Scanning of the Glucagon-Like Peptide-1 Receptor Localizes G Protein-Activating Determinants Primarily to the N Terminus of the Third Intracellular Loop
Swarna K. Mathi,
Yvonne Chan,
Xinfang Li and
Michael B. Wheeler
Departments of Medicine and Physiology University of
Toronto Toronto, Ontario, Canada M5S 1A8
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ABSTRACT
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It is well known that glucagon-like peptide-1
(736 amide) (tGLP-1) is a potent insulinotropic hormone with powerful
antidiabetogenic effects. In the present study we sought to determine
the precise regions of the intracellular domains of the tGLP-1 receptor
that are required for its efficient coupling to adenylyl cyclase
because cAMP is the primary candidate second messenger coupling tGLP-1
to insulin secretion. Recently, we identified an amino acid within the
third intracellular loop, K334, that was required for efficient
coupling of tGLP-1 receptor to adenylyl cyclase. A similar
mutagenesis-based strategy was employed here to examine the first and
second intracellular loops and to further define sequences in the third
loop required for the efficient coupling of the receptor to its second
messengers. Receptor mutants were expressed in COS-7 cells and examined
for tGLP-1 binding and cAMP stimulation. Three alanine substitution
mutations, V327A, I328A, and V331A, resulted in significantly lower
tGLP-1-stimulated cAMP production without reductions in receptor
expression. Analysis of the first and second intracellular loops
revealed only one mutation contained within the first loop, R176A,
where a significant reduction in cAMP activation was observed with
normal receptor expression. These studies suggest that specific
determinants of coupling for tGLP-1 receptor are primarily localized to
the predicted junction of the fifth transmembrane helix and the third
intracellular loop. We predict that V327, I328, and V331 form part of a
hydrophobic face that directly contacts the G protein.
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INTRODUCTION
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The incretin hormone, glucagon-like peptide-1 (GLP-1), is released
from the intestinal L cells to stimulate insulin secretion from
pancreatic ß-cells in a glucose-sensitive manner. The biologically
active forms secreted, truncated (t) GLP-1(737) and tGLP-1(736
amide), have been shown to be among the most potent insulinotropic
agents identified to date in mammals (1). The recent observation, that
mice expressing a null mutation in the GLP-1 receptor (R) are diabetic,
clearly establishes a major role for GLP-1 in mammalian carbohydrate
metabolism (2). A possible role for tGLP-1 in the central control of
feeding has also recently been suggested (3). Truncated GLP-1
administered via an intracerebroventricular injection was a powerful
inhibitor of feeding in fasted rats, a response proposed to be mediated
through tGLP-1 receptors in the hypothalamus. In addition, clinical
studies indicate that tGLP-1 not only stimulates insulin secretion in
normal subjects, but also in those with non-insulin-dependent diabetes
mellitus (4, 5, 6), supporting its therapeutic potential.
The insulinotropic properties of tGLP-1 are mediated through a
high-affinity tGLP-1 receptor on the insulin-secreting ß-cells of the
pancreas (7, 8). The receptor cDNA, initially cloned from rat
pancreatic islet cells, and subsequently from human pancreas (9, 10),
predicts a seven-transmembrane G protein-coupled receptor (GPCR) of the
glucagon/vasoactive intestinal peptide (VIP)/secretin, receptor
subfamily (11). Studies in monkey kidney COS cells have shown that the
recombinant tGLP-1R can couple to at least two G protein-coupled
signaling pathways, including adenylyl cyclase (AC) and phospholipase C
(12). Other work on the endogenous receptor in isolated ß-cells and
ß-cell lines, and with the recombinant receptor expressed in CHO
(Chinese hamster ovary) and CHL (Chinese hamster liver) cells, however,
strongly suggests that the cAMP-dependent activation of protein kinase
A and subsequent increase in free cytosolic Ca2+ is the
trigger for tGLP-1-stimulated insulin secretion (13, 14, 15).
Numerous investigations of ligand-activated signal transduction have
centered on the localization of sites of contact between the receptor
and proteins activated by the receptor. Structural studies on the GPCR
superfamily suggest that the predicted ends of the transmembrane
helices and the connecting intracellular loops present logical contact
points with G proteins. In keeping with this hypothesis, each of the
intracellular loops and the C-terminal domain have been implicated in
receptor activation (16). Within the biogenic amine receptor family
there is widespread consensus that the third intracellular domain (IC3)
contains a G protein activation region (17, 18, 19). There is, however,
significantly less information on potential G protein coupling within
the glucagon/GLP-1 receptor subfamily. At present, receptor mutagenesis
studies suggest that the C-terminal domain may be important for
receptor regulation/desensitization, but a direct role of this domain
in AC coupling is not supported (20, 21). The IC3 loop, however, has
recently been implicated in G protein activation by the tGLP-1
receptor. A single amino acid block in the predicted N-terminal portion
of the IC3 domain (K334-L335-K336) was shown to be required for the
efficient coupling of tGLP-1R to AC (22). Within this block, the K334
position was found to be most important for coupling. Because direct
interaction with G proteins most likely involves large protein
interfaces (16), we examined the predicted first and second loops of
the rat GLP-1 receptor and have further examined the junction between
the predicted fifth transmembrane (TM5) helix and the IC3 loop. Our
results suggest that residues within the cytoplasmic loops, the
majority of which are concentrated at the junction between the TM5
helix and the IC3 domain, are required for transducing the tGLP-1
signal to cAMP activation.
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RESULTS
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Analysis of the First Intracellular Loop
To examine the potential interaction between G proteins and the
tGLP-1 receptor, a series of deletion and substitution mutants spanning
the intracellular loops of the rat tGLP-1 receptor were generated,
expressed in COS-7 cells, and analyzed for [125I]tGLP-1
binding and cAMP formation in response to a tGLP-1 stimulation. In all
cases, basal cAMP levels (in the absence of tGLP-1 stimulation) were
found to be similar, suggesting that no mutation created a
constitutively active receptor. At a concentration of 10 nM
(determined previously to yield a maximal cAMP stimulation), tGLP-1
elicited an approximately 30-fold increase in cAMP accumulation with
the wild type (WT) tGLP-1 receptor, consistent with stimulation levels
reported previously (12). Strikingly lower cAMP responses, compared
with WT tGLP-1R, were observed with three deletion mutations, each
carrying a three-amino acid deletion (IC11-3) spanning the predicted
nine-amino acid IC1 domain (data not shown). For each mutation examined
in this loop and subsequent regions, binding displacement curves were
generated according to a one-site model, which described the data most
accurately in all cases. Key descriptors of the binding analysis, the
dissociation constant (Kd) and Bmax values,
were calculated as described in Materials and Methods. For
the WT tGLP-1R, the Kd value was in the range of 2.14.1
nM (see
Figs. 14


), which corresponds well with previous
studies (12, 22). The deletion mutations spanning IC1, however, failed
to demonstrate measurable specific binding, which likely explains the
lack of a cAMP response (data not shown). To reduce the possibility of
impairing the appropriate processing and/or expression of viable
receptors, an alanine replacement strategy was employed to allow
analysis of specific amino acid moieties with minimal alteration to
receptor conformation. Further analysis using single amino acid
substitutions showed varying levels of cAMP accumulation. COS cells
expressing the alanine substitution mutants F169A, R170A, H171A, L172A,
H173A, and T175A were capable of eliciting cAMP responses that were not
found to be significantly different (P > 0.05) from
the WT response (Fig. 1
). In contrast, cAMP responses
for C174A, R176A, and N177A were significantly different at 37 ±
5%, P < 0.05; 26 ± 8%, P <
0.01; and 43 ± 6%, P < 0.05, respectively,
compared with WT levels. Competitive binding experiments were employed
to determine whether appropriate cell surface expression could possibly
explain the reduced cAMP accumulation. With mutants C174A and N177A,
Bmax values were found to correlate well with reduced
receptor activation because significantly lower values compared with WT
(45 ± 6 and 22 ± 2% of WT, P < 0.01) were
observed. In contrast, with L172A, H173A, and T175A, receptor
expression (Bmax) was reduced, with little effect on cAMP
accumulation, suggesting that the levels of receptor expression are not
always linearly related to responsiveness. With mutation R176A, which
produced the lowest cAMP response of the substitution mutants examined
in IC1, the receptor number (111 ± 10% of WT) and the
Kd value (5.5 ± 1.2 vs. 3.9 ± 0.3
nM for the WT) were not considered significantly different.
Further, dose-dependent analysis of the cAMP response to tGLP-1
(10-12 through 10-6 M) with the
R176A mutant (Fig. 5b
, data for 10-6 M tGLP-1
not shown) indicated that the EC50 was approximately
10-fold higher (R176A = 22 nM vs. WT GLP-1R
= 1.7 nM), suggesting that a specific modification at this
basic residue in position 176 of IC1 impairs the stimulation of cAMP
production.

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Figure 1. Comparison of WT tGLP-1R and Substitution Mutants
of IC1 for Stimulation of cAMP Production and Receptor Expression
cAMP formation 72 h posttransfection in COS-7 cells stimulated by
10 nM tGLP-1 (as indicated by the vertical stippled
bar) is shown as a percentage of WT. The amino acid substituted
for alanine in each mutant is outlined within the sequence. The level
of expression (Bmax) and binding affinity (kd)
for each deletion mutant was calculated from binding displacement
curves and expressed relative to the WT GLP-1R (% WT). Results are
presented as the mean ± SEM of at least three
independent experiments. Receptor mutations which led to significant
alterations (P < 0.05) in cAMP
stimulationa or expressionb are indicated.
Mutations resulting in decreased cAMP production that are poorly
correlated to changes in receptor expression are indicated by
arrows.
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Figure 2. Comparison of WT tGLP-1R with Block Deletion and
Substitution Mutants of the IC2 Region
Binding characteristics and cAMP activation of block deletion mutants
(A) and alanine substitution mutants (B) of the IC2 region were
determined in transiently transfected COS-7 cells as described in Fig. 1 . Results are presented as the mean ± SEM of at
least three independent experiments. Receptor mutations which led to
significant alterations (P < 0.05) in cAMP
stimulationa or expressionb are indicated.
Detectable binding displacement was not observed with IC23 and 4 as
indicated.
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Figure 4. Comparison of WT tGLP-1R with Alanine Substitutions
Mutants at the Junction of the TM5 Domain and the IC3 Loop
cAMP formation in COS-7 cells and binding characteristics are expressed
as a percentage of WT. Values are shown as the mean ±
SEM from three or more independent experiments, and
significant differences are indicated as in Fig. 1 .
Arrows indicate mutations as described previously.
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Figure 5. Competitive Binding and cAMP Dose-Response Analyses
of Receptors with Impaired cAMP Responses
A, The displacement of [125I]tGLP-1 binding in COS-7
cells, transiently transfected with WT and receptor mutants R176A,
V327A, I328A, V331A, and DM-1 is expressed as specific binding. Results
are representative of at least three independent experiments. B,
Dose-dependent effects of tGLP-1 on cAMP accumulation in COS-7 cells.
One representative of two independent experiments is shown.
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Analysis of the Second Intracellular Loop
The second intracellular domain has also been implicated in G
protein activation within the biogenic amine receptor family (23),
further suggesting the possibility of multiple receptor/G protein
contact points. To test the possibility that the residues within the
tGLP-1 receptor IC2 domain contribute to the efficient coupling of the
receptor to cAMP, the block deletion strategy was initially employed.
Mutational analysis of the IC2 domain revealed that deletions in the
distal portion of the predicted loop, IC23 and IC24, were poorly
tolerated (Fig. 2a
). Block deletion mutants IC21 and
IC22 had reduced cAMP responses, the former reduction being
statistically significant (32 ± 3% vs. WT,
P < 0.01). Binding analysis, however, revealed a
strong correlation between ligand-binding efficiency and cAMP
production, suggesting that coupling was not specifically altered by
these mutations (Fig. 2a
). IC23 and IC24 were chosen for further
examination using single amino acid substitutions because deletions
were not tolerated in these blocks. Surprisingly, no substitution
mutations resulted in a reduction in the cAMP response (Fig. 2b
). There
appeared to be, in fact, a trend toward an exaggerated cAMP response
with two mutations, I265A and F266A. In each case the affinity and
expression level of the receptor mutants were not considered
significantly altered, relative to the unmodified receptor (Fig. 2b
).
Analysis of the N-Terminal Junction of the Third Intracellular
Loop
Previously, we reported that a single amino acid block deletion in
the predicted N-terminal portion of the rat tGLP-1 receptor IC3 domain
(K334-L335-K336) was required for the efficient coupling of tGLP-1R to
AC (22). Since the N-terminal region may contain several residues that
contribute to coupling, as predicted from studies by other members of
the GPCR superfamily (17, 18), we further examined the junction between
the predicted TM5 helix and the third cytoplasmic loop. Deletion
mutants spanning A333-F321 (DM1-DM6) were also assayed for
[125I]tGLP-1 binding and cAMP formation. Significantly
lower cAMP responses compared with WT tGLP-1R were observed with all DM
block deletion mutants (154% vs. WT, P <
0.01) with the exception of DM4 (Fig. 3
). The binding
affinities of mutants DM1-DM4 were not substantially altered, as
determined by Kd values, although mutants DM1-DM3 did have
significantly reduced receptor expression, with Bmax at
approximately 3825% of WT (P < 0.01). Importantly,
the reduced expression levels with DM-1 and DM-3 do not correlate with
the virtual lack of cAMP activation by these mutants, strongly
suggesting an uncoupling from AC and implicating the involvement of
residues within these deletions. The Kd and
Bmax values could not be determined for DM5 and DM6 because
appreciable displacement of the radioligand was not observed.
Further analysis by single amino acid substitution within this deleted
region revealed three residues that severely impaired cAMP activation
when modified, although not to the extent of the corresponding block
deletion mutants (Fig. 4
). The V327A, I328A, and V331A
substitutions resulted in a significantly lower cAMP response compared
with WT GLP-1 receptor (42 ± 6, 39 ± 5, and 45 ± 3%,
respectively, P < 0.01) (Fig. 4
). The reduced
stimulation was not correlated to significant alteration in binding
affinity or a reduction in the level of receptor expression relative to
the WT (Figs. 4
and 5a
), suggesting the importance of
these residues in coupling to AC. Further dose-dependent analysis of
the V327A, I328A, and V331A mutants for cAMP production were performed
to examine GLP-1-induced cAMP sensitivity (Fig. 5b
). For each mutation
examined, it was apparent that the EC50 value for cAMP
stimulation was approximately 10-fold greater than the WT GLP-1R
(V327 = 26 nM, I328 = 16 nM,
V331 = 23 nM vs. WT = 1.7
nM), despite the observation that the affinity of each
receptor for tGLP-1 binding was not significantly different (Fig. 5a
).
These data further suggest a less efficient coupling of the receptor
mutants to G protein activation.
Sufficient evidence exists to suggest that the proximal IC3 domain of
GPCRs may form an amphipathic
-helix (18, 24, 25, 26). To better
understand the relationship between the block deletion mutants and the
single substitution mutants, a helical representation of the N-terminal
IC3 loop was generated, following the model of Hill-Eubanks et
al. (18) for the muscarinic m5 receptor (Fig. 6
).
The critical amino acids, V327, I328, and V331, as well as K334
identified from our previous study, are localized to one face of the
predicted
-helix (Fig. 6a
). Because DM4 maintained normal phenotype
with respect to ligand binding and AC activation, this deletion mutant
is also represented in a helical projection that can be aligned with
the WT tGLP-1R (Fig. 6b
). The sequence of all deletion mutants is also
given for comparison (Fig. 6b
). DM2, -3, -5, and -6 mutants may be
expected to have normal activation because they have compatible amino
acid substitutions at the required positions. These mutants, however,
have significantly reduced receptor expression, which may lead, at
least in part, to significantly lowered activation.

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Figure 6. A Helical Projection of the IC3 N-Terminal Domain
Comparing WT and Mutant Receptors
A, A view of the tGLP-1 receptor IC3 loop from the membrane to the
interior of the cell is shown according to the model of Hill-Eubanks
et al. (18). The open line represents the
top of the view at the membrane surface. The sequence of the complete
IC3 domain and the predicted junctions of TM5 and TM6 are also given in
linear form. The critical residues indicated by circles
on the helical view are shown in boxes on the linear
view of IC3. The circled amino acid on the linear view
represents the only single amino acid substitution we generated with
completely abolished cAMP activation. B, A comparison of the WT
sequence with the block deletion mutants D1-D6 is given to determine
the effect of these deletions on the three-dimensional structure of the
IC3 N terminus. Positions corresponding to the four critical residues
of the WT receptor are also shown in a helical view of the DM4 mutant.
These positions are also underlined within the sequence
comparison of all deletion mutants. The symbol ++ indicates that DM4
elicits a normal cAMP response while all other deletions have blunted
cAMP responses.
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DISCUSSION
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The mechanism by which the binding of an agonist to a GPCR leads
to activation of intracellular signaling systems is the focus of
intense investigation. Evidence has accumulated indicating that the
predicted intracellular domains of the GPCRs contain specific regions
that provide contact points with G proteins. The C terminus (IC4) and
the third intracellular cytoplasmic loop, which display the highest
amount of heterogeneity among the GPCR family, have been implicated in
G protein activation (16). Previously, we characterized the IC3 loop of
the tGLP-1 receptor between residues K334-K351, using a series of
deletion and substitution mutations scanning the region (22). A single
amino acid, K334, in the N-terminal region of IC3, was required for the
efficient coupling of the tGLP-1 receptor to AC, supporting the general
model that the IC3 domain of GPCRs contains specific residues required
to activate G proteins. Since K334 is located near the junction with
the predicted TM5 region, and several amino acids are likely involved
in direct protein-protein contact with G proteins (16), we have
continued our analysis of this area into the predicted transmembrane
domain. Analysis at this junction, spanning F321-A333, identified five
deletion mutations that significantly lowered the cAMP response and
receptor expression (Fig. 3
). The reduced stimulation of cAMP
accumulation with the deletion mutations DM-1 and DM-3, however, was
far more dramatic than the corresponding reduction in receptor
expression, suggesting that this region may contain key residues
involved in G protein contact. Further analysis by single alanine
substitution revealed three critical residues that, when modified,
resulted in receptors with impaired signaling properties. V327A, I328A,
and V331A substitutions resulted in a significant reduction in cAMP
response without appreciable alteration in binding affinity or reduced
expression relative to the WT receptor (Figs. 4
and 5
). Interestingly,
these amino acids, along with K334 identified previously (22), are
concentrated at the junction between TM5 and IC3.
Although it will not be possible to unequivocally define the precise
junction between IC3 and TM5 without structural data, the existence of
amphipathic
-helices at the N and C termini that are continuous with
the predicted transmembrane helices is supported (24, 25, 26). Evidence
that the N-terminal region of IC3 in the m5 muscarinic receptor exists
as an
-helix with functional importance was provided using random
saturation mutagenesis (18). This region within the m5 receptor
contained four critical amino acids that are predicted to face one side
of the amphipathic helix, terminating in a completely conserved,
charged residue (Arg). If the IC3 region of tGLP-1 receptor is
initiated at I325, the four residues identified in our studies are
analogous, in position and type, with those of the m5 receptor (18).
When this model is applied to the tGLP-1 receptor, it can be predicted
to form a continuous
-helix that terminates at the charged lysine
residue, suggesting that V327, I328, V331, and K334 form an analogous
hydrophobic face, which couples directly with the G protein (Fig. 6a
).
In the m5 receptor, the outer residues of the hydrophobic face were
proposed to be involved in direct ionic interaction with the G protein,
whereas the inner residues were involved in maintaining the hydrophobic
interactions that define the structure of the coupling pocket (27).
Analysis of the amino acid sequence alignment for the entire family of
glucagon/VIP/secretin receptors revealed only identical amino acids or
conservative substitutions for the critical hydrophobic residues,
terminating in a completely invariant lysine residue. Therefore, this
subfamily of GPCRs predicts a three-dimensional structure for the
N-terminal IC3 region consistent with the model proposed for the
muscarinic receptors. The C-terminal region of IC3, as well as the
N-terminal region, has also been implicated in direct G protein
coupling with the muscarinic receptors (18, 26). We have not identified
similar C-terminal residues within the tGLP-1R. Two separate mutations
in this region at position 350, however (A350E or A350K), were not
tolerated and thus could not be tested (22). Interestingly, Heller
et al. (28) described a tGLP-1 receptor substitution, R348G,
rather than the R348A mutant we described earlier (22), which resulted
in a receptor with significantly reduced affinity for tGLP-1 and an
abolished cAMP response. In our experience, mutations that dramatically
compromise receptor expression or binding affinity are difficult to
interpret with respect to alterations in G protein coupling. Thus,
R348-L349-A350 and other residues within the intracellular domains may
indeed play important roles in coupling and require further study.
The possibility that further G protein coupling determinants within the
glucagon/tGLP-1 receptor family involve loops in addition to IC3 has
been the focus of several recent studies. Deletion of the majority of
the IC4 domain of the glucagon receptor did not alter the ability of
glucagon to activate AC (20). Similar work with the tGLP-1R (21), and
with the structurally related GIP (29) and PTH (30) receptors,
demonstrate that large blocks of the predicted IC4 domain can be
eliminated without uncoupling the receptor from AC. Although, these
studies do not support the presence of direct G protein coupling within
this domain, it appears that this region in each receptor may be
important for receptor regulation through homologous and heterologous
desensitization mediated through phosphorylation and receptor
internalization (21). The involvement of the IC1 and IC2 domains in G
protein activation within the glucagon receptor family has received
little attention. We have generated a series of deletion and
substitution mutations that encompasses the IC1 and IC2 domains (Figs. 1
and 2
). These studies revealed only one potential site within IC1,
R176, where a notable reduction in tGLP-1-mediated cAMP stimulation was
observed without a corresponding effect on receptor expression (Fig. 1
). It is unclear how R176 may play a role in coupling along with
residues in the proximal IC3 region; however, one can speculate that
intracellular domains may be in close proximity and therefore could
interact with the G protein. Another recently described mutation in the
GLP-1R (H180R) (28), caused a dramatic shift in affinity and a parallel
decrease in cAMP activation. We predict, however, that H180 is probably
positioned within TM-2 and is not likely to provide a direct contact
point for G proteins. The IC2 region appeared to lack any determinants
of coupling and activation because no amino acid substitutions resulted
in significantly impaired cAMP accumulation. This domain, in fact, had
uncharacteristically exaggerated cAMP responses with some substitution
mutations in the distal part of the loop.
In conclusion, we have extended our characterization of the junction
between the fifth transmembrane domain and the third intracellular loop
of the rat tGLP-1 receptor using a mutagenesis-based strategy. We have
identified several mutations that produced receptors with reduced
responsiveness to tGLP-1. Although it was not possible to fully
ascertain whether the reduced activity of all mutants examined (
Fig. 14


) was the result of reduced receptor expression and/or increased
EC50 values, three amino acids localized to IC3 (V327,
I328, and V331) displayed significantly decreased cAMP activation when
mutated, without significant alterations to receptor expression. These
residues, along with K334 (identified in our earlier work), therefore,
may be required for binding G proteins and activating AC. Our findings
are consistent with recent studies of the m5 muscarinic receptor, which
provide strong evidence that the IC3 N-terminal region exists as an
-helix continuous with the TM5 domain, terminating with a conserved
basic residue (18, 27). The four amino acids of the GLP-1 receptor that
were critical for G protein coupling and activation of AC were found in
analogous positions to those of the m5 receptor and could form a
hydrophobic face when viewed in a helical arrangement of the region.
The presence of highly conservative substitutions in corresponding
positions of all other members of the glucagon/VIP/secretin subfamily,
as well as receptors for various biogenic amines (27), suggests that a
common mechanism of activation may be widely shared by diverse seven-
transmembrane receptors coupled to G proteins. Based on the prediction
that these residues are required for direct side chain interactions
with the G protein, as well as the structural integrity of the
hydrophobic pocket, we intend to test amino acid substitutions that
specifically alter these properties in the future. In addition, since
the levels of receptor expression are not always linearly related to
responsiveness (35), several mutations with reduced Bmax
and AC activation identified in the present study should receive
further examination as well.
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MATERIALS AND METHODS
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Generation of Receptor Mutants and Cell Transfection
The WT rat tGLP-1 receptor DNA (9) was introduced into the
HindIII-XbaI sites of the vector pBS (Stratagene,
La Jolla, CA) before mutagenesis. Mutations were introduced into the
GLP-1 receptor coding sequence by oligonucleotide-directed mutagenesis
using double-stranded template (Stratagene), or single-stranded
template (31, 32). Mutated sites were verified by dideoxy sequencing
with T7 polymerase (Pharmacia, Uppsala, Sweden). Mutant receptor cDNAs
were subcloned into the expression vector pcDNA3 (Invitrogen, San
Diego, CA) for transient expression in COS-7 cells. Approximately
3 x 106 cells were seeded in 10-cm dishes and
cultured in DMEM supplemented with 10% FBS. Cells were transfected
with WT tGLP-1R or receptor mutants (5 or 10 µg for cAMP and binding
assay, respectively), using the diethylaminoethyl-dextran method as
previously described (12).
Iodination of GLP-1 and Binding Assays
Synthetic human tGLP-1(736) amide (Bachem, Torrance, CA) was
used in all binding studies. Radioiodination was accomplished by the
chloramine T method as previously described (12, 33). The
[125I]tGLP-1(736 amide) product was purified by reverse
phase adsorption to a C-18 Sep-pak column (Waters Associates, Milford,
MA) and had a specific activity of approximately 125250 µCi/µg.
Whole cell binding assays were performed as previously described (12, 33). Briefly, COS-7 cells expressing the WT and mutant receptors were
cultured for 72 h posttransfection, washed twice in PBS, and
recovered from plates with 2 mM EDTA in PBS. Cells
(
5 x 105/tube) were incubated for 45 min at 37 C
in binding buffer (DMEM containing 0.4% glucose, 1% BSA, and 0.1
mg/ml bacitracin, pH 7.4) with radiolabeled tracer
[125I]tGLP-1 amide (50,000 cpm,
270 pM)
and unlabeled peptide at concentrations of 10-14 to
10-6 M, in a final volume of 200 µl. Cell
suspensions were centrifuged at 12,000 x g and the
cell-associated radioactivity was counted (Cobra II, Canberra Packard,
Meriden, CT). Specific binding (total binding minus nonspecific binding
measured in the presence of excess (1 µM) tGLP-1) was
determined for the WT and each mutant receptor. Binding
characteristics, including specific binding, Bmax, and
Kd, were calculated according to Keen and MacDermot (34)
from competitive binding-displacement curves generated using Prism
(GraphPad Software, San Diego, CA).
cAMP RIAs
COS-7 cells expressing the WT and mutant receptors were passaged
into six-well plates 24 h posttransfection. After culture for an
additional 48 h, cells were washed in PBS, followed by assay
buffer (DMEM containing 0.4% glucose, 1% BSA, and 0.1 mg/ml
bacitracin, pH 7.4), and preincubated 30 min at 37 C, followed by a
30-min stimulation period with tGLP-1 (10 nM) and
3-isobutyl-1-methylxanthine (1 mM). For dose-response
curves in the 24-well format, cells were stimulated with tGLP-1
(10-12 to 10-6 M). The cAMP was
extracted with 80% ethanol, and lyophilized samples were reconstituted
in sodium acetate buffer (pH 6.2). Cyclic AMP production was measured
by RIA (Biomedical Technologies, Stoughton, MA) as previously described
(12, 33). All test agents were prepared from lyophilized samples on the
day of assay and added from concentrated stocks.
Statistics
All values are expressed as the mean ± SEM of
at least three independent observations unless stated otherwise.
Statistical analysis was performed using ANOVA followed by Tukeys
post analysis (InStat, GraphPad Software, San Diego, CA), comparing
each test condition to those values obtained with the WT tGLP-1R.
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ACKNOWLEDGMENTS
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We would like to thank Dr. Patricia Brubaker for her expert
advice with several technical aspects of the study and for supplying
[125I]tGLP-1.
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FOOTNOTES
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Address requests for reprints to: Michael B. Wheeler, Department of Physiology, University of Toronto, 1 Kings College Circle, Toronto, Ontario, Canada M5S 1A8.
This work was funded by grants to M.B.W. from the Medical Research
Council of Canada (MT-12898) and the Canadian Diabetes Association in
memory of the late Marjorie and Willis E. Montgomery
Received for publication December 16, 1996.
Revision received January 23, 1997.
Accepted for publication January 23, 1997.
 |
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