©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Interaction of fluorescein-TrpGlucagon with the Human Glucagon Receptor Expressed in Drosophila Schneider 2 Cells (*)

(Received for publication, March 23, 1995; and in revised form, August 4, 1995)

Michael R. Tota (§) Lei Xu Anna Sirotina (1) Catherine D. Strader Michael P. Graziano

From the Department of Molecular Pharmacology and Biochemistry, and the Department of Research Immunology, Merck Research Laboratories, Rahway, New Jersey 07065

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The human glucagon receptor was expressed at high density in Drosophila Schneider 2 (S2) cells. Following selection with G418 and induction with CuSO(4), 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.


INTRODUCTION

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 beta-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.


EXPERIMENTAL PROCEDURES

Materials

Synthetic human glucagon was purchased from Sigma or Peninsula Laboratories or synthesized on an Applied Biosystems 432A peptide synthesizer. [2-thio-Trp]Glucagon was prepared from bovine/porcine glucagon (Sigma) as described previously (12) and then reacted with 5-iodoacetamidofluorescein (Molecular Probes, Eugene, Oregon) to prepare [fluorescein-Trp]glucagon, as described in (11) . An extinction coefficient of 26,000 M cm at 496 nm (11) was used to determine the concentration of [fluorescein-Trp]glucagon. Schneider's Drosophila media(11720-018) was purchased from Life Technologies, Inc. [I]Glucagon was purchased from Dupont; Gpp(NH)p (^1)tetralithium salt was from Boehringer Manheim, and anti-fluorescein antibody was from Molecular Probes. The concentration of antibody was adjusted so that 10 µl quenched 90% of the fluorescence of 1 ml of a 5 nM fluorescein solution at pH 8.0.

Expression of the Human Glucagon Receptor in S2 Cells

Recombinant plasmid pVE2702 (4) was incubated with restriction endonuclease NheI, and the 5` ends were filled in by incubation with the Klenow fragment of DNA polymerase I and deoxynucleotide triphosphates (dNTPs). A 1.4-kilobase pair fragment encoding the human glucagon receptor was isolated by further cleavage of pVE2702 with XbaI. The 1.4-kilobase pair fragment was subcloned into Bluescript SK via the SmaI and XbaI sites (blue-hGlu). blue-hGlu was restricted with NotI, and the ends were filled in by incubation with Klenow polymerase and dNTPs. The 1.4-kilobase pair fragment containing the glucagon receptor cDNA was isolated by restriction with EcoRI and was subcloned into the expression plasmid pRmHa3 ((10) , kindly provided by Dr. L. S. B. Goldstein, University of Arizona) via the SmaI and EcoRI sites. This plasmid construct is subsequently referred to as pRm-hGlu.

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 times 10^7 cells) were cotransfected with 1 µg of pUChsneo (gift of Dr. Herman Steller) (13) and 20 µg of pRm-hGlu using the CaPO(4) 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 times 10^6 cells/ml) were induced to express receptor by the addition of 1 mM CuSO(4).

Fluorescence Flow Cytometry and Cell Sorting

Cells were analyzed for single cell fluorescence on a FACScan/Vax flow cytometeter (Benton Dickinson Immunocytometry Systems, San Jose, CA). The cells were sorted based on fluorescein intensity using a FACStar cell sorter-consort/Vax (FACS, Benton Dickinson) following standard procedures.

Determination of Glucagon Binding and Whole Cell cAMP Levels in S2 Cells

S2 membranes were prepared by hypotonic lysis, frozen in liquid N(2) and stored at -80 °C as described previously(14) . [fluorescein-Trp]Glucagon binding was performed in Buffer A (20 mM Tris-Cl, pH 7.4, 2.5 mM MgCl(2), 1 mM dithiothreitol, 25 µM phenylmethylsulfonyl fluoride, 3 µMo-phenathroline). [I]Glucagon binding assays were performed in Buffer A plus 0.05% bovine serum albumin. Membranes and [I]glucagon (100 pM) were incubated for 1 h at 22 °C. Membranes were harvested by filtration over GF/C filters (Whatman) that had been presoaked in 0.5% polyethylenimine. Due to high levels of nonspecific binding, a direct determination of the K(d) for [I]glucagon by saturation binding was not possible. At the low concentrations of [I]glucagon employed in the competition binding experiments, the IC values observed for each state should approximate the K(d) values. For the measurement of cAMP, cells were resuspended in ACC buffer (75 mM Tris-HCl, pH 7.4, 250 mM sucrose, 12.5 mM MgCl(2), 1.5 mM EDTA, 0.2 mM Na(2)S(2)O 0.1 mM Ro 20-1724 (4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone, from BioMol, Inc.)) and incubated with ligand for 45 min at 22 °C as described previously(15) . cAMP was quantified by radioimmunoassay using an Attoflow automated radioimmunoassay machine (ATTO Instruments, Inc.). Protein was determined using the method of Bradford as amended by Bio-Rad.

Immunological Methods

Four branch multiple antigenic peptides (16) corresponding to sequences in the C terminus of the human glucagon receptor were synthesized using Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry. The sequence of peptides LX1 (NH(2)-SELRRRWHRWRLGKVLWEER-COOH) and LX2 (NH(2)-SQDSSAETPLAGGLPRIAESP-COOH) were consistent with amino acid analysis. Polyclonal sera were raised in New Zealand White rabbits (Cocalico Biologicals Inc.).

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`)(2) fragment, Amersham Corp.) was used at a 1:3000 dilution.

Fluorescence Spectroscopy

Fluorescence measurements were performed on an SLM 48000 spectrofluorometer at 20 °C. Excitation was achieved with an argon laser, where the 488-nm line was selected by a sharp bandpass filter and attenuated with a 0.3-1.0 optical density neutral density filter. In order to obtain a correction factor to compensate for differences between photomultipliers, the sample was excited with both vertical and horizontal light. This was achieved by first depolarizing the laser light and then selecting either vertical or horizontal light with a polarizer. Emission light was selected with a 530-nm bandpass filter (Corion) followed by either a parallel or perpendicular polarizer. Using the T format, parallel and perpendicular emissions were collected simultaneously. Cuvettes were filled with 1.5 ml of buffer and were continuously stirred. Background and correction factors were recorded prior to the relevant portion of the data collection for time-based acquisition. All signals were recorded in the ratio mode using rhodamine as the standard. The background fluorescence (S2 cell membranes in the absence of [fluorescein-Trp]glucagon) was subtracted from each signal, and then the correction factor was applied to the vertical intensity. The data were then analyzed as follows.

Data Analysis

The fraction of bound and free ligand were determined by (17)

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(1), A(2), (1), (2) 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(max) values (the concentration of ligand bound at saturation) were determined from the fitted competition curves as described previously(19) .


RESULTS

Expression of the Human Glucagon Receptor in S2 Cells

The human glucagon receptor cDNA was subcloned into vector pRmHa3 and cotransfected with pUChsneo into S2 cells. pRmHa3 contains a Drosophila metallothionein promoter that is tightly regulated in S2 cells(10) . After 3 weeks of growth in 0.3 µg/ml G418, cells were induced to express glucagon receptor by incubation with 1 mM CuSO(4). After 3 days of induction, the cells bound [I]glucagon with a B(max) of 110 pmol/mg of membrane protein, whereas mock-transfected S2 cells displayed no specific [I]glucagon binding.

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(4), 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(4), no significant loss of receptor expression has been observed.

Immunoblotting of the Human Glucagon Receptor Expressed in S2 Cells

Antisera from rabbits injected with either peptide LX1 or LX2 recognized a protein with an apparent molecular mass of 48 kDa in transfected S2 cells induced with CuSO(4) (Fig. 1, lanes C and F). This protein was absent in nontransfected or transfected but uninduced S2 cells (Fig. 1). The M(r) = 48 kDa observed in this system differs from that measured by affinity labeling of the glucagon receptor in rat liver membranes (M(r) =63 kDa, (20) ), most likely reflecting differences in glycosylation between the receptor expressed in Drosophila versus mammalian cells.


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(4) for 3 days. Blot was exposed to Hyperfilm-ECL for 30 s.



Binding of [fluorescein-Trp]Glucagon to the Receptor

The displacement of [I]glucagon by glucagon or [fluorescein-Trp]glucagon was best fit to a two-site model with IC values of 0.34 and 37 nM for glucagon and 0.31 and 111 nM for [fluorescein-Trp]glucagon (Fig. 2). When a similar experiment was performed in the presence of the nonhydrolyzable GTP analog Gpp(NH)p, which uncouples receptors from G proteins, a single low affinity site for glucagon was observed (IC = 19 nM, data not shown), suggesting that the high affinity site observed in the absence of Gpp(NH)p represents binding of the agonist to the receptor-G protein complex. Competition of [I]glucagon by [fluorescein-Trp]glucagon demonstrated that the affinity of the fluorescein-labeled glucagon was similar to that of unmodified glucagon (Fig. 2), as previously reported by Heithier et al.(11) .


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(4) for 3 days, membranes were prepared, and binding of 0.1 nM [I]glucagon determined in competition with glucagon (circle) or [fluorescein-Trp]glucagon (bullet). 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 + spanbulletfraction1/(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.



Determination of the Anisotropy and Fluorescence Intensity of Bound [fluorescein-Trp]Glucagon

The observed anisotropy is a combination of signals from the bound and free ligand. The anisotropy of the bound ligand alone could be measured if receptor were present in excess or if free ligand were removed from the solution. Adding excess receptor was not practical under these experimental conditions. However, the signal from free ligand could rapidly be removed by adding anti-fluorescein antibody, which quenched up to 95% of the fluorescein fluorescence upon binding(21, 22) . As shown in Fig. 4A, the addition of anti-fluorescein antibody to the [fluorescein-Trp]glucagon-receptor complex resulted in a rapid quenching of the fluorescence of the free ligand, while that of the bound ligand was protected from the antibody. As the ligand dissociated from the receptor, its fluorescence was quenched by the anti-fluorescein antibody, as observed by a slow decrease in intensity after the rapid quenching phase. While the intensity was decreasing during this period, the anisotropy remained stable (Fig. 4B). This anisotropy signal originated from the bound ligand and was determined to be 0.281 ± 0.001 (n = 2). Once the anisotropy of the bound ligand was determined, could be used to estimate the fraction of ligand bound to the receptor. Applying to the data in Fig. 4(r = 0.176, r = 0.096 (data not shown), r = 0.281) indicated that 43% of the ligand is bound under these conditions, consistent with the amount of fluorescence protected from quenching by the antibody (Fig. 4A).


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.



Dissociation Kinetics of [fluorescein-Trp]Glucagon from Its Receptor

The dissociation time course of [fluorescein-Trp]glucagon was measured in the absence of Gpp(NH)p using low concentrations (2-10 nM) of the agonist. Under these conditions, two distinct relaxation phases could be observed (Fig. 5, Table 1), suggestive of agonist binding to two affinity states of the receptor. The slowly dissociating phase corresponds to the high affinity guanine nucleotide sensitive site noted in the competition binding experiments shown in Fig. 2, whereas the fast phase corresponds to the low affinity state of the receptor. These experiments, in which approximately 10 nM ligand and receptor were used, indicate that most of the glucagon receptor in the S2 cells is in the low affinity uncoupled state, as would be expected for a system in which the receptor is significantly overexpressed and G protein may be limiting. In contrast, at the submaximal concentration of [I]glucagon used in the binding assays, most of the radioligand would bind to the high affinity state of the receptor, so that the high affinity state would be significantly overrepresented in the competition binding experiments (Fig. 2).


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 times 10 s; amplitude 2 = 25.5% with a rate of 6.6 times 10 s; and an end point of 0.113. For a single phase fit, the data were fit with a rate of 3.4 times 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 times 10 s was obtained from the anisotropy transformation and 6.8 times 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 times 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(i) 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 times 10 s) was similar to that determined by a local fit of the anisotropy data (5.8 times 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.

Association Kinetics of [fluorescein-Trp]Glucagon with its Receptor

Association rates for [fluorescein-Trp]glucagon were measured in a range of 5-150 nM ligand in the presence of 100 µM Gpp(NH)p (Fig. 7). Gpp(NH)p was included to simplify the analysis by converting the receptor to a single population of binding sites. Pilot experiments showed that Gpp(NH)p had minimal effects on the association rate of 10 nM [fluorescein-Trp]glucagon with the receptor, lowering the total binding by 18% and increasing the rate of binding by about 20% (data not shown). The association rate data were adequately fit by a single relaxation time using and could be evaluated by a reversible one-step binding mechanism


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(1) 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(1) and the intercept equivalent to k(25). As shown in Fig. 7, inset, the data fit well to this model, with k(1) = 7.9 ± 1.1 times 10^4M s and k = 5.5 ± 0.01 times 10 s (n = 2). The dissociation rate in the presence of Gpp(NH)p measured directly (5.9 ± 0.5 times 10 sn = 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(d) derived from this association experiment (k/k(1) = 69 ± 10 nM, n = 2) with the thermodynamic K(d) 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(d) of 36 ± 9 nM (n = 2, data not shown). Both of these values are in reasonable agreement with the K(d) value for the low affinity site derived from thermodynamic analysis of the displacement of [I]glucagon (111 nM, Fig. 2).


DISCUSSION

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^3] 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(1) (7.9 times 10^4M 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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by 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 Molecular Pharmacology and Biochemistry, Merck Research Laboratories, Box 2000, Rahway, NJ 07065. Tel.: 908-594-3768; Fax: 908-594-3337; mike_tota@merck.com.

(^1)
The abbreviations used are: Gpp(NH)p, guanyl-5`-yl imidodiphosphate; FACS, fluorescence-activated cell sorting.


ACKNOWLEDGEMENTS

We thank Dr. Dennis Zaller for invaluable advice and comments regarding the S2 expression system, Hollis Williams for performing amino acid analysis, and Dr. Maria Bednarek for synthesizing glucagon. We also thank Dr. Margaret Cascieri, Dr. Jeff Toney, and Nancy Thornberry for helpful discussions and Dr. Robert Boltz for assistance with sorting the S2 cells.


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