From the Center for Molecular Recognition,
§ Departments of Psychiatry and Pharmacology, Columbia
University College of Physicians and Surgeons, New York, New
York 10032
Received for publication, December 6, 2002
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ABSTRACT |
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Considerable evidence suggests that
G-protein-coupled receptors form homomeric and heteromeric dimers
in vivo. Unraveling the structural mechanism for cross-talk
between receptors in a dimeric complex must start with the
identification of the presently unknown dimer interface. Here, by using
cysteine cross-linking, we identify the fourth transmembrane segment
(TM4) as a symmetrical dimer interface in the dopamine D2 receptor.
Cross-linking is unaffected by ligand binding, and ligand binding and
receptor activation are unaffected by cross-linking, suggesting that
the receptor is a constitutive dimer. The accessibility of adjacent residues in TM4, however, is affected by ligand binding, implying that
the interface has functional significance.
G-protein-coupled receptors
(GPCRs)1 comprise a large
superfamily of receptors that couple binding of a diverse group of
ligands to activation of heterotrimeric G-proteins (1). A number of class C GPCRs have been shown to form dimers in the plasma membrane, including the calcium-sensing receptor (2), the GABAB
receptor (3-5), and the metabotropic glutamate receptors (6). In the case of the GABAB receptor, heterodimerization is essential
for proper trafficking to the cell surface (7). Furthermore, the evidence supports a scenario in which the binding of GABA to the R1
subunit causes the R2 subunit to bind to and activate G-protein (8, 9).
In addition, mounting evidence supports the hypothesis that many class
A rhodopsin-like receptors, including the dopamine D2 receptor (D2R)
(10-13), in membrane and in some cases in detergent, are dimeric as
well (reviewed in Refs. 14-16).
Unraveling the structural mechanism for cross-talk between receptors in
a dimeric complex must start with the identification of the presently
unknown dimer interface. Two hypotheses have been proposed for GPCR
dimerization: domain swapping and contact dimerization (17, 18),
although recent experimental results seem inconsistent with domain
swapping being the dominant form of dimerization (19, 20).
Synthetic peptides of TM6 block dimerization and activation of the
Computational studies have proposed a number of different potential
interfaces (22-24), but these have not yet been experimentally verified. By using cysteine cross-linking, we have now explored the
possibility that the D2R exists as a dimer or higher order oligomer in
the plasma membrane. We show that the D2R is at least a homodimer, with
the extracellular end of TM4 at a symmetrical dimer interface.
Numbering of Residues and Site-directed
Mutagenesis--
Residues are numbered according to their positions in
the human dopamine D2short receptor sequence. We also index
residues relative to the most conserved residue in the TM in which it
is located (32). The most conserved residue is assigned the position index "50," e.g. Trp-1604.50, and therefore
Val-1594.49 and Val-1614.51. Mutations were
generated as described previously (30) and were confirmed by DNA
sequencing. Mutants are named as (wild-type residue)-(residue
number)position index (mutant residue), where the residues
are given in the single-letter code.
Transfection--
The cDNA encoding the dopamine
D2short receptor or the appropriate cysteine mutant was
epitope-tagged at the amino terminus with the cleavable
influenza-hemagglutinin signal sequence followed by the FLAG epitope
(DYKDDDK) and inserted into the bicistronic expression vector pcin4
(30), creating SFD2-pcin4. Cells were maintained, and stable
transfections were performed as described previously (30). For
coimmunoprecipitation studies, an NH2-terminal signal
sequence Myc epitope-tagged D2R was created and subcloned in the
bicistronic vector pCIhyg (33), creating SMD2-pcihyg. Double stable
cells were created by transfecting stable cells expressing SFD2-pcin4
with the SMD2-pcihyg construct and selecting with 250 µg/ml
hygromycin. EM4 cells, an adherent clonal line of HEK 293 cells, were
maintained as described (33) and were used for cAMP experiments (34).
Cross-linking--
HEK 293 cells or EM4 cells stably transfected
with the appropriate D2R construct were washed and then reacted for 10 min at room temperature with the stated concentrations of copper
sulfate and 1,10-phenanthroline (CuP) as described previously (35). Phenanthroline was used at a 4-fold molar excess over copper. After
removal of the CuP solution, the cells were washed twice with 4 ml of
PBS and reacted for 20 min at room temperature with 10 mM
N-ethylmaleimide (NEM) in PBS++ buffer (11 mM
Na2HPO4, 154 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 0.1 mM CaCl2) to block free sulfhydryl groups. The
cells were scraped into PBS++/PI buffer (PBS++ supplemented with 2 µg/ml Pefabloc, 2 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin A, and 10 mM NEM) and pelleted at
800 × g for 5 min at 4 °C. The pellet was suspended
and incubated in 0.5% dodecyl maltoside (DM) for 60 min at 4 °C.
The mixture was centrifuged at 20,000 × g for 30 min
at 4 °C. Twenty µl of extract was mixed with an equal volume of
4× Laemmli sample buffer without reducing agent.
Coimmunoprecipitation--
HEK 293 cells, stably transfected
with SFD2 or SMD2 or both were reacted with CuP and solubilized in DM
as above. To 0.3 ml of DM extracts was added 1.5 µl anti-FLAG M1
mouse monoclonal antibody (Sigma), and the mixture was incubated for
1 h on ice. Thirty µl of prewashed rec-protein G-Sepharose
(Zymed Laboratories Inc.) were added, and the mixture
was incubated 1 h at 4 °C, washed four times in 1 ml of lysis
buffer, and eluted at room temperature for 30 min in 50 µl of 2×
Laemmli sample buffer without reducing agent.
Immunoblotting--
Samples were applied to 1.5-mm, 10-well
7.5% acrylamide gels prepared and run according to Laemmli (36). The
bands were transferred to a polyvinylidene difluoride membrane
(Millipore), which was blocked for 1 h at room temperature in 5%
nonfat milk, 1% bovine serum albumin, 0.1% Tween 20 in TBS (50 mM Tris-HCl, 150 mM NaCl, pH 7.4). To detect
FLAG-D2R, we typically incubated the blot with an anti-FLAG rabbit
polyclonal antibody (Sigma) diluted 1:10,000 in blocking buffer for
1 h at room temperature. For Myc-D2R we incubated with an anti-Myc
rabbit polyclonal (Santa Cruz Biotechnology) diluted 1:400 in blocking
buffer for 1 h at room temperature. The membranes were washed
three times for 10 min in TBS containing 0.1% Tween 20, incubated in
horseradish peroxidase-conjugated anti-rabbit-antibody (Santa Cruz
Biotechnology), diluted 1:15,000 in blocking buffer for 1 h at
room temperature, washed three times for 10 min with TBS containing
0.1% Tween, and reacted with ECL-Plus reagent (Amersham
Biosciences). Luminescence was detected and quantitated on a
FluorChem 8000 (Alpha Innotech Corporation).
[3H]N-Methylspiperone Binding and cAMP
Assay--
Intact cells were harvested for binding and
[3H]N-methylspiperone (PerkinElmer Life
Sciences) binding was performed as described previously (30).
EM4 cells stably expressing the D2R mutants were used for assay of cAMP
accumulation as described previously (37) except that 10 µM forskolin was used to stimulate adenylyl cyclase, and
increasing concentrations of dopamine were used to activate the D2R and
inhibit the forskolin-stimulated cyclase.
Our strategy to identify residues forming the dimer interface in
the D2R was to use cysteine cross-linking. Thus, it was essential to
develop a system that would allow non-cross-linked receptor to run as a
monomer on non-reducing SDS-PAGE. In preliminary experiments using
transient transfection of D2R, we found, consistent with previous
reports (12), that a substantial amount of receptor migrated as dimer
or higher-order oligomer (data not shown). Stably expressed D2R
produced substantially less of higher-order species on SDS-PAGE.
Moreover, we found that mutation to Ser of Cys-3716.61 and
Cys-3736.63 in the third extracellular loop, between TM6
and TM7, which leaves only two extracellular cysteines (the highly
conserved disulfide-bonded Cys-1073.25 and
Cys-182E2) further decreased oligomeric SDS-resistant
species (data not shown). The resulting background construct
(C1183.36S/C3716.61S/C3736.63S) ran
almost exclusively as a heterogeneously glycosylated monomer of ~65
kDa on non-reducing SDS-PAGE (Fig.
1A). (As shown below, this
background construct, subsequently referred to as D2R, was fully
functional and bound multiple ligands normally.) Thus, if the D2R is
oligomeric, the oligomer dissociates in SDS. In addition, the D2R is
not an obligatory disulfide-linked dimer in the plasma membrane, unlike
some class C receptors (2, 6).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
2-adrenergic receptor (21), and it has been suggested that the GXXXG motif in TM6 may be involved in dimerization
of this receptor, although this motif is not highly conserved in other
family A GPCRs. Synthetic peptides of TM6 and of TM7 also blocked
dimerization of the D2 receptor (D2R) (12). Although these findings
were specific to these particular synthetic peptides, the data do not
necessarily establish TM6 and/or TM7 as the dimer interface, as the
peptide-receptor interactions might modulate the ability of the
receptor to form dimers at a different interface. In addition, if the
receptors form higher order oligomers, multiple interfaces must exist.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Cross-linking of D2R to a homodimer by copper
phenanthroline. A, treatment of FLAG-D2R with 0, 10/40,
40/160, 100/400, 400/1600, and 1000/4000 µM CuP
(lanes 1-6, respectively). B, exponential
association fit of dimer/total density plotted against CuP from
A. C, anti-Myc blot of anti-FLAG
immunoprecipitation of FLAG-D2R, Myc-D2R, and coexpressed FLAG-D2R and
Myc-D2R (see "Experimental Procedures"). The sharp ~130-kDa band
present in each of the extract lanes is a nonspecific band labeled by
the anti-Myc antibody. The molecular masses of protein standards are
given in kDa. Representative data from n = 3 experiments are shown.
Using cells stably coexpressing two D2R constructs, one FLAG-tagged and the other Myc-tagged, we attempted to coimmunoprecipitate Myc-D2R with an anti-FLAG monoclonal antibody and protein G. After solubilization with DM we detected no or only trace coimmunoprecipitation of Myc-D2R under these conditions (Fig. 1C), although the FLAG-D2R was immunoprecipitated as expected (data not shown). Thus, if the D2R is oligomeric in the membrane, the oligomer does not survive DM solubilization.
As a control before introducing engineered cysteines into FLAG-D2R for disulfide cross-linking experiments we reacted FLAG-D2R in intact cells with copper phenanthroline (CuP), an oxidizing reagent that promotes the formation of disulfide bonds directly between cysteines (25, 26). Reaction with CuP produced a new band of ~133 kDa (Fig. 1A), approximately twice that of monomer. The fraction of total density that was present in the ~133 kDa band was plotted against increasing CuP (Fig. 1, A and B), giving half-maximal cross-linking at 60 ± 10 µM CuP and maximal cross-linking of 80 ± 14% (n = 3).
The cross-linked species size was consistent with it being a homodimer of D2R, but it was possible that it might represent D2R cross-linked to another protein of similar size. The partners in the cross-linked species were definitively identified by coimmunoprecipitation of Myc-D2R stably coexpressed with FLAG-D2R. After reaction with CuP, immunoprecipitation with anti-FLAG antibody produced an ~133-kDa band that was recognized by anti-Myc antibody (Fig. 1C). In contrast when FLAG-D2R and Myc-D2R were expressed separately and then cross-linked, precipitation with anti-FLAG antibody did not produce a ~133-kDa species recognized by anti-Myc antibody (Fig. 1C), demonstrating the specificity of the antibodies. These results establish that the ~133-kDa band is a D2R homodimer that is disulfide cross-linked via one of the remaining endogenous cysteines.
Mutation of Cys-1684.58 in the fourth transmembrane segment
(TM4) to Ser (as well as to Ala or Phe, data not shown), but not
mutation of Cys-561.54, Cys-1263.44, or
Cys-3566.47, completely prevented CuP-induced cross-linking
(Fig. 2A), demonstrating that
this Cys at the extracellular end of TM4 forms the disulfide cross-link
at a symmetrical homodimer interface.
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To assess ligand binding effects on the dimerization state of the receptor, we treated cells expressing the background construct with the agonists, quinpirole or bromocriptine, or the antagonists, sulpiride or butaclamol. Neither acute nor 24-h treatment with these ligands significantly impacted cross-linking under conditions in which nearly all the receptor was cross-linked (Fig. 2B). This suggests that the receptor is a constitutive dimer (or possibly a higher order oligomer) in the plasma membrane and that ligand interaction does not lead to dissociation of the dimer.
To study the effects of cross-linking Cys-1684.58 on ligand
binding, we oxidized with 1 mM/4 mM CuP to
ensure that essentially all D2R was cross-linked (see Fig. 1). We
observed no change in the KD or Bmax for
the antagonist [3H]N-methylspiperone (Fig.
3C), or in the
KI for sulpiride or dopamine (Fig. 3, A
and B). We also assessed the effects of cross-linking
Cys-1684.58 on dopamine-mediated inhibition of adenylyl
cyclase by the D2R. In the absence of D2R, treatment with 1 mM/4 mM CuP significantly inhibited
forskolin-stimulated adenylyl cyclase through a direct effect on
adenylyl cyclase (data not shown). This resulted in an apparent
decrease in the potency and efficacy of dopamine in both the
Cys-1684.58 and C1684.58S constructs
after treatment with CuP (Fig. 3D). Nonetheless, after
treatment with CuP, dopamine was equally efficacious at inhibiting
cyclase in a Cys-1684.58 construct that was highly
cross-linked and in a C1684.58S construct that did not
cross-link. We observed no dopamine-mediated inhibition of cAMP levels
in untransfected EM4 cells. This argues that the dopamine-mediated
inhibition of cyclase occurred only via the stably expressed
D2R.
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Because cross-linking requires that only one of the two cysteines involved is modified initially by the reagent, and the derivatized cysteine then reacts by collision with the second unmodified cysteine, the rate of collision must be much faster than the rate of initial modification. This is consistent with the cysteines being very close initially. The very high fraction of receptor that can be cross-linked, the apparent specificity of the cross-linking, based on the appearance of a single homodimer band, and the lack of cross-linking of Cys-561.54, which based on the bovine rhodopsin structure has a similar lipid accessibility as Cys-1684.58, all argue for the proximity of the TM4 cysteines in the native state. Thus, it is likely that in the membrane, untreated with CuP, D2R exists as a homodimer but that this dimer does not survive detergent solubilization.
Cross-linking does not impair the ability of dopamine to inhibit
cyclase via the D2R (Fig. 3), demonstrating that the receptor can bind
dopamine and activate Gi with a disulfide between
Cys-1684.58 in each "subunit" of the dimer. Thus,
significant movement of the dimer interface at the
Cys-1684.58 position is not necessary for function.
Moreover, ligand recognition was unaltered by cross-linking, and the
extent of cross-linking was unaltered by treatment with agonists or
antagonists, suggesting that the receptor is a constitutive dimer.
These results are consistent with the recent findings of Mercier
et al. (27). Using quantitative bioluminescence resonance
energy transfer (BRET), they showed that more than 82% of
2-adrenergic receptor in the plasma membrane exists as a
constitutive dimer (or higher order oligomer) over a broad range of
expression levels. They also failed to observe a change in BRET upon
addition of ligand and suggested that the dimer does not form or
dissociate upon activation.
Bovine rhodopsin was crystallized in detergent as a non-physiological
dimer in which the cytoplasmic face of one molecule is in the same
plane as the extracellular face of the second molecule (28). It is
possible, however, that the native oligomeric structure of rhodopsin is
disrupted in detergent and is therefore not seen in the crystal
structure. Indeed, our findings that the dopamine receptor is a dimer
in the plasma membrane but cannot be immunoprecipitated from a DM
extract argues that the D2 receptor native quaternary structure is not
preserved in this detergent. Interestingly, squid rhodopsin in
detergent forms two-dimensional crystals that show a TM4 dimer
interface (29) (Fig. 4A).
These results suggest that the TM4 dimer interface may be common to
other GPCRs as well as the D2R.
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Using the substituted cysteine accessibility method in the D2R, we
found that cysteines in TM4 substituted for the highly conserved
Trp-1604.50 as well as for Phe-1644.54 and
Leu-1714.61 were accessible to charged sulfhydryl reagents
and that this reaction was protected by the antagonist sulpiride (30).
We originally inferred that these residues in TM4 faced the binding site crevice. Surprisingly, in the bovine rhodopsin structure, the
aligned residues Trp-1614.50, Leu-1654.54, and
Leu-1724.61 face outward and not into the binding site
crevice (28). Our observations regarding the anomalous conservation and
accessibility of this "back face" of TM4 suggested to us the
possibility that this surface might be at the interface between two D2R
subunits (31). Our finding that the site of cross-linking in D2R is
Cys-1684.58 at the extracellular end of TM4 directly
adjacent to these accessible residues (Fig. 4B) is
consistent with this hypothesis that TM4 forms a symmetrical dimer
interface. We are currently extending these studies to include other
residues in TM4 in an attempt to map the entire interface and to assess
whether conformational changes occur at this interface in different
functional states of the receptor. Given that cysteines substituted for
Trp-1604.50, Phe-1644.54, and
Leu-1714.61 were protected from reaction by sulpiride, we
think it likely that such conformational changes do occur and may
mediate cross-talk between receptor subunits.
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ACKNOWLEDGEMENTS |
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We thank Myles Akabas, Juan A. Ballesteros, Marta Filizola, Arthur Karlin, and Harel Weinstein for helpful discussion and Gebhard F. X. Schertler and Helen R. Saibil for providing the photograph used to create Fig. 4A.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants MH57324 and MH54137 and by the Lebovitz Trust.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: Center for Molecular Recognition, Columbia University, P & S 11-401, 630 West 168th St., Box 7, New York, NY 10032. Tel.: 212-305-7308; Fax: 212-305-5594; E-mail: jaj2@columbia.edu.
Published, JBC Papers in Press, December 19, 2002, DOI 10.1074/jbc.C200679200
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ABBREVIATIONS |
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The abbreviations used are: GPCR, G-protein-coupled receptor; TM, transmembrane segment; D2R, dopamine D2 receptor; PBS, phosphate-buffered saline; NEM, N-ethylmaleimide; DM, dodecyl maltoside; BRET, bioluminescence resonance energy transfer; CuP, copper phenanthroline.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Gether, U.
(2000)
Endocr. Rev.
21,
90-113 |
2. |
Bai, M.,
Trivedi, S.,
and Brown, E. M.
(1998)
J. Biol. Chem.
273,
23605-23610 |
3. | White, J. H., Wise, A., Main, M. J., Green, A., Fraser, N. J., Disney, G. H., Barnes, A. A., Emson, P., Foord, S. M., and Marshall, F. H. (1998) Nature 396, 679-682[CrossRef][Medline] [Order article via Infotrieve] |
4. | 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] |
5. | 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] |
6. |
Romano, C.,
Yang, W. L.,
and O'Malley, K. L.
(1996)
J. Biol. Chem.
271,
28612-28616 |
7. | Margeta-Mitrovic, M., Jan, Y. N., and Jan, L. Y. (2000) Neuron 27, 97-106[Medline] [Order article via Infotrieve] |
8. |
Havlickova, M.,
Prezeau, L.,
Duthey, B.,
Bettler, B.,
Pin, J. P.,
and Blahos, J.
(2002)
Mol. Pharmacol.
62,
343-350 |
9. |
Duthey, B.,
Caudron, S.,
Perroy, J.,
Bettler, B.,
Fagni, L.,
Pin, J. P.,
and Prezeau, L.
(2002)
J. Biol. Chem.
277,
3236-3241 |
10. |
Armstrong, D.,
and Strange, P. G.
(2001)
J. Biol. Chem.
276,
22621-22629 |
11. |
Lee, S. P.,
O'Dowd, B. F., Ng, G. Y.,
Varghese, G.,
Akil, H.,
Mansour, A.,
Nguyen, T.,
and George, S. R.
(2000)
Mol. Pharmacol.
58,
120-128 |
12. | 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] |
13. | Zawarynski, P., Tallerico, T., Seeman, P., Lee, S. P., O'Dowd, B. F., and George, S. R. (1998) FEBS Lett. 441, 383-386[CrossRef][Medline] [Order article via Infotrieve] |
14. | Rios, C. D., Jordan, B. A., Gomes, I., and Devi, L. A. (2001) Pharmacol. Ther. 92, 71-87[CrossRef][Medline] [Order article via Infotrieve] |
15. | George, S. R., O'Dowd, B. F., and Lee, S. P. (2002) Nat. Rev. Drug Discov. 1, 808-820[CrossRef][Medline] [Order article via Infotrieve] |
16. | Angers, S., Salahpour, A., and Bouvier, M. (2002) Annu. Rev. Pharmacol. Toxicol. 42, 409-435[CrossRef][Medline] [Order article via Infotrieve] |
17. | Gouldson, P. R., Higgs, C., Smith, R. E., Dean, M. K., Gkoutos, G. V., and Reynolds, C. A. (2000) Neuropsychopharmacology 23, S60-S77[CrossRef][Medline] [Order article via Infotrieve] |
18. | Gouldson, P. R., Snell, C. R., Bywater, R. P., Higgs, C., and Reynolds, C. A. (1998) Protein Eng. 11, 1181-1193[Abstract] |
19. |
Schulz, A.,
Grosse, R.,
Schultz, G.,
Gudermann, T.,
and Schoneberg, T.
(2000)
J. Biol. Chem.
275,
2381-2389 |
20. | Hamdan, F. F., Ward, S. D., Siddiqui, N. A., Bloodworth, L. M., and Wess, J. (2002) Biochemistry 41, 7647-7658[CrossRef][Medline] [Order article via Infotrieve] |
21. |
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 |
22. |
Gouldson, P. R.,
Dean, M. K.,
Snell, C. R.,
Bywater, R. P.,
Gkoutos, G.,
and Reynolds, C. A.
(2001)
Protein Eng.
14,
759-767 |
23. | Filizola, M., Olmea, O., and Weinstein, H. (2002) Protein Eng. 15, in press |
24. | Filizola, M., and Weinstein, H. (2002) Biopolymers (Peptide Science) 66, 317-325 |
25. | Careaga, C. L., and Falke, J. J. (1992) J. Mol. Biol. 226, 1219-1235[Medline] [Order article via Infotrieve] |
26. |
Wu, J.,
and Kaback, H. R.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
14498-14502 |
27. |
Mercier, J. F.,
Salahpour, A.,
Angers, S.,
Breit, A.,
and Bouvier, M.
(2002)
J. Biol. Chem.
277,
44925-44931 |
28. |
Palczewski, K.,
Kumasaka, T.,
Hori, T.,
Behnke, C. A.,
Motoshima, H.,
Fox, B. A., Le,
Trong, I.,
Teller, D. C.,
Okada, T.,
Stenkamp, R. E.,
Yamamoto, M.,
and Miyano, M.
(2000)
Science
289,
739-745 |
29. | Davies, A., Gowen, B. E., Krebs, A. M., Schertler, G. F., and Saibil, H. R. (2001) J. Mol. Biol. 314, 455-463[CrossRef][Medline] [Order article via Infotrieve] |
30. | Javitch, J. A., Shi, L., Simpson, M. M., Chen, J., Chiappa, V., Visiers, I., Weinstein, H., and Ballesteros, J. A. (2000) Biochemistry 39, 12190-12199[CrossRef][Medline] [Order article via Infotrieve] |
31. |
Ballesteros, J. A.,
Shi, L.,
and Javitch, J. A.
(2001)
Mol. Pharmacol.
60,
1-19 |
32. | Visiers, I., Ballesteros, J. A., and Weinstein, H. (2002) Methods Enzymol. 343, 329-371[Medline] [Order article via Infotrieve] |
33. |
Saunders, C.,
Ferrer, J. V.,
Shi, L.,
Chen, J.,
Merrill, G.,
Lamb, M. E.,
Leeb-Lundberg, L. M.,
Carvelli, L.,
Javitch, J. A.,
and Galli, A.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
6850-6855 |
34. | Robbins, A. K., and Horlick, R. A. (1998) BioTechniques 25, 240-244[Medline] [Order article via Infotrieve] |
35. |
Hastrup, H.,
Karlin, A.,
and Javitch, J. A.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
10055-10060 |
36. | Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve] |
37. |
Liapakis, G.,
Ballesteros, J. A.,
Papachristou, S.,
Chan, W. C.,
Chen, X.,
and Javitch, J. A.
(2000)
J. Biol. Chem.
275,
37779-37788 |