ACCELERATED PUBLICATION
Functionally Different Agonists Induce Distinct
Conformations in the G Protein Coupling Domain of the
2
Adrenergic Receptor*
Pejman
Ghanouni
§,
Zygmunt
Gryczynski¶,
Jacqueline J.
Steenhuis
,
Tae Weon
Lee
,
David L.
Farrens
,
Joseph R.
Lakowicz¶, and
Brian K.
Kobilka
§**
From the § Department of Molecular and Cellular
Physiology and ** Division of Cardiovascular Medicine,
Howard Hughes Medical Institute, Stanford University
Medical School, Stanford, California 94305, the ¶ Department of
Biochemistry and Molecular Biology, Center for Fluorescence
Spectroscopy, School of Medicine, University of Maryland, Baltimore,
Maryland 21201, and the
Departments of Biochemistry and
Molecular Biology, Oregon Health Sciences University, Portland, Oregon
97201
Received for publication, April 4, 2001
 |
ABSTRACT |
G protein-coupled receptors represent the
largest class of drug discovery targets. Drugs that activate G
protein-coupled receptors are classified as either agonists or partial
agonists. To study the mechanism whereby these different classes of
activating ligands modulate receptor function, we directly monitored
ligand-induced conformational changes in the G protein-coupling domain
of the
2 adrenergic receptor. Fluorescence
lifetime analysis of a reporter fluorophore covalently attached to this
domain revealed that, in the absence of ligands, this domain oscillates
around a single detectable conformation. Binding to an antagonist does
not change this conformation but does reduce the flexibility of
the domain. However, when the
2 adrenergic receptor is
bound to a full agonist, the G protein coupling domain exists in two
distinct conformations. Moreover, the conformations induced by a full
agonist can be distinguished from those induced by partial agonists.
These results provide new insight into the structural consequence of
antagonist binding and the basis of agonism and partial agonism.
 |
INTRODUCTION |
G protein-coupled receptors
(GPCRs)1 are remarkably
versatile biological sensors. They are responsible for the majority of cellular responses to hormones and neurotransmitters, as well as for
the senses of sight, smell, and taste. Our current models of the
mechanism of GPCR activation by diffusible agonists have been deduced
from indirect measures of receptor conformation, such as G protein or
second messenger activation (1-4). These indirect assays of GPCR
activity provide only limited insight into the agonist-induced
structural changes that define the active state of the receptor.
To elucidate the mechanism of GPCR activation by diffusible agonists,
we developed a means for directly monitoring the active conformation of
purified, detergent-solubilized
2 adrenergic receptor
(
2AR) by site-specific labeling of an endogenous
cysteine (Cys265) with fluorescein maleimide
(FM-
2AR) (5). Based on homology with rhodopsin (6),
Cys265 is located in the third intracellular loop (IC3) at
the cytoplasmic end of the transmembrane 6 (TM6)
helix (Fig.
1A). Mutagenesis studies have
shown this region of IC3 to be important for G protein coupling (7, 8).
An environmentally sensitive fluorophore covalently bound to
Cys265 is therefore well positioned to detect
agonist-induced conformational changes relevant to G protein
activation. The effect of agonists and partial agonists on the
fluorescence intensity of FM-
2AR correlates well with
their biological properties (5). Binding of the full agonist
isoproterenol induces a conformational change that decreases the
fluorescence intensity of FM bound to Cys265 by ~15%
(Fig. 1B), whereas binding of partial agonists results in
smaller changes in intensity, and binding of an antagonist has no
effect (5).

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Fig. 1.
A, schematic diagram of the secondary
structure of 2AR illustrating the FM labeling site at
Cys265. B, effect of the full agonist ISO on
fluorescence intensity of FM- 2AR. Purified,
detergent-solubilized 2AR was labeled with FM at
Cys265 and examined by fluorescence spectroscopy. Change in
intensity of FM- 2AR in response to the addition of ISO
followed by the reversal by the neutral antagonist ALP.
|
|
Agonist-induced movement of FM bound to Cys265 was
characterized by examining the interaction between the fluorescein at
Cys265 and fluorescence quenching reagents localized to
different molecular environments of the receptor. The results of these
experiments are most consistent with either a clockwise rotation of TM6
and/or a tilting of the cytoplasmic end of TM6 toward TM5 (5). Our findings suggest that the conformational changes associated with
2AR activation are similar to those in rhodopsin (9) and
indicate a shared mechanism of GPCR activation.
These results provide insight into the nature of the structural changes
that occur upon agonist binding. However, several mechanistic
questions remain. Using conventional spectroscopy, we observe no change
in the fluorescence intensity from FM-
2AR upon
antagonist binding. This could indicate that antagonists do not alter
receptor structure or that the structural changes are not detectable by
FM bound to Cys265. Of greater interest is the structural
basis of partial agonism. Partial agonists induce a smaller change in
intensity of FM-
2AR than do full agonists. Two models
could explain this observation. If we assume that the receptor exists
in two functional conformational states, inactive or active, then a
partial agonist may simply induce a smaller fraction of receptors to
undergo the transition to the active state than does the full agonist.
Alternatively, partial agonists may induce a conformation distinct from
that induced by full agonists. Conventional fluorescence spectroscopy, which represents an average intensity over a population of fluorescent molecules, does not allow us to distinguish between these two models.
We therefore used fluorescence lifetime spectroscopy, which is capable
of distinguishing substates within a population of fluorescent
molecules, to look for ligand-specific conformational states. Our
results indicate that partial agonists and agonists induce distinct
conformations. Moreover, we observe structural effects of antagonist
binding that could not be detected by conventional spectroscopy. These
results help elucidate the structural mechanisms that underlie
ligand efficacy.
 |
EXPERIMENTAL PROCEDURES |
Fluorescence Spectroscopic Studies of the
2AR--
Construction, expression, and purification of
human
2AR were performed as described (10). Purified,
detergent-solubilized, wild-type receptor was labeled with FM
(Molecular Probes) as described previously (5). The labeling procedure
resulted in incorporation of 0.6 mol of FM per mol of receptor.
Fluorescence spectroscopy experiments were performed on a SPEX
Fluoromax spectrofluorometer with photon counting mode using an
excitation and emission bandpass of 4.2 nm. Approximately 25 pmol of
FM-labeled
2AR was diluted into 500 µl of 200 mM Tris, pH 7.5, 500 mM NaCl, 0.1%
n·dodecyl-
-D-maltoside, 100 mM mercaptoethanolamine. Excitation was at 490 nm, and
emission was measured from 500 to 599 nm with an integration time of
0.3 s/nm for emission scan experiments. For time course experiments, excitation was at 490 nm, and emission was monitored at 517 nm. For
anisotropy studies, fluorescence intensities were measured with
excitation and emission polarizers in horizontal (H) and vertical (V) combinations. The G factor was calculated from
the ratio of the intensities (I) of
IHV/IHH, and the anisotropy
(r) was calculated from Equation 1.
|
(Eq. 1)
|
For studies measuring ligand effects, no difference was observed
when using polarizers in magic angle conditions. Unless otherwise
indicated, all experiments were performed at 25 °C, and the sample
always underwent constant stirring. The volume of the added ligands
was
1% of total volume, and fluorescence intensity was
corrected for this dilution in all experiments shown. All of the
compounds tested had an absorbance of less than 0.01 at 490 and 517 nm
in the concentrations used, excluding any inner filter effect in the
fluorescence experiments.
Fluorescence Lifetime Analysis of Fluorescein-labeled
2AR--
To determine fluorescence lifetimes, ~250
pmol of FM-
2AR was diluted in 1.5 ml of 200 mM Tris, pH 7.5, 500 mM NaCl, 0.1% n·dodecyl-
-D-maltoside, 100 mM
mercaptoethanolamine and incubated for 10 min at 25 °C with or
without ligand. Fluorescence lifetimes were measured using a frequency
domain 10-GHz fluorometer equipped with a Hamamatsu 6-µm microchannel
plate detector as described previously (11). The instrument covered a
wide frequency range (4-5000 MHz), which allowed detection of
lifetimes ranging from several nanoseconds to a few picoseconds.
Samples were placed in a 10-mm path length cuvette. The excitation was
provided by the frequency-doubled output of a cavity-dumped pyridine-2
dye laser tuned at 370 nm synchronously pumped by a mode-locked argon ion laser. Sample emission was filtered through Corning 3-72 and 4-96
filters. For the reference signal,
4-(dimethylamino)-4'-cyanostilbene in methanol (463-ps
fluorescence lifetime) was observed through the same filter
combination. The governing equations for the time-resolved intensity
decay data were assumed to be a sum of discrete exponentials as in
Equation 2,
|
(Eq. 2)
|
where I(t) is the intensity decay,
i is the amplitude
(pre-exponential factor), and
i is
the fluorescence lifetime of the i-th discrete component or
a sum of Gaussian distribution functions as in Equation 3,
|
(Eq. 3)
|
and Equation 4,
|
(Eq. 4)
|
where
is the center value of the lifetime
distribution, and
is the standard deviation of the Gaussian, which
is related to the full width at half maximum by 2.354
. In the
frequency domain, the measured quantities at each frequency
are the
phase shift (
) and demodulation factor
(m
) of the emitted light versus
the reference light. Fractional intensity, amplitude, and lifetime
parameters were recovered by a non-linear least squares procedure using
the software developed at the Center for Fluorescence Spectroscopy. The
measured data were compared with calculated values
(
c
,
mc
), and the goodness of fit was
characterized by Equation 5,
|
(Eq. 5)
|
where
is the number of degrees of freedom, and

and
m are the
uncertainties in the measured phase and modulation values, respectively. The sum extends over all frequencies (
).
 |
RESULTS AND DISCUSSION |
Using Fluorescence Lifetime Spectroscopy to Study Ligand-induced
Conformational Changes in the
2AR--
The
2AR was purified and labeled at Cys265 with
fluorescein maleimide to generate FM-
2AR as described
previously (5). We examined ligand-dependent changes in
fluorescence lifetime of FM-
2AR in an effort to identify
the existence of agonist-specific conformational states. Fluorescence
lifetime analysis can detect discrete conformational states in a
population of molecules, whereas fluorescence intensity measurements
reflect the weighted average of one or more discrete states. Based on
the changes in steady-state fluorescence intensity we observed, we
predicted that ligand-induced conformational changes in the receptor
would alter the fluorescence lifetime of the fluorophore. Fluorescence
lifetime,
, refers to the average time that a fluorophore that has
absorbed a photon remains in the excited state before returning to the
ground state. The lifetime of fluorescein (nanoseconds) is much faster
than the predicted off-rate of the agonists we examined (µs-ms) and much shorter than the half-life of conformational states of
bacteriorhodopsin (µs) (12) or rhodopsin (ms) (13, 14) or of ion
channels (µs-ms) (15). Therefore, lifetime analysis of fluorescein
bound to Cys265 is well suited to capture even short-lived,
agonist-induced conformational states.
Antagonist Binding Narrows the Distribution of Fluorescence
Lifetimes--
Data from fluorescence lifetime experiments on
FM-
2AR bound to different drugs at equilibrium were
analyzed in two ways. Traditionally, fluorescence decays are fit to
single and multiple discrete exponential functions, and the best
fit is determined by
2 analysis (Table
I). This discrete component analysis
assumes that the receptor exists in one or a few rigid protein
conformations and does not accurately reflect the dynamic nature of
proteins. Proteins that are functionally in a single conformational
state actually undergo small conformational fluctuations around a
minimum energy state (16), and these small structural perturbations can
lead to small changes in the environment around an attached fluorophore. These perturbations are thought to reflect local unfolding
reactions within the three-dimensional structure of proteins (17). Such
flexibility in protein structure can be modeled using fluorescence
lifetime distributions (18), wherein the width of the distributions
reflects the conformational flexibility of the protein (Fig.
2). The mobility of fluorescein relative to the receptor is minimal, as determined by its high measured anisotropy (r = 0.30 ± 0.02; n = 3) and therefore would be expected to contribute little to the width of
the lifetime distribution. Thus, the width of the distribution can be
attributed to conformational flexibility in the receptor itself.
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Table I
Fluorescent lifetime data for FM- 2AR in the presence and
absence of drugs fit to discrete exponential functions
The observed fluorescence decay was resolved into one or more
exponential components, with each component, i, being
described by i and i, where i
represents the fractional contribution of i to the overall
decay. The best fit to single or multiple components was determined by
2 analysis. If different agonists induce a single active
state, then the fluorescence lifetime associated with that state
( R*) should be the same for different drugs, and only the
fractional contributions ( DRUG) should differ. However, if
there are agonist-specific conformational states we should observe
unique, agonist-specific lifetimes (e.g. ISO,
SAL, and DOB).
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Fig. 2.
Effect of drugs on fluorescence
lifetime distributions of
FM- 2AR. Fluorescence
lifetimes were determined by phase modulation, and lifetime
distributions of FM- 2AR were calculated in the absence
of ligand (black trace), with the neutral antagonist ALP
(red trace), or in the presence of the full agonist ISO
(green trace). The mean lifetime and the full width at half
maximum for the distributions are as follows: No Drug,
= 4.21 ± 0.01 ns; FWHM = 1.1 ± 0.1;
2 = 2.8. ALP, = 4.21 ± 0.01 ns; FWHM = 0.7 ± 0.2; 2 = 2.9. ISO: LONG = 4.36 ± 0.08 ns;
FWHMLONG = 0.5 ± 1.1; SHORT = 0.76 ± 0.33 ns; FWHMSHORT = 1.7 ± 1.2;
2 = 3.2.
|
|
Lifetime analysis of unliganded FM-
2AR reveals a single,
flexible state. This is indicated by both the single, broad Gaussian distribution of lifetimes centered around 4.2 ns (Fig. 2, black trace) and the discrete component analysis, where the fluorescence decay rate of FM-
2AR in the absence of any drug is best
fit by a single exponential function (Table I). Binding of the neutral antagonist ALP to FM-
2AR does not significantly change
the fluorescent lifetime (Table I) but does narrow the distribution of
lifetimes (Fig. 2, red trace), suggesting that ALP
stabilizes the receptor and reduces conformational fluctuations. This
interpretation is consistent with the results of experiments
demonstrating that the
2AR is more resistant to protease
digestion when bound to ALP (19).
Agonists and Partial Agonists Induce Distinct
Conformations--
Unexpectedly, binding of the full agonist ISO
promotes conformational heterogeneity. In the presence of saturating
concentrations of ISO, FM-
2AR has two distinguishable
fluorescence lifetimes (see Fig. 2, green trace, and Table
I) representing at least two distinct conformational states. The long
lifetime component is only slightly longer than the lifetime observed
in the absence of drugs; however, the distribution is narrower than
that observed in the presence of the antagonist ALP (Fig. 2,
green and red traces). In contrast, the
distribution of the short lifetime component observed in the presence
of ISO is relatively broad, suggesting that there is considerable
flexibility around Cys265 in this agonist-induced conformation.
We next examined the effect of the partial agonists salbutamol (SAL)
and dobutamine (DOB) on the fluorescence lifetime of FM-
2AR. Similar to ISO, we observed two lifetimes when
the receptor was bound to saturating concentrations of SAL and DOB (see
Table I and Fig. 3). The long lifetime
component found in the presence of these two partial agonists is
indistinguishable from that observed in the ISO-bound receptor;
however, the short lifetime component found in both the SAL- and
DOB-bound receptor is statistically different from that for the
ISO-bound receptor. We observe a strong correlation between a reduction
in fluorescence intensity of FM bound to Cys265 and drug
efficacy (5), and shortening of the average fluorescence lifetime is
associated with a reduction in fluorescence intensity. Therefore, the
short lifetime, found only in the presence of agonists, likely
represents the G protein-activating conformation of
FM-
2AR.

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Fig. 3.
Comparison of the effects of full and partial
agonists on the fluorescence lifetime distributions of
FM- 2AR. A, the
effect of the full agonist ISO and partial agonists SAL and DOB on the
lifetime distributions of FM- 2AR are compared.
B, expanded view of the short lifetime distributions shown
in A. The mean lifetime and the full width at half maximum
for the new distributions are as follows: SAL,
LONG = 4.37 ± 0.04 ns; FWHMLONG = 0.7 ± 0.3; SHORT = 1.93 ± 0.24 ns;
FWHMSHORT = 0.7 ± 0.3; 2 = 2.1. DOB, LONG = 4.38 ± 0.01 ns;
FWHMLONG = 0.4 ± 0.4; SHORT = 1.78 ± 0.01; FWHMSHORT = 0.9 ± 0.6;
2 = 2.0.
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The different short lifetimes for the full agonist (ISO) and the
partial agonists (SAL and DOB) indicate different molecular environments around the fluorophore and therefore represent different, agonist-specific active states. The narrowing and rightward shift of
the long lifetime component following binding of both agonists and
partial agonists indicate that this lifetime also reflects an
agonist-bound state but most likely represents a more abundant intermediate state that would not be expected to alter greatly the
intensity of FM bound to Cys265. It is possible that the
number of conformations that we observe in these experiments represent
only a few of the possible conformations that can be stabilized by
drugs. Moreover, whereas the overlapping short lifetime distributions
of SAL and DOB (see Fig. 3B and Table I) suggest that they
induce similar conformations, it is possible that a conformationally
sensitive probe positioned elsewhere on the receptor could distinguish
between DOB- and SAL-bound receptors states.
Models of GPCR Activation--
According to the prevailing
two-state model of GPCR activation, receptors exist in an equilibrium
between a resting (R) state and an active (R*) state that stimulates
the G protein (20-22). Agonists preferentially enrich the R* state,
whereas inverse agonists select for the R state of the receptor.
Neutral antagonists possess an equal affinity for both states and
function simply as competitors. In this simple system, functional
differences between drugs can be explained by their relative affinity
for the single active R* state (Fig.
4A). Alternatively,
differences in efficacy between drugs can be because of ligand-specific
receptor states (23-25). Based on our lifetime experiments, we propose
a model with multiple, agonist-specific receptor states, wherein
activation occurs through a sequence of conformational changes. Upon
agonist binding, the receptor undergoes a conformational change to an
intermediate state (R') that is associated with a narrowing and
rightward shift in the long lifetime distribution. The less abundant
active state, represented by the short lifetime, is different for the
full agonist ISO (R*) and the partial agonists DOB and SAL
(Rx). The relatively slow,
temperature-dependent rate of change of fluorescence
intensity following agonist binding and the rapid rate of reversal by
antagonist (see Ref. 5 and Fig. 1B) suggest that transitions
from the intermediate state to the active state are relatively rare
high energy events. It is likely that in vivo the active
conformation is further stabilized by interactions between the receptor
and its cognate G protein Gs. Thus, one might expect the
proportion of receptor in the active state to be greater when the
receptor is coupled with Gs.

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Fig. 4.
Diagram of the two-state model of GPCR
activation (20). A, R is the inactive conformation, and
R* is the active conformation capable of activating the G protein. The
equilibrium between R and R* is influenced differently by agonists
(ISO) and partial agonists (DOB). The width of the arrows
reflects the rate constant. B, diagram of a multistate model
of GPCR activation. The agonist ISO and the partial agonist DOB both
induce an intermediate state, R', as well as distinct G
protein-activating conformations R* and Rx, respectively.
The neutral antagonist ALP induces a conformation R° that is
functionally equivalent to R at activating the G protein Gs
but can be distinguished from R by susceptibility to digestion by
proteases.
|
|
Conclusions--
Our results have implications for drug discovery
and efforts to obtain high resolution crystal structures of GPCRs. The
conformational flexibility observed in the ligand-free receptor (Fig.
2) may make it particularly challenging to obtain crystals in the
absence of a bound ligand. Of greater concern, the existence of two
conformational states in the presence of saturating concentrations of
full and partial agonists (Fig. 3) will impact efforts to obtain a high resolution structure of the active, agonist-bound receptor. If such an
agonist-bound structure is obtained, it will likely represent the more
energetically stable of the two conformations and may not be the
maximally active conformation. Thus, the use of such a structure for
rational drug design efforts may have significant limitations. In
support of this contention, recent structural analyses of the
intermediate conformational states of the proton pump bacteriorhodopsin
have revealed discrepancies attributed to energetic inhibition of
conformational states within the three-dimensional crystal (12, 26).
Finally, our results indicate that GPCRs are relatively plastic. The
number of conformations that we observed in these experiments may
represent only a few of a larger spectrum of possible conformations
that could be stabilized by drugs. Thus, it may be possible to identify
even more potent agonists or agonists that can alter G protein coupling specificity.
 |
ACKNOWLEDGEMENTS |
Brian Kobilka thanks Robert Lefkowitz,
HenryBourne, Lee Limbird, and Jurgen Wess for helpful comments on the
manuscript. Pejman Ghanouni acknowledges Gayathri Swaminath and
Zhiping Yao for assistance with protein purification.
 |
FOOTNOTES |
*
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. Tel.: 650-723-7069;
Fax: 650-498-5092; E-mail: kobilka@cmgm.stanford.edu.
Published, JBC Papers in Press, April 24, 2001, DOI 10.1074/jbc.C100162200
 |
ABBREVIATIONS |
The abbreviations used are:
GPCR(s), G
protein-coupled receptors;
2AR,
2
adrenergic receptor;
FM, fluorescein maleimide;
TM, transmembrane;
ISO, (
)-isoproterenol;
ALP, (
)-alprenolol;
SAL, salbutamol;
DOB, dobutamine;
FWHM, full width at half maximum.
 |
REFERENCES |
1.
|
Tota, M. R.,
and Schimerlik, M. I.
(1990)
Mol. Pharmacol.
37,
996-1004[Abstract]
|
2.
|
Selley, D. E.,
Sim, L. J.,
Xiao, R.,
Liu, Q.,
and Childers, S. R.
(1997)
Mol. Pharmacol.
51,
87-96[Abstract/Free Full Text]
|
3.
|
Krumins, A. M.,
and Barber, R.
(1997)
Mol. Pharmacol.
52,
144-154[Abstract/Free Full Text]
|
4.
|
Perez, D. M.,
Hwa, J.,
Gaivin, R.,
Mathur, M.,
Brown, F.,
and Graham, R. M.
(1996)
Mol. Pharmacol.
49,
112-122[Abstract]
|
5.
|
Ghanouni, P.,
Steenhuis, J. J.,
Farrens, D. L.,
and Kobilka, B. K.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
5997-6002[Abstract/Free Full Text]
|
6.
|
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[Abstract/Free Full Text]
|
7.
|
O'Dowd, B. F.,
Hnatowich, M.,
Regan, J. W.,
Leader, W. M.,
Caron, M. G.,
and Lefkowitz, R. J.
(1988)
J. Biol. Chem.
263,
15985-15992[Abstract/Free Full Text]
|
8.
|
Liggett, S. B.,
Caron, M. G.,
Lefkowitz, R. J.,
and Hnatowich, M.
(1991)
J. Biol. Chem.
266,
4816-4821[Abstract/Free Full Text]
|
9.
|
Farrens, D. L.,
Altenbach, C.,
Yang, K.,
Hubbell, W. L.,
and Khorana, H. G.
(1996)
Science
274,
768-770[Abstract/Free Full Text]
|
10.
|
Gether, U.,
Lin, S.,
and Kobilka, B. K.
(1995)
J. Biol. Chem.
270,
28268-28275[Abstract/Free Full Text]
|
11.
|
Laczko, I. G. G.,
Gryczynski, Z.,
Wiczk, W.,
Malak, H.,
and Lakowicz, J. R.
(1990)
Rev. Sci. Instrum.
61,
2331-2337[CrossRef]
|
12.
|
Subramaniam, S.,
and Henderson, R.
(2000)
Nature
406,
653-657[CrossRef][Medline]
[Order article via Infotrieve]
|
13.
|
Farahbakhsh, Z. T.,
Hideg, K.,
and Hubbell, W. L.
(1993)
Science
262,
1416-1419[Medline]
[Order article via Infotrieve]
|
14.
|
Arnis, S.,
Fahmy, K.,
Hofmann, K. P.,
and Sakmar, T. P.
(1994)
J. Biol. Chem.
269,
23879-23881[Abstract/Free Full Text]
|
15.
|
Hoshi, T.,
Zagotta, W. N.,
and Aldrich, R. W.
(1994)
J. Gen. Physiol.
103,
249-278[Abstract]
|
16.
|
Frauenfelder, H.,
Sligar, S. G.,
and Wolynes, P. G.
(1991)
Science
254,
1598-1603[Medline]
[Order article via Infotrieve]
|
17.
|
Freire, E.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
11680-11682[Free Full Text]
|
18.
|
Gratton, E.,
Alcala, R.,
and Prendergast, F. G.
(1989)
in
Fluorescent Biomolecules: Methodologies and Applications
(Jameson, D. M., ed)
, pp. 17-32, Plenum Press, New York
|
19.
|
Kobilka, B. K.
(1990)
J. Biol. Chem.
265,
7610-7618[Abstract/Free Full Text]
|
20.
|
Samama, P.,
Cotecchia, S.,
Costa, T.,
and Lefkowitz, R. J.
(1993)
J. Biol. Chem.
268,
4625-4636[Abstract/Free Full Text]
|
21.
|
Lefkowitz, R. J.,
Cotecchia, S.,
Samama, P.,
and Costa, T.
(1993)
Trends Pharmacol. Sci.
14,
303-307[CrossRef][Medline]
[Order article via Infotrieve]
|
22.
|
Leff, P.
(1995)
Trends Pharmacol. Sci.
16,
89-97[CrossRef][Medline]
[Order article via Infotrieve]
|
23.
|
Kenakin, T.
(1997)
Trends Pharmacol. Sci.
18,
416-417[CrossRef][Medline]
[Order article via Infotrieve]
|
24.
|
Tucek, S.
(1997)
Trends Pharmacol. Sci.
18,
414-416[CrossRef][Medline]
[Order article via Infotrieve]
|
25.
|
Strange, P. G.
(1999)
Biochem. Pharmacol.
58,
1081-1088[CrossRef][Medline]
[Order article via Infotrieve]
|
26.
|
Sass, H. J.,
Buldt, G.,
Gessenich, R.,
Hehn, D.,
Neff, D.,
Schlesinger, R.,
Berendzen, J.,
and Ormos, P.
(2000)
Nature
406,
649-653[CrossRef][Medline]
[Order article via Infotrieve]
|
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.