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INTRODUCTION |
The biological effects of glucagon are initiated by high affinity
binding to its membrane-bound receptor, a member of a distinct class
within the superfamily of G protein-coupled receptors
(GPCRs)1 (1-3). Upon
glucagon binding, the glucagon receptor (GR) interacts with the
heterotrimeric G protein, Gs, to catalyze GDP/GTP exchange. Two predominant splice variants of G
s, a short and a
long form (Gs-S and Gs-L) result from
differential splicing of a single precursor mRNA (4, 5).
Gs-S and Gs-L are present in most cells;
however, their relative proportions vary (6). The functional significance of the multiple forms of Gs is unclear, but
the conservation of two predominant isoforms suggests that the
efficiency of interactions between receptors and Gs
might be modulated by differences in structure between the isoforms as
well as the stoichiometry of these proteins in cells (7-10). X-ray
crystallographic studies have revealed that G subunits consist of a
GTPase domain that resembles the oncogene protein
p21ras and a helical domain (11-13). In the
structures, the guanine-nucleotide binding site lies in a cleft between
these two distinct domains. A 15-amino acid insert in
Gs-L following residue 71 is located in the first of two
linkers that connect the Ras-like domain and the helical domain. Linker
1 lies near two of three "switch" regions in the G structure
that undergo conformational rearrangement to allow nucleotide exchange.
Thus, the 15-residue insert in Gs-L may influence the
kinetics of receptor-mediated nucleotide exchange.
Glucagon is known to exhibit both high and low binding affinities for
GRs, which is thought to be due to the existence of a heterogeneous
population of receptors in cells, one having a high affinity and the
other a low affinity for ligand (14-16). An expanded ternary complex
model of receptor activation postulates that GPCRs exist in an
equilibrium between two interconverting states that are stabilized by
association with G proteins, even prior to ligand binding. The
differential expression of the two forms of Gs in
various tissues suggests the possibility for functional consequences
for glucagon-dependent activation of GRs that may account
for the observed differences in receptor affinities.
To test this hypothesis, we prepared fusion constructs of GR with
Gs-S and Gs-L and assessed the fusion
proteins for signal transduction properties in transiently transfected
COS-1 cells. The abilities of glucagon and the glucagon antagonist
desHis1[Nle9,Ala11,Ala16]glucagon
amide to bind to the expressed GR-Gs-S and
GR-Gs-L were measured. In addition, cAMP measurements
were carried out to evaluate the ability of GR-Gs-S and
GR-Gs-L to couple to adenylyl cyclase. Both
GR-Gs-S and GR-Gs-L caused
glucagon-dependent adenylyl cyclase activation and were
constitutively active. The antagonist bound to GR-Gs-S
and GR-Gs-L with equal affinities, but behaved as a weak
partial agonist when bound to GR-Gs-L but not with
GR-Gs-S. Finally, a non-signaling GR mutant (G23D1R) in
which the second (i2) and third (i3) intracellular loops were each
replaced with the first (i1) intracellular loop sequence from the D4
dopamine receptor, was fused to Gs-L and studied. Interestingly, the G23D1R-Gs-L was not constitutively
active, but did display high affinity glucagon binding. Our results
using GR-Gs fusion proteins demonstrate that
Gs-S and Gs-L have different effects on
GR-mediated signaling, and suggest that receptors pre-coupled to
Gs-L account for GTP-sensitive high affinity glucagon binding.
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EXPERIMENTAL PROCEDURES |
Materials--
The cDNA for the rat Gs long
splice variant (Gs-L) was kindly provided by Dr. Randall
R. Reed (17). The construction of the rat GR synthetic gene
(GenBankTM/EMBL Data Bank accession no. U14012) was
reported previously (18). Affinity-purified anti-Gs
antibody RM, raised against the C-terminal decapeptide RMHLRQYELL of
Gs was kindly provided by Dr. Allen M. Spiegel (19). The
preparation and characterization of the anti-GR antibody DK-12 was
reported previously (20, 21). The synthesis by solid phase methods and
characterization of the glucagon antagonist
desHis1[Nle9,Ala11,Ala16]glucagon
amide have been described (22, 23). 125I-Glucagon (507 Ci/mmol) was from NEN Life Science Products. N-Glycosidase F, endoglycosidase H, and GTP
S were from Roche Molecular Biochemicals.
Construction of the GR-Gs Fusion DNAs--
The
GR-Gs fusion DNAs were generated using a combination of
restriction fragment replacement and PCR-based techniques in a modified
pGEM-2 (Invitrogen) cloning vector. All nucleotide sequences were
verified by fluorescence-based sequencing (Perkin Elmer/Applied
Biosystems model 377A DNA sequencer). GR-Gs-L was assembled in a four-part ligation consisting of: (i) the large AvrII-NotI restriction fragment from the GR
synthetic gene in pGEM-2, which deletes the last eight codons (position
478-485) and the termination codon from the C terminus of GR; (ii) a
BseRI-NsiI restriction fragment encoding residues
20-385 of Gs-L; (iii) a 78-base pair synthetic duplex
with cohesive ends for AvrII and BseRI encoding
the C-terminal residues 478-485 of GR and the N-terminal residues
2-19 of Gs-L; and (iv) a 30-base pair synthetic duplex with cohesive ends for NsiI and NotI encoding the
C-terminal 386-393 residues of Gs-L, two successive
stop codons, and a NotI site at the 3'-end of the open
reading frame in the fusion. GR-Gs-S DNA was generated
by the overlap extension PCR protocol using Pfu polymerase
(24) and the GR-Gs-L construct as template. A set of
primers was synthesized to remove codons for amino acids 72-86 and to
replace Glu71 by Asp. In the first round of PCR, two
overlapping DNA sequences were amplified independently. The sense
primer at the deletion site and an antisense primer from the
NsiI site of Gs-L were used in one PCR
reaction, and the antisense deletion primer and an extension primer
from the SacII site of GR were used in another reaction. In
a second round of PCR, the two overlapping sequences were spliced
together to form a single product using the terminal extension primers
from the complementary strands. The resulting PCR-amplified fragment
was digested with AccI and BglII and ligated into
GR-Gs-L in pGEM-2. After DNA sequencing, a
SpeI-NotI fragment with the correct
Gs-S sequence was introduced in place of the analogous
restriction fragment in GR-Gs-L in the eukaryotic
expression vector pMT (25). In GR mutant G23D1R, the i2 loop
(Ser252-Glu261) and the i3 loop
(Leu334-Ala349) were replaced by an 11-amino
acid peptide derived from the dopamine D4 receptor sequence (26). To
prepare G23D1R-Gs-L, a KpnI-SacII restriction fragment from GR-Gs-L in pGEM-2 was replaced
by the analogous restriction fragment from G23D1R in pcDNA3 (26). A KpnI-NotI restriction fragment was then
transferred to GR-Gs-L in pMT. To transfer
Gs-L into pMT, a BseRI-NotI
restriction fragment from GR-Gs-L in pGEM-2,
corresponding to residues 20-393 of Gs-L, and a 67-base
pair EcoRI-BseRI synthetic duplex containing the codons for N-terminal residues 1-19 of Gs-L were joined
with an EcoRI-NotI restriction fragment from pMT
in a three-part ligation reaction.
Characterization of GR-Gs
Fusions--
GR-Gs fusion genes were expressed in COS-1
cells by transient transfection using LipofectAMINE (Life Technologies,
Inc.). In addition, GR was independently co-expressed with
Gs-L by transfecting COS-1 cells with equimolar ratios
of vectors. Preparation of membranes from transfected COS-1 cells was
carried out as described previously (18). Membrane protein
concentration was determined using the Bio-Rad DC protein assay kit.
N-Glycosidase F digestion and immunoblot analyses of
expressed GR-Gs protein fusions were carried out essentially as described (18, 20). Competition binding experiments with
125I-glucagon and adenylyl cyclase activity assays to
determine intracellular cAMP levels as a function of glucagon
concentration were performed as previously reported (18). Competitive
binding studies with 125I-glucagon were also performed in
membranes expressing GR and Gs fusion proteins in the
absence or presence of 100 µM GTPS. Binding and
adenylyl cyclase activities were also measured in COS-1 cells that had
been co-transfected with GR and Gs-L. Ligand binding and
adenylyl cyclase studies were performed at least three times to verify
reproducibility. The data were fit to a four-parameter logistic
function and the IC50 and EC50 values
calculated from the inflection point of the best-fit curve with Sigma
Plot version 4 (Jandel Scientific Software).
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RESULTS |
Characterization of Expressed GR-Gs
Fusions--
The rat GR was fused with the short and long forms of
Gs (Gs-S and Gs-L) to
construct GR-Gs-S and GR-Gs-L. The
3'-terminal codon of the open reading frame of the synthetic GR gene
was joined directly to codon 2 of the cDNAs encoding the two
Gs variants. The nonsense codon of the GR gene and the
initiator-Met codon of the Gs cDNAs were removed.
Gs-L differs from Gs-S by 16 amino acids.
Gs-L contains a Glu in place of Asp at position 71 and
an insert of 15 amino acids starting with position 72 (17). The
15-amino acid insert in Gs-L is located within the first of two linkers connecting the Ras and helical domains in the structure of Gs (Fig. 1).
Gs-L was also fused to GR mutant G23D1R, in which the i2
and i3 loops were both replaced with the i1 loop of the D4 dopamine
receptor, to form G23D1R-Gs-L. G23D1R did not couple to
cellular Gs as described previously (26). In addition,
experiments were carried out in which GR and Gs-L were co-expressed from separate vectors in COS-1 cells.
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Fig. 1.
Schematic representation of the
GR-Gs fusion proteins. The
extracellular N-terminal tail of the GR-Gs fusion
protein is toward the top of the figure. Seven putative
transmembrane helices are depicted within the plasma membrane
(PM). The intracellular C-terminal tail of the GR is
covalently joined to the N terminus of Gs.
Gs consists of two structural domains - a helical domain
and a Ras domain. The guanine-nucleotide binding site lies in a pocket
between these domains, which are connected by two linker sequences. The
predominant splice variants of Gs differ in the amino
acid sequence of linker 1. Linker 1 of Gs-L contains a
15-amino acid insert at position 72 and a glutamic acid instead of an
aspartic acid at position 71. The C-terminal tail of
GR-Gs is oriented toward the cytoplasmic domain of GR.
Agonist-dependent guanine-nucleotide exchange is thought to
require the interaction of the C-terminal tail of Gs
with intracellular loops i2 and i3 of activated GR.
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Immunoblot analysis of the membrane preparations from cells expressing
GR-Gs-L was carried out with DK-12 anti-GR antibody (Fig. 2A) and RM
anti-Gs antibody (Fig. 2B). The fusion
protein GR-Gs-L immunoreacted with DK-12 to yield a band
with an apparent molecular mass of 102 kDa after
N-glycosidase treatment (Fig. 2A) (20, 21). This
result was expected since the apparent molecular mass of
Gs-L is 52 kDa (4), and the GR migrates as a broad band
with an apparent molecular mass of 55-75 kDa that collapsed to a band
at 50 kDa after N-glycosidase F treatment. GR-Gs-S migrated with an apparent molecular mass of 95 kDa, which is consistent with the apparent molecular mass of
Gs-S (45 kDa) plus GR (Fig. 2A) (4).
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Fig. 2.
Immunoblot analysis of the
GR-Gs fusions expressed in
transiently transfected COS-1 cells. Membranes from COS-1 cells
that had been transfected with GR, GR-Gs-S, and
GR-Gs-L were treated with N-glycosidase F (N-Gase
F) to remove N-linked carbohydrates. Samples (10 µg
of protein/lane) were separated by SDS-polyacrylamide gel
electrophoresis, transferred to Immobilon-P membranes, and probed with
anti-GR DK-12 antibody (A) or anti-Gs
antibody RM (B) as described under "Experimental
Procedures." Immunoreactive bands were visualized by
chemiluminescence. Lanes labeled ( ) and (+) correspond to
samples untreated and treated with N-glycosidase F,
respectively. A, GR was visualized as a broad band at an
apparent molecular mass of 55-75 kDa (lane 1, ). The
deglycosylated GR migrated with an apparent molecular mass of about 48 kDa (lane 2, +). The glycosylated (lanes 3 ()
and 5 ()) and deglycosylated (lanes 4 (+) and 6 (+)) forms of
GR-Gs-L and GR-Gs-S migrated with the
expected molecular masses. Higher molecular weight bands appeared to be
dimers of the GR or fusion proteins. Dimerization of GR was described
previously (18). B, the identical sample as in
panel A was probed with anti-Gs
antibody RM. In COS-1 membranes expressing GR (lanes 1 and
2), the endogenous Gs splice variants,
Gs-S and Gs-L were visualized at apparent
molecular masses of 45 and 52 kDa, respectively. Gs-S
was more abundant than Gs-L. GR-Gs-L and
GR-Gs-S (lanes 3 and 5) migrated
as broad bands that collapsed to single bands with apparent molecular
masses of 100 and 93 kDa, respectively, after digestion with
N-glycosidase F (lanes 4 and 6).
Probable fusion protein dimers were visible with molecular masses above
115 kDa. The endogenous Gs splice variants were also
visible in lanes 3-6. Other RM immunoreactive bands were
presumably derived from proteolysis of the fusion proteins.
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The RM antibody, which is directed against a C-terminal peptide of
Gs, did not react with GR. However, as expected it
detected in COS-1 cells the endogenous short and long forms of
Gs, which migrate with apparent molecular masses of 45 and 52 kDa, respectively (Fig. 2B, lanes
1 and 2). The immunoblots consistently showed that the COS-1 cell membranes contained a greater proportion of the
short form relative to the long form of Gs. Bands at
apparent molecular masses of 102 and 95 kDa corresponding to
deglycosylated GR-Gs-L and GR-Gs-S (Fig.
2B, lanes 4 and 6),
respectively, were also detected by RM antibody. The membrane
preparations also contained minor bands that were immunoreactive with
RM antibody at 35, 40, and 60 kDa that could be degradation products
derived from the fusion proteins. The fusion proteins were not
recognized by anti-GR antibody ST-18, which was generated against a
C-terminal peptide of GR. This suggests that the ST-18 epitope is
eclipsed in the fusion or that the free C-terminal carboxylic acid
group is required for immunoreactivity (data not shown) (18). Both GR
and GR-Gs-L were insensitive to endoglycosidase H
digestion, indicating that they were properly processed and transported
to the cell surface (20) (Fig.
3A).
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Fig. 3.
Glycosidase analysis of
GR-Gs fusions expressed in
transiently transfected COS-1 cells. Membranes from COS-1 cells
that had been transfected with GR, GR-Gs-S, and
GR-Gs-L were treated with N-glycosidase F
(N-Gase F) to remove N-linked carbohydrates, or
endoglycosidase H (Endo H) to remove immature, high mannose
N-linked carbohydrates. Samples (10 µg of protein/lane)
were separated by SDS-polyacrylamide gel electrophoresis, transferred
to Immobilon-P membranes, and probed with anti-GR DK-12 antibody
(A) or anti-Gs antibody RM (B) as
described under "Experimental Procedures." Immunoreactive bands
were visualized by chemiluminescence. Lanes labeled () and
(+) correspond to samples untreated and treated with the labeled
glycosidase, respectively. A, broad bands arising from the
glycosylated forms of GR and GR-Gs-L were visible in
lanes 1 and 4. GR and GR-Gs-L were
sensitive to N-Gase F digestion (lanes 3 and
6). However, most of expressed GR and GR-Gs-L
were insensitive to endoglycosidase H digestion (lanes 2 and
5), suggesting that they were properly transported to the
cell surface. B, COS-1 cell membranes transfected with
non-fused GR, GR-Gs-L, GR-Gs-S,
G23D1R-Gs-L, and vector alone were treated with
N-Gase F and probed with RM antibody. Only endogenous
Gs-S and Gs-L were immunoreactive in
membranes from COS-1 cells transfected with GR (lane 1) or
with vector (lane 5). Deglycosylated forms of
GR-Gs-L, GR-Gs-S,
G23D1R-Gs-L were visible in lanes 2-4
migrating with the expected relative molecular masses.
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Competitive Ligand Binding Experiments--
Increasing
concentrations of glucagon were used to compete with
125I-glucagon for binding to membranes from COS-1 cells
containing the expressed fusion proteins. GR bound glucagon with the
expected average IC50 value of 46 nM (18, 20,
26) (Table I). GR-Gs-S displayed an affinity for glucagon that was essentially the same as
non-fused GR (Fig. 4). In contrast,
GR-Gs-L bound glucagon with an IC50 value of
3.2 nM (Fig. 4, Table I). GR and Gs-L were
co-expressed in COS-1 cells to determine if simply increasing the
proportion of Gs-L to GR increased glucagon-binding
affinity. The overexpression of Gs-L was confirmed by
immunoblot analysis using RM antibody to detect an increase in the
intensity of the 52-kDa band, which represents total combined
endogenous and recombinant Gs-L expression (data not
shown). The expression of GR in combination with overexpression of
Gs-L resulted in an increase in glucagon binding
affinity. However, the IC50 value obtained for the fusion protein GR-Gs-L (IC50 value = 3.2 nM) was still about 5-fold lower than that obtained for GR
in the presence of excess Gs-L (IC50
value = 16 nM) (Table I).
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Fig. 4.
Competitive displacement assay of
125I-glucagon binding to COS-1 cell membranes expressing GR
and GR-Gs fusion proteins.
Membranes from COS-1 cells expressing GR, GR-Gs-L, and
GR-Gs-S were incubated with 125I-glucagon
and the indicated concentrations of unlabeled glucagon as described
under "Experimental Procedures." Data are presented as percentage
of total binding of radiolabeled hormone versus the log of
glucagon concentration. Each symbol represents the mean of duplicate
determinations. The concentration of unlabeled glucagon required to
displace 50% of receptor-bound 125I-glucagon
(IC50) was calculated from the curve (Table I) and
represents the average of at least three independent
determinations.
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Mutation of the loops i2 and i3 of GR has little effect on the
ligand-binding pocket of the receptor. GR mutant G23D1R bound glucagon
with an affinity similar to that of the wild-type GR (26). However,
G23D1R was unable to activate endogenous Gs in COS-1
cells as measured by cAMP accumulation, or in HEK-293T cells as
measured by calcium flux (26). Despite the inability of G23D1R to
activate Gs, tethering G23D1R to Gs-L in
the fusion protein G23D1R-Gs-L led to a 10-fold increase
in affinity for glucagon. The IC50 value for competitive
displacement of labeled glucagon from G23D1R-Gs-L
(IC50 value = 6.3 nM) was similar to that
of the wild-type GR (Table I).
Competitive binding of glucagon by the fusion proteins
GR-Gs-S and GR-Gs-L was also measured in
the presence of GTPS, a non-hydrolyzable GTP analogue. The affinity
of glucagon for GR and for GR-Gs-S was not affected by
the presence of 100 µM GTPS. This result suggests that
GR-Gs-S displayed a single low affinity state that was
independent of GTP. However, GR-Gs-L displayed two
glucagon-binding affinities. In the presence of 100 µM
GTPS, the binding affinity of glucagon for GR-Gs-L
shifted to an IC50 value about 10-fold higher than that
observed in the absence of GTPS (Fig.
5).
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Fig. 5.
GTP sensitivity of 125I-glucagon
binding to COS-1 cell membranes expressing
GR-Gs fusion proteins.
Membranes from COS-1 cells expressing GR-Gs-L and
GR-Gs-S were incubated with
125I-glucagon and the indicated concentrations of
unlabeled glucagon in the absence and presence of 100 µM
GTPS. Data are presented as percentage of total binding of
radiolabeled hormone versus the log of glucagon
concentration. Each symbol represents the mean of duplicate
determinations. The IC50 value was calculated from the
curve (Table I) and represents the average of at least three
independent determinations.
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The binding affinity of the glucagon analogue
desHis1[Nle9,Ala11,Ala16]glucagon
amide for the GR-Gs fusion proteins was also evaluated. The glucagon analogue binds to native GRs in rat liver membranes with
an affinity similar to that of glucagon and effectively inhibits glucagon action (22). In COS-1 cell membranes expressing recombinant GRs,
desHis1[Nle9,Ala11,Ala16]glucagon
amide displayed a slightly lower binding affinity (IC50 = 71 nM) than glucagon. The glucagon analogue had the same
binding affinity for GR-Gs-S, GR-Gs-L,
and GR (Table I). Bringing the GR and Gs signaling
partners in close proximity in the fusion proteins did not lead to
higher binding affinities for the antagonist even in the absence of GTP.
Glucagon-dependent Adenylyl Cyclase Activity--
The
fusion proteins were assayed for the ability to mediate an increase in
intracellular cAMP upon stimulation with glucagon. The fusion proteins
GR-Gs-S and GR-Gs-L caused
glucagon-dependent adenylyl cyclase activation, which led
to an increase in intracellular cAMP concentrations in transfected
COS-1 cells. The EC50 value of
glucagon-dependent adenylyl cyclase activation in COS-1
cells expressing GR was 5.8 nM (Table I). Essentially the
same EC50 value was obtained for GR-Gs-L
(5.1 nM). Consistent with a lower glucagon binding
affinity, GR-Gs-S exhibited an EC50 value
that was 4 times higher than that measured for GR-Gs-L
or non-fused GR (22.4 nM).
In addition, expression of both fusion proteins GR-Gs-S
and GR-Gs-L produced elevated basal levels of cAMP in
the absence of glucagon, which is characteristic of constitutive
receptor signaling (Fig. 6). The
constitutive activity of the fusion proteins GR-Gs-S and
GR-Gs-L was not suppressed by
desHis1[Nle9,Ala11,Ala16]glucagon
amide. The fact that
desHis1[Nle9,Ala11,Ala16]glucagon
amide had no effect on the constitutive activity of the fusion proteins
confirms that the elevated basal activity is not caused by endogenous
glucagon. In the classification of antagonists, this result would mean
that
desHis1[Nle9,Ala11,Ala16]glucagon
amide is a neutral antagonist, not a negative antagonist or inverse
agonist (27, 28).
DesHis1[Nle9,Ala11,Ala16]glucagon
amide showed no measurable adenylyl cyclase activity (<0.001%
relative to glucagon), when assayed in liver membrane preparations.
However, in COS-1 cells expressing recombinant GRs, a small cAMP
elevation, 0.01% relative to glucagon, was observed upon treatment
with
desHis1[Nle9,Ala11,Ala16]glucagon
amide, consistent with very weak partial agonist activity (Fig.
7). Interestingly, the glucagon analogue
induced adenylyl cyclase activity above baseline only in cells
expressing GR-Gs-L, but not in cells expressing
GR-Gs-S (Fig. 7). This observation suggests that the
partial agonist activity in intact cells induced by
desHis1[Nle9,Ala11,Ala16]glucagon
amide may be attributed to receptors that are coupled to
Gs-L, and not Gs-S.
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Fig. 6.
Adenylyl cyclase activity of COS-1 cells
expressing GR and GR-Gs
fusions. COS-1 cells were transiently transfected with GR,
GR-Gs-L, GR-Gs-S, or
G23D1R-Gs-L fusion genes and incubated with increasing
concentrations of glucagon as described under "Experimental
Procedures." Cell extracts were assayed for cAMP using a method that
measures the ability of cAMP in each sample to compete with
[8-3H]cAMP for a high affinity cAMP-binding protein. The
pmol of cAMP produced by 105 transfected COS-1 cells are
plotted versus the log of peptide concentration. Each symbol
represents the mean from duplicate experiments, and the curve
represents a single experiment. The concentration required to elicit
50% full agonist response (EC50) was determined from the
inflection point of the best-fit curve derived from at least three
experiments and is given in Table I. Experiments were repeated at least
three times to verify reproducibility. GR-Gs-L and
GR-Gs-S were constitutively active. GR and the fusion
G23D1R-Gs-L displayed normal basal adenylyl cyclase
activities.
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Fig. 7.
Adenylyl cyclase activity of COS-1 cells
expressing GR and GR-Gs fusion
genes in the presence of glucagon antagonist. COS-1 cells were
transiently transfected with GR, GR-Gs-L, or
GR-Gs-S and incubated with increasing concentrations of
the glucagon antagonist,
desHis1[Nle9,Ala11,Ala16]glucagon
amide. Cell extracts were assayed for cAMP levels as described for the
agonist glucagon under "Experimental Procedures." The pmol of cAMP
produced by 105 transfected COS-1 cells are plotted
versus the log of antagonist concentration. Each symbol is a
mean of duplicate samples and the curve is a representative of one
experiment. The antagonist
desHis1[Nle9,Ala11,Ala16]glucagon
amide showed weak partial agonist activity in COS-1 cells expressing
GR. Increasing concentrations of the antagonist did not inhibit the
constitutive activity of GR-Gs-L and
GR-Gs-S. The antagonist did not stimulate adenylyl
cyclase activity in COS-1 cells expressing GR-Gs-S, but
did display partial agonist activity in cells expressing
GR-Gs-L.
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Glucagon-dependent cyclase activity was also assayed in
COS-1 cells expressing the mutant receptor-Gs fusion,
G23D1R-Gs-L. G23D1R was shown to be incapable of
signaling due to the replacement of its second and third intracellular
loops with the first intracellular loop of the unrelated D4 dopamine
receptor (26). G23D1R-Gs-L bound glucagon with a higher
affinity than G23D1R and displayed an IC50 value (6.3 nM) close to that of GR-Gs-L. However,
unlike GR-Gs-L, G23D1R-Gs-L was not
constitutively active in the absence of glucagon, and treatment with
glucagon did not cause adenylyl cyclase activation (Fig. 6).
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DISCUSSION |
The effect of GTP to lower the affinity of certain hormones for
membrane receptors was described in the pioneering work of M. Rodbell
(29). The molecular basis of the "GTP effect" is now generally
understood. Heterotrimeric G proteins bind to GPCRs to stabilize a high
affinity agonist-binding conformation. In the presence of agonist, GTP
induces the dissociation of G protein from its receptor, which then
assumes a low affinity agonist-binding conformation. GRs in hepatocyte
membranes display the classical GTP effect, but they are also known to
exist in two interconverting ligand affinity conformations that are
modulated by Gs even prior to glucagon binding (30).
Gs exists in two predominant splice variants,
Gs-S and Gs-L. We aimed to test whether
differences in GR coupling to these splice variants may account for the
observed heterogeneity in GR ligand-binding affinity.
One useful approach to study the interactions of GPCRs and G proteins
has been to join a G subunit directly to the C terminus of a GPCR
and express a single fusion protein signaling complex (31-37). This
strategy allows unambiguous assignment of the functional consequences
of agonist binding and subsequent receptor activation and signaling.
Ideally, the fusion proteins ensure a defined 1:1 stoichiometry of GPCR
to a specific G subtype and enhance the efficiency and specificity
of activation by constraining two predetermined signaling partners in
relatively close proximity. Early attempts to find differences between
the long and the short forms of Gs in its interaction
with the 
2-adrenoreceptor (2-AR) led to
conflicting observations (38, 39). However, fusion of the
2-AR with each of the two forms of Gs
demonstrated that the properties of either protein in the fusion did
not change, and revealed that the subtle structural differences in the
two variants of Gs had significant effects on
2-AR-mediated signaling (33). When expressed in Sf9 cells, the 2-AR in
2-AR-Gs-L, but not in
2-AR-Gs-S, possessed characteristics of a
constitutively active receptor that could be inhibited by an inverse
agonist of 2-AR (33).
The GR belongs to a unique class of receptors within the GPCR family
and bears little sequence resemblance to the 2-AR, which falls within the rhodopsin-like group of receptors (1-3). We previously demonstrated that the i2 and i3 loops of the GR are essential for G protein coupling (26). Replacement of both cytosolic domains with the i1 loop sequence of the D4 dopamine receptor in the
receptor mutant, G23D1R, resulted in complete loss of G protein
signaling, as measured by cAMP accumulation and calcium flux assays
(26). The expressed GR can mediate both cAMP elevation and calcium flux
by coupling to Gs (26).
Fusion of GR to Gs-S and to Gs-L allowed
efficient coupling between both of the tethered partners, as
demonstrated by the ability of transfected cells expressing the fusion
to undergo glucagon-stimulated adenylyl cyclase activity (Fig. 6). This
established that the intrinsic properties of either protein were
preserved within the fusion construct, and that the physical connection did not hinder or restrict the conformational changes and domain movements that must accompany the activation process in both proteins. In the case of the 2-AR-Gs fusion, it was
shown that downstream post-activation events such as desensitization
and the repalmitoylation mechanisms were compromised, presumably
because complete separation of receptor from G protein was not possible
in the fusion protein (32, 36). Moreover, another study reported that
restricting the mobility of Gs relative to receptor in
2-AR-Gs affected its GTPase activity
(40).
The C-terminal tail of GR is relatively long compared with that of
2-AR. We preserved the long C-terminal tail of GR in the design of the fusion, even though mutant receptors lacking most of the
C-terminal extension still coupled efficiently to Gs
(20). Since signaling requires Gs to contact
determinants within the i2 and i3 loops of GR simultaneously, the
Gs portion of the fusion requires enough flexibility and
mobility to align its C terminus close to the cytosolic loops of GR, to
dissociate after GDP/GTP exchange, and to interact with adenylyl
cyclase. The long C-terminal extension presumably serves as a flexible
tether to minimize steric hindrance during intraprotein contact.
Transfected non-fused wild-type GR bound glucagon with the expected
average dissociation constant (IC50 value) of 46 nM (18, 20, 26) (Table I). The IC50 was
significantly higher than that of GRs in liver membranes, but was
typical of recombinant receptors expressed transiently in COS cells or
stably in HEK-293 cells (18, 41). This low affinity binding is
presumably caused by inefficient coupling to G proteins, or a lack of G
proteins relative to overexpressed receptors (41, 42). Fig. 4 shows that the GR in GR-Gs-L bound glucagon with a higher
affinity than non-fused GR. The IC50 for glucagon binding
shifted more than 10-fold closer to values observed for high affinity
native receptors in hepatocytes (Table I). Consistent with this
observation, co-expression of GR and Gs-L also resulted
in improved ligand-binding affinity (Table I). While these results
supported the assumption that an increase in the proportion of
Gs-L to GR shifted the binding affinity, the
IC50 value measured for GR-Gs-L was still 5-fold lower than that obtained for co-expressed GR and
Gs-L, suggesting that the physical proximity of the
signaling partners in the fusion also contributed to the improved
ligand-binding affinity. In marked contrast to GR-Gs-L,
GR-Gs-S had an affinity for glucagon that was
essentially the same as non-fused GR, with IC50 values at
least 10-fold higher than that measured for GR-Gs-L (Fig. 4, Table I).
In the classical view, the GR exists in the "off" state, R, until
glucagon docks into its binding site and induces a change of
conformation in R to the active form R*, which initiates the signaling
cascade by interacting with G proteins. The extended ternary model of
receptor activation considers that receptors are in close contact with
G proteins to form a complex with some basal activity even in the
absence of agonist (43-45). In the fusion, the signaling partners were
constrained to pre-couple, and should be in the activated form R*G even
prior to ligand binding. It is reasonable to assume that the
conformational change in the receptor induced by interaction with
Gs-S in GR-Gs-S, must be different from
that induced by association with Gs-L in
GR-Gs-L. Our results show that glucagon was able to
discriminate between GR-Gs-S and GR-Gs-L
and suggest that glucagon stabilized the active state of
Gs-L-coupled GRs more than
Gs-S-coupled GRs.
The high affinity state of GPCRs is known to be sensitive to guanine
nucleotides, and only agonist binding to high affinity receptors are
expected to be GTP-sensitive. Upon GDP/GTP exchange, GTP-occupied
Gs dissociates from GR, and the affinity of receptor for
hormone is decreased. The binding experiments were carried out using
membranes isolated from cells expressing the fusion proteins and all
Gs domains would be expected to be either GDP-bound or
nucleotide-free and primed for activation by glucagon binding to GR. As
shown in Fig. 5, in the presence of 100 µM GTPS, the displacement curve for glucagon binding to GR-Gs-L
shifted to the right to a higher IC50 value. In contrast,
the "GTP shift" was not observed with glucagon binding to membranes
expressing GR-Gs-S or non-fused GR (Fig. 5).
Following transient transfection in COS-1 cells, the GR in both fusion
proteins GR-Gs-S and GR-Gs-L activated
adenylyl cyclase in response to glucagon (Fig. 6). In addition, both
fusion proteins displayed 3-fold elevations in the basal levels of cAMP
over that of expressed GR in the absence of glucagon. Glucagon
treatment caused a further increase in adenylyl cyclase stimulation.
Certain mutations in GPCRs have been shown to increase the likelihood of agonist-independent signal transduction and are associated with
specific familial diseases (46, 47). A His178 
Arg
mutation in the i1 loop of GR was also reported to cause constitutive
activity (48). Constitutive activity of the fusion proteins suggests
that pre-coupling of the signaling partners stabilizes the active
conformation to cause GDP/GTP exchange and alter the basal activity
even in the absence of agonist. In contrast, G23D1R-Gs-L
was not constitutively active even though it displayed high affinity
glucagon binding. In addition, it did not support additional cAMP
production in response to glucagon.
Interestingly, glucagon bound to the fusion G23D1R-Gs-L
with an IC50 value similar to that obtained for
GR-Gs-L, a 10-fold increase in affinity from that
obtained for G23D1R (26). This result shows that the high affinity
glucagon-binding conformation in the G23D1R-Gs-L complex
does not require the involvement of the i2 and i3 loops of GR. This
observation is striking because the G23D1R receptor itself does not
display high affinity glucagon binding and does not couple to
Gs. The result with G23D1R-Gs-L suggests
that the GR mutant G23D1R is analogous to rhodopsin mutants that bind,
but fail to activate, the retinal G protein, transducin (Gt) (49). Certain rhodopsin mutants with alterations of
their i2 and i3 loops bound Gt in the GDP form, but failed
to induce nucleotide exchange or Gt release in the presence
of GTP (49, 50). Even though these mutants did not activate
Gt, they were stabilized in the metarhodopsin II
conformation, which is analogous to the high affinity agonist-binding
state of GPCRs with diffusible ligands. To our knowledge, the GR mutant
G23D1R in the context of the G23D1R-Gs-L fusion is the
first example of such a phenotype in a group II GPCR.
Extensive structure-activity analysis allowed us to identify residues
in glucagon that contribute a specific structural determinant to either
binding or transduction (51-53). Residues at positions 1, 9, and 16 of
glucagon are essential for receptor activation, but less important for
binding. Replacement of these residues led to the analogue
desHis1[Nle9,Ala11,Ala16]glucagon
amide, which has been shown to be a potent competitive glucagon
antagonist (22, 54). In the present study,
desHis1[Nle9,Ala11,Ala16]glucagon
amide displayed similar binding affinity for GR and the fusion
proteins. This property is characteristic of antagonists that are
unable to distinguish between different receptor agonist-affinity conformations. The fact that both fusion proteins exhibited elevated basal levels of adenylyl cyclase activation even in the presence of
increasing concentrations of
desHis1[Nle9,Ala11,Ala16]glucagon
amide confirms that the elevated basal cAMP levels are not caused by
glucagon (Fig. 7). In the classification of antagonists, desHis1[Nle9,Ala11,Ala16]glucagon
amide would be referred to as a neutral antagonist and not a negative
antagonist or inverse agonist (27, 28). Although the antagonist showed
no measurable adenylyl cyclase activation when assayed in liver
membrane preparations, very weak partial agonist activity was
detectable in COS-1 cells expressing recombinant GRs (Fig. 7). Taken
together, the results indicate that the adenylyl cyclase activation
observed in intact cells when stimulated by desHis1[Nle9,Ala11,Ala16]glucagon
amide may be attributable to Gs-L-coupled
GRs, since partial agonist activity was observed only in cells
expressing GR-Gs-L, and not in cells expressing
GR-Gs-S.
In addition, membranes containing expressed GR-Gs-L were
able to support full adenylyl cyclase activation in response to increasing concentrations of glucagon, while membranes expressing only
non-fused GR did not. This suggests that either the COS-1 membranes
were stripped of most of the endogenous Gs during
membrane preparation or that, after one activation cycle, most of the
endogenous Gs that dissociates from the membranes were
unable to participate in ensuing rounds of reactivation (55, 56).
Despite pertussis toxin pretreatment to eliminate potential interaction
with endogenous Gi, there was no indication that the
2-AR in the fusion
2-AR-C351G-Gi-1 had the capacity to
activate both its fusion partner Gi-1 and endogenous
Gs in an agonist-dependent manner when
expressed in COS cells (37).
Whether the 15-amino acid difference in linker 1 between the Ras-like
and helical domains of Gs variants has direct influence on agonist efficacy is unclear since it is remote from the C terminus of Gs, which is known to interact with receptor.
However, linker 1 is situated near the switch 1 and switch 2 regions of
Gs, which border the nucleotide binding cleft and
are known to change conformation upon GTP binding. The
flexibility afforded by the 15-residue peptide insert in linker 1 of
Gs-L may facilitate GDP/GTP exchange (11, 58, 59). The
IC50 values of the antagonist show that there was
essentially no difference in the ability of
desHis1[Nle9,Ala11,Ala16]glucagon
amide to stabilize either GR-Gs-S or
GR-Gs-L, but the subsequent signal transfer to effector
via receptors coupled to Gs-L appears to be favored.
Previous work with 2-AR-Gs fusion proteins
showed that Gs-L had a lower affinity for GDP and was
more easily activated, if not already nucleotide-free (33). Based on
those observations, it appears that
Gs-L-coupled GR is more primed for
activation than GR coupled to Gs-S. Thus, it is likely
that after the initial recognition, even small conformational changes
in the receptor brought about by the interaction of
desHis1[Nle9,Ala11,Ala16]glucagon
amide with activating residues on the extracellular face of the
receptor might be sufficient to transduce a signal via the already
empty Gs-L in the fusion, but not readily with GDP-bound
Gs-S. Despite its partial activity on
Gs-L-coupled receptors,
desHis1[Nle9,Ala11,Ala16]glucagon
amide is a potent inhibitor of glucagon action and may not only compete
for hormone binding sites, but also prevent the receptor from assuming
the conformation required for full agonist activity (22, 54).
The chemical or conformational features of a glucagon analogue that
determine whether it is an agonist, partial agonist, or antagonist
remains the object of investigation in drug development (22, 51-53,
57, 60, 61). It is clear that the efficacy and potency of ligands are
regulated not only by their recognition sites in the receptor, but also
by the type and relative amounts of G proteins expressed within the
cell. Evidence from the GR-Gs fusions shows that
pre-coupling of GR to different Gs isoforms can generate
multiple conformations with distinct ligand-binding affinities
resulting in diverse cellular responses. Glucagon-binding affinity for
the GR-Gs-L fusion is GTP-sensitive and simulates pharmacological responses of native receptors in hepatocytes. Thus, it
may be a useful model for pharmacological and biophysical studies in
recombinant systems. The enhanced basal activity of GR-Gs-L will be useful in assessing novel glucagon
analogues for inverse agonist activity. More importantly, a GR
mutant-Gs-L fusion protein revealed the existence of an
active intermediate in the glucagon signaling cascade analogous to the
metarhodopsin II species of the rhodopsin pathway and should facilitate
further analysis of GR interaction with Gs.
s*
§,
TOP
ABSTRACT
INTRODUCTION
