayama, H.
(1988)
BioTechniques
6,
632-638[Medline]
[Order article via Infotrieve] |
| 18. |
Laemmli, U. K.
(1970)
Nature
227,
680-685[CrossRef][Medline]
[Order article via Infotrieve] |
| 19. |
Guest, P. C.,
Ravazzola, M.,
Davidson, H. W.,
Orci, L.,
and Hutton, J. C.
(1991)
Endocrinology
129,
734-740[Abstract] |
| 20. |
Fink, K.,
Zentner, J.,
and Gothert, M.
(1995)
Naunyn-Schmiedeberg's Arch. Pharmacol.
352,
451-454[Medline]
[Order article via Infotrieve] |
| 21. |
Bonaventure, P.,
Voorn, P.,
Luyten, W. H.,
Jurzak, M.,
Schotte, A.,
and Leysen, J. E.
(1998)
Neuroscience
82,
469-484[CrossRef][Medline]
[Order article via Infotrieve] |
| 22. |
Barnes, N. M.,
and Sharp, T.
(1999)
Neuropharmacology
38,
1083-1152[CrossRef][Medline]
[Order article via Infotrieve] |
| 23. |
Durkin, M. M.,
Gunwaldsen, C. A.,
Borowsky, B.,
Jones, K. A.,
and Branchek, T. A.
(1999)
Mol. Brain Res.
71,
185-200[Medline]
[Order article via Infotrieve] |
| 24. |
Lee, D. K.,
Lynch, K. R.,
Nguyen, T., Im, D. S.,
Cheng, R.,
Saldivia, V. R.,
Liu, Y.,
Liu, I. S.,
Heng, H. H.,
Seeman, P.,
George, S. R.,
O'Dowd, B. F.,
and Marchese, A.
(2000)
Biochim. Biophys. Acta
149,
311-323 |
| 25. |
Yamaguchi, F.,
Tokuda, M.,
Hatase, O.,
and Brenner, S.
(1996)
Biochem. Biophys. Res. Commun.
227,
608-614[CrossRef][Medline]
[Order article via Infotrieve] |
| 26. | Zhang, G., Contos, J. J., Weiner, J. A., Fukushima, N., and Chun, J. (1999) Gene (AMST.) 227, 89-99[CrossRef][Medline] [Order article via Infotrieve] |
| 27. |
Ng, G. Y.,
O'Dowd, B. F.,
Lee, S. P.,
Chung, H. T.,
Brann, M. R.,
Seeman, P.,
and George, S. R.
(1996)
Biochem. Biophys. Res. Commun.
227,
200-204[CrossRef][Medline]
[Order article via Infotrieve] |
| 28. |
Ciruela, F.,
Casado, V.,
Mallol, J.,
Canela, E. I.,
Lluis, C.,
and Franco, R.
(1995)
J. Neurosci. Res.
42,
818-828[CrossRef][Medline]
[Order article via Infotrieve] |
| 29. |
Singer, I. I.,
Scott, S.,
Kawka, D. W.,
Chin, J.,
Daugherty, B. L.,
DeMartino, J. A.,
DiSalvo, J.,
Gould, S. L.,
Lineberger, J. E.,
Malkowitz, L.,
Miller, M. D.,
Mitnaul, L.,
Siciliano, S. J.,
Staruch, M. J.,
Williams, H. R.,
Zweerink, H. J.,
and Springer, M. S.
(2001)
J. Virol.
75,
3779-3790 |
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