(Received for publication, March 23, 1995; and in revised form, August 4, 1995)
From the
The human glucagon receptor was expressed at high density in Drosophila Schneider 2 (S2) cells. Following selection with
G418 and induction with CuSO, the cells expressed the
receptor at a level of 250 pmol/mg of membrane protein. The glucagon
receptor was functionally coupled to increases in cyclic AMP in S2
cells. Protein immunoblotting with anti-peptide antibodies revealed the
expressed receptor to have an apparent molecular mass of 48 kDa,
consistent with low levels of glycosylation in this insect cell system.
Binding of [fluorescein-Trp]glucagon to S2
cells expressing the glucagon receptor was monitored as an increase in
fluorescence anisotropy along with an increase in fluorescence
intensity. Anisotropy data suggest that the mobility of the fluorescein
is restricted when the ligand is bound to the receptor. Kinetic
analysis indicates that the binding of glucagon to its receptor
proceeds via a bimolecular interaction, with a forward rate constant
that is several orders of magnitude slower than diffusion-controlled.
These data would be consistent with a conformational change upon the
binding of agonist to the receptor. The combination of
[fluorescein-Trp
]glucagon with the S2 cell
expression system should be useful for analyzing glucagon receptor
structure and function.
Glucagon is a 29-amino acid peptide produced by proteolytic
cleavage of the proglucagon gene product in the A cells of the
pancreas. The peptide acts at the liver to increase the rate of
gluconeogenesis and glycogenolysis, in this regard serving as the major
counterregulatory hormone of insulin (for review, see (1) ).
Glucagon binds to specific receptors on the surface of hepatocytes to
stimulate increases in cyclic AMP, inositol phosphate, and
intracellular calcium. The rat and human glucagon receptors have been
cloned (2, 3, 4) and shown to contain seven
putative transmembrane domains characteristic of the G protein-coupled
family of receptors(5) . The glucagon receptor shares
significant sequence homology with the subfamily of G protein-coupled
receptors that includes receptors for glucagon-like peptide-1 and
parathyroid hormone (for review, see (6) ). This subclass of G
protein-coupled receptors bears little sequence homology to the well
characterized -adrenergic/rhodopsin subfamily, and relatively
little is known about the molecular interactions of ligands with
receptors in this class.
Glucagon itself has been the subject of a wide variety of physical studies such as x-ray crystallography(7) , NMR(8) , and circular dichroism(9) . However, these studies have been performed in various lipid or detergent solutions, which affect the conformation of the peptide. In addition, it has been observed that the secondary structure of glucagon is dependent on the concentration of peptide used in the experiment(9) . For these reasons, the structure determined by these techniques may not reflect the physiological state of glucagon as it interacts with its receptor.
As is the case for
most G protein-coupled receptors, biophysical and structural
characterization of the glucagon receptor has been hampered by an
inability to produce sufficient quantities of receptor protein. In the
present study, we have expressed the glucagon receptor at high density
using the Drosophila Schneider 2 (S2) cell system (10) . [fluorescein-Trp]Glucagon (11) has been used as a tool for obtaining kinetic and
structural information on the human glucagon receptor. The high levels
of expression of human glucagon receptor obtained in S2 cells have
allowed us to directly monitor changes in the fluorescence properties
of this ligand as it binds to the receptor. This system has proven
useful in understanding the environment of the ligand binding site of
the human glucagon receptor and in monitoring conformational changes in
both the receptor and the ligand during the binding interaction.
S2 cells were maintained and
induced to express recombinant protein essentially as described by
Bunch et al.(10) . S2 cells (provided by Dr. L. S. B.
Goldstein, University of Arizona) were maintained at 27 °C in
Schneider media supplemented with 10% heat-inactivated fetal calf
serum, two mM glutamine, and 50 µg/ml gentamycin. S2 cells
(1 10
cells) were cotransfected with 1 µg of
pUChsneo (gift of Dr. Herman Steller) (13) and 20 µg of
pRm-hGlu using the CaPO
precipitation method. pUChsneo
encodes a gene for G418 resistance whose expression is driven by the Drosophila heat shock promoter. Twenty-four hours after
transfection, G418 was added to a final concentration of 300 µg/ml.
Cells were split approximately every 5-7 days. A stable
population of G418-resistant cells was obtained in 3-4 weeks. S2
cells (1-2
10
cells/ml) were induced to
express receptor by the addition of 1 mM CuSO
.
Western blot analysis was performed on
nitrocellulose (BA85, 0.4-µm pore size (Schleicher and Schuell
Inc). Immunodetection of glucagon receptor was performed in TBS-T (20
mM Tris-Cl, pH 7.6, 137 mM NaCl, 0.1% Tween 20)
utilizing the ECL kit (Amersham Corp.). The secondary antibody
(anti-rabbit IgG, horseradish peroxidase-linked F(ab`) fragment, Amersham Corp.) was used at a 1:3000 dilution.
where r is the observed anisotropy of a
mixture of free ligand and ligand bound to the receptor. assumes that there is no quantum yield change upon ligand
binding. In order to evaluate the fraction bound by anisotropy with a
quantum yield change, the following modification was used (17)
where Q is the relative quantum yield change upon binding. Association time courses were fit to using the Marquardt's algorithm as described by Press et al.(18) .
where r(t) is the anisotropy at time t, and the initial value is assumed to be the anisotropy of
the free ligand. The fitted values A is the amplitude and
is the relaxation time for the exponential increase. Dissociation
time courses were fit as follows
where A, A
,
,
are the amplitudes and relaxation
times for two kinetic phases. The same equation was used for fitting
intensity data (I(t)).
For radioligand and functional
assays, IC (the concentration of ligand displacing 50% of
the labeled ligand from the binding site) and EC
(the
half-maximal effective concentration of ligand in a functional assay)
values were calculated using the Prism program (Graphpad Software). B
values (the concentration of ligand bound at
saturation) were determined from the fitted competition curves as
described previously(19) .
In an attempt to select
a subpopulation of cells with higher expression, cells induced to
express the glucagon receptor were incubated with
[fluorescein-Trp]glucagon and sorted by FACS.
The 5% of the cells displaying the highest levels of binding were
collected and expanded. Upon induction with 1 mM CuSO
, cells displayed a time-dependent increase in
[
I]glucagon binding with maximal levels of 250
pmol/mg of protein achieved at 3-4 days after induction (data not
shown). This represents about a 2-fold increase in receptor expression
levels following FACS. After 4 months of continuous culture in the
absence of CuSO
, no significant loss of receptor expression
has been observed.
Figure 1:
Immunoblot analysis of
membranes prepared from S2 cells. Membranes were prepared from S2
cells, and 10 µg of protein were loaded/lane on a 10%
SDS-polyacrylamide gel. Following electrophoresis, proteins were
transferred to nitrocellulose, and Western blotting was performed as
detailed under ``Experimental Procedures'' with antisera to
LX1 (lanes A-C) or LX2 (lanes D-F) at a
1:8000 dilution. Lanes A and D, nontransfected cells; lanes B and E, cells transfected with pRm-hGlu; lanes C and F, cells transfected with pRm-hGlu and
incubated with 1 mM CuSO for 3 days. Blot was
exposed to Hyperfilm-ECL for 30 s.
Figure 2:
Competition binding of
[I]glucagon with glucagon and
[fluorescein-Trp
]glucagon. G418-resistant S2
cells transfected with pRm-hGlu were incubated with 1 mM CuSO
for 3 days, membranes were prepared, and binding
of 0.1 nM [
I]glucagon determined in
competition with glucagon (
) or
[fluorescein-Trp
]glucagon (
). Data shown
are the mean ± S.D. from duplicate determinations and are
representative of three similar experiments. The curves are fit to a
two-site competition model using the Graphpad Prism program. The
equation was cpm bound = background +
span
fraction1/(1+ 10
When expressed in S2 cells, the human
glucagon receptor was functionally coupled to increases in cAMP.
Incubation of S2 cells expressing the receptor with glucagon or
[fluorescein-Trp]glucagon led to a 30-fold
increase in cAMP levels, with an EC
of 1.6 nM for
glucagon and 1.9 nM for
[fluorescein-Trp
]glucagon (data not shown).
[fluorescein-Trp
]Glucagon thus functions as an
agonist in the S2 cells, with an efficacy and potency similar to that
of unlabeled glucagon.
[fluorescein-Trp]Glucagon binding to membrane
preparations from S2 cells expressing the human glucagon receptor was
detected by monitoring changes in fluorescence anisotropy and
intensity. Fig. 3A shows the time-dependent increase in
anisotropy that was observed following the addition of
[fluorescein-Trp
]glucagon to these
membranes and the time-dependent reversal of this increase upon
displacement with one µM unlabeled glucagon. The increase
in anisotropy was not observed with S2 membranes from nontransfected
cells (Fig. 3C) and was blocked by preincubation with
unlabeled glucagon (Fig. 3A). An increase in
fluorescence intensity was also observed upon binding of
[fluorescein-Trp
]glucagon to its receptor, which
was reversed by the addition of unlabeled glucagon (Fig. 3B). Nonspecific interactions were examined by
adding [fluorescein-Trp
]glucagon to membranes
prepared from nontransfected S2 cells not expressing the glucagon
receptor (Fig. 3C). The intensity transformation
displayed an initial decrease followed by a gradual return to a
base-line level that was not affected by the subsequent addition of
unlabeled glucagon. The initial decrease was observed frequently, but
not always, and is assumed to result from a mixing artifact and/or
absorption to the membranes of
[fluorescein-Trp
]glucagon. Because anisotropy
measurements were less sensitive to nonspecific binding or mixing
artifacts than were the intensity changes (Fig. 3C),
changes in anisotropy were used as a readout to examine the kinetic
parameters of the interaction of
[fluorescein-Trp
]glucagon with the human
glucagon receptor.
Figure 3:
Binding of
[fluorescein-Trp]glucagon detected by an
increase in anisotropy and intensity. A, detection of binding
by anisotropy. Membranes were diluted to 0.02 mg/ml (containing 2.6
nM [
I]glucagon binding sites), and 2
nM [fluorescein-Trp
]glucagon was added
at time = 0. Anisotropy was recorded as described under
``Experimental Procedures.'' Glucagon was added at the
indicated time by a 1000-fold dilution from a 1 mM stock in
0.1 N acetic acid.
[fluorescein-Trp
]Glucagon was also added to
sample to which 1 µM glucagon was added first (Glucagon Blocked). B, detection of binding by
fluorescence intensity. Data from the anisotropy experiment shown in panel A were analyzed to monitor intensity, as described under
``Experimental Procedures.'' C, control experiment
in which [fluorescein-Trp
]glucagon was added to
membranes prepared from nontransfected S2 cells. Intensity (I)
and anisotropy (r) data are shown.
Figure 4:
Glucagon receptor protects
[fluorescein-Trp]glucagon from fluorescence
quenching by an anti-fluorescein antibody. Membranes were diluted to a
receptor concentration of 5.3 nM in
[
I]glucagon binding sites.
[fluorescein-Trp
]Glucagon was added to the
membranes to a final concentration of 2 nM. After binding was
complete (15 min), 10 µl of anti-fluorescein antibody (see
``Experimental Procedures'') was added (time = 0). The
data were processsed as described under ``Experimental
Procedures'' except that the residual fluorescence at the end of
the reaction was subtracted from the parallel and perpendicular
fluorescence intensities collected after the introduction of the
antibody. The antibody was also added to membranes and
[fluorescein-Trp
]glucagon where the receptor was
blocked by 1 µM glucagon (Glucagon blocked). Panel A shows the intensity recording for the addition of
anti-fluorescein antibody to the glucagon receptor and
[fluorescein-Trp
]glucagon mixture and the
mixture preblocked with glucagon. Panel B shows the anisotropy
recording for the addition of anti-fluorescein antibody to the glucagon
receptor and [fluorescein-Trp
]glucagon
mixture.
Figure 5:
Dissociation of
[fluorescein-Trp]glucagon detected by anisotropy
in the absence of Gpp(NH)p. Ten nM
[fluorescein-Trp
]glucagon was added to membranes
diluted to 0.09 mg/ml (10.4 nM
[
I]glucagon binding sites). Unlabeled glucagon
(1 µM) was added 15 min after the addition of
[fluorescein-Trp
]glucagon (time 0). The data
were fit to a single or two-phase exponential decay using .
The fitted parameters for a two-phase fit were amplitude 1 =
74.5% with a rate of 4.7
10
s
; amplitude 2 = 25.5% with a rate of 6.6
10
s
; and an end point of
0.113. For a single phase fit, the data were fit with a rate of 3.4
10
s
and an end point of
0.120. Both fits are presented in the figure; however, the two-phase
fit is obscured by the experimental data points. Residuals for the one
and two phase analyses are shown in the insets.
For more detailed kinetic analysis,
Gpp(NH)p was included in the incubation to simplify the kinetics by
converting all of the receptor to the low affinity state and to reduce
the time needed for the reaction to come to completion. The addition of
excess glucagon caused a decrease in anisotropy and intensity as the
[fluorescein-Trp]glucagon dissociated from the
receptor. The intensity decrease was about 9% (4.02 to 3.65) and was
minimally affected by changes in pH (data not shown). Using the
anisotropy of the bound ligand (0.281) determined from Fig. 4,
the fraction of ligand bound in Fig. 6was determined to be 30%
from . Knowing the fraction of ligand bound and the
intensity change upon dissociation, the quantum yield increase of
[fluorescein-Trp
]glucagon upon binding to the
receptor was determined to be 1.34-fold.
Figure 6:
Dissociation of
[fluorescein-Trp]glucagon detected by anisotropy
and intensity. Two nM
[fluorescein-Trp
]glucagon was added to membranes
diluted to 0.09 mg/ml (10.4 nM
[
I]glucagon binding sites). Gpp(NH)p (100
µM) was added after about 10 min. Unlabeled glucagon was
added after an additional 10 min (time = 0) and the dissociation
time course was measured. A, anisotropy and intensity were fit
separately to a single exponential decay using . A rate of
5.8
10
s
was obtained
from the anisotropy transformation and 6.8
10
s
from the intensity
transformation. B, global analysis using and . Each data set was weighted by its standard deviation. The
standard deviation was estimated by a linear regression analysis of the
plateau regions before the addition of glucagon. The bound anisotropy
(0.281, determined from Fig. 4) was constrained as a constant
during the fit. The calculated values were 23.3% of the ligand bound
with a relative fluorescence increase of 1.42. The free anisotropy (or
end point) was calculated to be 0.109, and the fitted dissociation rate
was 6.6
10
s
. Residuals
(fit value - experimental value) are shown in the insets.
must be
applied with some caution in this situation since the intensity of the
ligand is not constant during the binding reaction. The kinetics of the
dissociation of [fluorescein-Trp]glucagon when
monitored by intensity changes were similar, but not identical, to the
kinetics when monitored by changes in anisotropy (Fig. 6). This
difference has been postulated to arise from an optical effect on the
anisotropy resulting from changes in fluorescence intensity. The
relationship between intensity and anisotropy can be described by the
following equations (23)
where x is the fraction of the ligand bound
or free, q
is the relative increase in
intensity of that component and I(t) and r(t) are the observed intensity and anisotropy,
respectively. Using these equations, a global analysis was performed in
which the anisotropy and intensity data were analyzed together (23, 24) (Fig. 6B). The q
for the free ligand was 1.00, and the fitted
value for q
for the bound ligand was 1.42, which
agreed fairly well with the value of 1.34 determined using . The global fit indicated that 23.3% of the ligand was
bound under these conditions, compared with 30% bound ligand calculated
using . The fitted value for the dissociation rate
determined by global analysis (6.6
10
s
) was similar to that determined by a local fit of
the anisotropy data (5.8
10
s
). Because the magnitude of the effect of the
intensity change on the anisotropy measurement was small and the
intensity data were less precise than the anisotropy data, subsequent
analysis used only the local fit of the anisotropy data for
determination of rate constants.
Figure 7:
Association time courses of several
concentrations of [fluorescein-Trp]glucagon with
2.6 nM receptor. Inset, plot of observed rate
constant versus [fluorescein-Trp
]glucagon concentration
monitored by anisotropy. Membranes were diluted to a concentration of
2.6 or 5.2 nM receptor (determined by
[
I]glucagon binding) in the presence of 100
µM Gpp(NH)p. The higher receptor concentrations were used
only at high (>80 nM) ligand concentration in order to
yield a larger amplitude. All relaxation times were fit to a single
phase (), giving approximately the same initial value for
the ansitropy of free ligand. The calculated parameters are summarized
in Table 1.
where k and k
are the forward and reverse rate constants, respectively. The
inverse relaxation time of ligand association
(
) would be defined as
A plot of (also defined as the observed
rate, k
) versus L should yield a
straight line with the slope equivalent to k
and
the intercept equivalent to k
(25). As shown in Fig. 7, inset, the data fit well to this model, with k
= 7.9 ± 1.1
10
M
s
and k
= 5.5 ± 0.01
10
s
(n = 2). The
dissociation rate in the presence of Gpp(NH)p measured directly (5.9
± 0.5
10
s
n = 4; Fig. 6; Table 1) was similar to the k
value determined in this experiment, thus
supporting the above model. The model was further supported by the
agreement of the K
derived from this association
experiment (k
/k
= 69 ± 10 nM, n = 2) with
the thermodynamic K
derived from the amplitudes of
the association kinetics fit to . The fitted amplitudes and
the quantum yield change upon binding were used in to
calculate the fraction of bound and free
[fluorescein-Trp
]glucagon. The data were then
analyzed by a Scatchard plot to determine the thermodynamic K
of 36 ± 9 nM (n = 2, data not shown). Both of these values are in
reasonable agreement with the K
value for the low
affinity site derived from thermodynamic analysis of the displacement
of [
I]glucagon (111 nM, Fig. 2).
Biophysical and structural characterization of the glucagon
receptor, like that of other G protein-coupled receptors, has been
hampered by the lack of availability of sufficient quantities of active
receptor protein. Using a Drosophila S2 system, we have
isolated a polyclonal cell population that expresses the human glucagon
receptor to a level of 250 pmol/mg as measured by
[I]glucagon binding. Using this system, the
interaction of the agonist
[fluorescein-Trp
]glucagon with its receptor
could be monitored via an increase in fluorescence anisotropy and
intensity during the binding reaction. The anisotropy was more stable
and less sensitive to nonspecific binding than the quantum yield
increase and was therefore used as the primary means to monitor the
binding of [fluorescein-Trp
]glucagon to the
receptor.
The anisotropy of the ligand bound to the receptor was
examined by using anti-fluorescein antibody to quench the free ligand
in solution. The bound anisotropy was determined to be 0.281 ±
0.001, indicating that the fluorescein moiety is relatively immobile
when anchored in the binding pocket of the glucagon receptor. For
comparison, the anisotropy of fluorescein in
[fluorescein-Lys] substance P bound to the NK1
neurokinin receptor was only 0.17 (22) and that for fluorescein
labeled epidermal growth factor bound to the epidermal growth factor
receptor was determined to be 0.18(26) .
It is apparent from
the equilibrium binding titrations (Fig. 2) and the dissociation
rate studies (Fig. 5; Table 1) that
[fluorescein-Trp]glucagon binds with two classes
of receptor binding sites and that the conversion from high to low
affinity is stimulated by Gpp(NH)p. These results indicate that the
expressed glucagon receptor is coupled to G protein(s) in the S2 cells,
consistent with the ability of glucagon to stimulate cAMP accumulation
in these cells. In order to simplify the analysis of association rates,
the receptor was converted to a single class of binding sites by
preincubation with Gpp(NH)p. Gpp(NH)p had only a minimal effect on the
observed association rate for
[fluorescein-Trp
]glucagon. Monophasic
association kinetics were observed in both the presence and absence of
the guanine nucleotide, with only a small (<20%) increase in the
association rate in the presence of Gpp(NH)p. Monophasic association
kinetics were also observed in radioligand binding studies by Horwitz et al.(27) where association of
[
I-Tyr
]glucagon was described by a
single relaxation time.
A kinetic analysis of
[fluorescein-Trp]glucagon binding was performed
in the presence of 100 µM Gpp(NH)p. The data were
consistent with the model described in and ,
implying a simple bimolecular reaction between the ligand and the
receptor. However, the association rate constant k
(7.9
10
M
s
) was much slower than that expected for a
diffusion-controlled reaction, suggesting a more complex mechanism
involving a slow conformational change in either the receptor or the
ligand. Further biophysical studies will be required to address this
possibility.
The increase in fluorescence intensity observed upon
binding [fluorescein-Trp]glucagon to the
receptor is atypical, as the association of fluorescein with a protein
is often accompanied by a decrease in intensity. These data suggest
that the fluorescence of
[fluorescein-Trp
]glucagon is quenched in
solution by some internal quenching mechanism and that quenching is
relieved upon receptor binding, perhaps reflecting the conformational
change implied by the slow association kinetics. Thus, the receptor may
actively ``hold'' the fluorescein away from the rest of the
ligand, which would also be consistent with the high anisotropy
determined for the bound ligand.
Previous structural studies of
glucagon in solution have been performed at high (nonphysiological)
concentrations of glucagon, or have used detergents or lipids to model
receptor binding. The fluorescence analysis in the present study was
performed at much lower ligand concentrations and gives direct
information about the conformation of glucagon bound to its receptor.
The combination of the S2 expression system with
[fluorescein-Trp]glucagon should allow further
biophysical analysis of this receptor ligand interaction.