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Laboratory for Molecular Pharmacology (S.A.H., C.Ø., T.W.S.)
Department of Pharmacology
Department of Anatomy (C.Ø.)
The Panum Institute DK-2200 Copenhagen N, Denmark
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Structure-functional analysis of the glucagon/VIP/calcitonin receptor family is still at a relatively early stage as compared with the quantitatively much larger family of rhodopsin-like 7TM peptide receptors for which a more detailed picture of important ligand-receptor interactions has emerged. For instance, for the rhodopsin-like receptors binding of small agonist ligands such as monoamines appears to involve distinct mechanisms from those involved in binding of the larger peptide ligands (5). Thus, while binding of the small ligands is believed to occur through high-affinity interaction with ‘binding pockets’ buried relatively deeply in the transmembrane domain, the peptide ligands seem to acquire their binding energy predominantly through interactions with residues present in the extracellular domain of the receptor. No common mechanism therefore seems to account for the initial recognition events between individual agonist ligands and the corresponding 7TM receptors, although the signaling mechanism appears to be shared (6). For the glucagon/VIP/calcitonin receptors, binding of the peptide ligands appears to be critically dependent on major parts of the extracellular N-terminal extension, although this receptor domain by itself does not suffice for high-affinity peptide binding and/or receptor activation (7, 8, 9, 10, 11, 12, 13, 14, 15).
Constitutive receptor activity, i.e. signaling by receptors in the absence of ligand binding, has been described extensively for the rhodopsin-like 7TM receptor family. Here, constitutive activity has been observed in the form of naturally occurring mutants associated with human diseases (reviewed in Ref.16) and in some wild-type/unmutated monoamine receptors (17, 18), as well as for engineered substitutions (19, 20). For several of these mutant forms the substitutions are located in the intracellular (IC) loop segments, in particular the IC loop 3, although examples of mutations located in essentially any subdomain of the rhodopsin-like receptor structure have been demonstrated to cause increased signaling activity; these include exchanges at positions located in the extracellular loops, e.g. in the MSH receptor (21), and the thrombin receptor (22). Recently the first examples of constitutively active receptor mutants in the glucagon/VIP/calcitonin family were reported (23, 24). The mutation of a highly conserved histidine residue present at the presumed junction between TM-II and IC loop 1 was identified in the PTH receptor gene of patients suffering from a rare form of dwarfism, Janssen-type chondrodysplasia (23). This mutation [H223R], which appears to form (part of) the molecular basis for the disease, was shown by expression studies to be accompanied by elevated levels of ligand-independent levels of adenylyl cyclase activity. Subsequently, the exchange of a another highly conserved residue located at the cytoplasmic end of TM-VI (T410P) was shown also to be associated with this syndrome (24).
In the present study we have exchanged the corresponding conserved
residues in the rat glucagon receptor, His178 and
Thr352, respectively (Fig. 1
), and characterized receptor
constructs carrying different substitutions at each of these positions.
The highly diverse phenotypes exhibited by these mutants shed further
light on the molecular mechanisms that cause and accompany constitutive
activity of the glucagon receptor and underlines similarities as well
as differences between the glucagon/VIP/calcitonin receptor family and
the quantitatively dominating 7TM family, the rhodopsin-like
receptors.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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, indicating the
substituted conserved residues His178 and
Thr352, located at the membrane-cytosol interphase of TM-II
and TM-VI, respectively. The receptor constructs ([H178R], [H178K],
[H178A], [H178E], [T352P], [T352S], and [T352A]) were
expressed transiently in COS-7 cells and characterized by competition
binding analysis using [125I]glucagon and with regard to
basal and stimulated levels of cAMP accumulation.
Radioligand Binding Experiments with
His178
In homologous competition binding analysis, each of the four
mutant His178 receptors bound glucagon with an improved
affinity compared with the wild-type receptor (Table 1), up to a 10-fold increase for the
[H178E] construct. The receptor density (Bmax) estimated
on the basis of [125I]glucagon competition binding
experiments indicated a reduced level of receptor expression for all
mutant constructs, the [H178E] receptor in particular (Table 1). The
glucagon analog des-His1-[Glu9]glucagon
amide, which both in vivo and in vitro has been
characterized as a glucagon receptor antagonist (25, 26), bound the two
His178-substituted glucagon receptors that were analyzed
with affinities similar to that seen for the wild-type receptor (Table 1).
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), while for each of the other
His178 constructs ligand-independent adenylyl cyclase
activity was indistinguishable from the wild-type response. The basal,
i.e. ligand- independent level of adenylyl cyclase activity
observed for the [H178R] construct was elevated 4.9 ± 1.2-fold
relative to that observed for the wild-type receptor (Figs. 2 and 3) and further stimulated, in response to
glucagon, with an increment of approximately half that obtained for the
wild-type receptor. Adenylyl cyclase activity reached a maximal level
at 
10-7 M glucagon, with a slight decrease
in activity occurring at even higher concentrations (Fig. 3). This
biphasic response, which has been also reported previously in liver
membranes (25), was seen both for the wild-type receptor and for the
[H178R] mutant (Fig. 3). Surprisingly, the potency of glucagon was
reduced 12-fold for the constitutively active [H178R] receptor
construct (from 0.30 ± 0.04 nM to 3.6 ± 1.2
nM, Fig. 3 and Table 2). Thus
for this mutant the IC50 for glucagon binding and the
EC50 for glucagon-mediated stimulation of adenylyl cyclase
were shifted in opposite directions.
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).
Moreover, and in contrast to glucagon,
des-His1-[Glu9]glucagon amide showed a
similar potency for adenylyl cyclase stimulation on the wild-type and
the [H178R] receptor, respectively [EC50 (wild-type) =
52.8 ± 6.8 nM; EC50 [H178R] = 35.3
± 9.2 nM]. The maximal stimulation attained in
response to des-His1-[Glu9]glucagon amide was
reduced in comparison with the response exerted by glucagon on the
wild-type receptor (Fig. 2B). Consequently the analog would be
classified as a partial agonist. However, when acting on the
constitutively active glucagon receptor [H178R] maximal activity
exerted by the analog exceeded that obtained with the full agonist,
glucagon. Thus on this receptor the glucagon analog would be
characterized rather as a full agonist (or even a superagonist).
All four constructs mutated at the His178 position were
expressed at reduced levels compared with the wild-type receptor
(Bmax values in Table 1) and exhibited, accordingly, a
lower maximal level of stimulation (Fig. 2A). To further examine the
basal levels of cAMP production in relation to expression levels we
performed a gene dosage experiment (Fig. 4). For the wild-type and the [H178A]
receptors there was no detectable change (increase) in the basal level
of cAMP activity even at high expression levels, thus indicating a very
low inherent signaling activity of these receptors. This is in contrast
to the steep increment in basal cAMP level observed for the
constitutively active [H178R] receptor construct. Only the lower
range of expression levels could be assessed for the two other
constructs substituted at the His178 position, [H178K] and [H178E],
but no indication of constitutive activity was observed for these
receptors (data not shown).
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). This
resulted in rather pleiotropic effects, as reflected in distinctly
different phenotypes with regard to 1) ligand binding affinity, 2)
basal signaling activity, and 3) potency of glucagon for adenylyl
cyclase stimulation. Thus, for each of the three constructs, at least
one parameter differed distinctly from the corresponding wild-type
value. For the [T352S] construct, ligand binding affinity was reduced
to 45 nM (Table 1). For the [T352P] construct, the
potency of glucagon for cAMP signaling was decreased from 0.3
nM to 1.6 nM (Table 2), while the [T352A]
substitution was accompanied by 3.7-fold enhancement of the basal level
of adenylyl cycase activity (Fig. 2A). Analysis of these constructs at
increasing levels of expression affirmed the constitutive activity of
the [T352A] construct, albeit less so than that seen for the
[H178R] mutant (Figs. 2 and 4). However, the basal level of cAMP
production similar to the wild-type for the two constructs [T352S)
(Figs. 2 and 4) or [T352P] (Fig. 2).
Effect of [H178R] Substitution in N-Terminally Truncated
Receptors
To assess further the mechanism responsible for the constitutive
activity conferred by the [H178R] substitution, a set of receptors
was constructed in which the N-terminal extracellular domain had been
deleted. These constructs, 
N-term and N-term-[H178R], were, as
expected, deficient in peptide binding (Fig. 5, top panel) and showed no
glucagon-inducible accumulation of cAMP. However, the construct that
included the [H178R] mutation exhibited a constant and elevated basal
level of cAMP (Figs. 2 and 5), suggesting that the receptor domain
consisting of the TM segment and the intervening loops can mediate
signaling by itself, and that the His178 
Arg
substitution promotes this activity even in the absence of the presumed
main ligand-binding receptor domain of the receptor.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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According to the allosteric ternary complex model (19), 7TM receptors
exist in equilibrium between inactive (R) and active (R*)
conformational states, and the pharmacological property of a given
ligand may be viewed simply as the ability of that ligand, upon binding
to the receptor, to stabilize either the R or the R* state. Agonists
(A) thus act by virtue of stabilizing the receptor R* state.
Importantly, in this model active receptor complexes include bound
(R*-A) as well as unbound (R*) species, the latter representing the
constitutively active forms. In this two-state model, an increased
prevalence of active receptor states (as in constitutive active
mutants) is inseparably linked to an enhanced agonist affinity as was
indeed originally observed for the adrenergic receptors (19, 20).
However, as pointed out by Cotecchia, Costa and co-workers (28) in a
recent analysis of a series of 
1B-adrenergic receptor
mutants, the two parameters, basal activity and agonist affinity, may
in fact change independently upon mutagenesis. In that study, enhanced
agonist affinity was observed in a number of mutants, but not
necessarily accompanied by constitutive activation. To fully account
for these observations, a multi-state extension of the two-state model
was proposed. Central to this new model is the assumption that the
(macroscopic) functional states that are discerned experimentally could
be composed of ensembles of multiple different (microscopic) receptor
conformations, and importantly that the subsets of agonist-preferring
and active conformations, respectively, within this ensemble need not
be identical although they must clearly overlap. In the multi-state
model the preferential binding of an agonist therefore does not
explicitly imply the formation of an active state as reflected in, for
example, stimulation of adenylyl cyclase. The multi-state model thus,
in contrast to the two-state model, accommodates independent
changes in agonist affinity and basal and ligand-stimulated activity.
In the present analysis we have observed not only a lack of correlation
between agonist affinity and basal (constitutive) activity but, in
addition, disparate changes in agonist affinity and the potency for
receptor stimulation. The data therefore, when viewed in the context of
the multi-state model, suggest the existence of receptor conformations
that, although characterized by an enhanced agonist affinity, exhibit
no increase in basal activity and even a decreased ability to interact
productively with the G-protein as reflected in the rightward shift in
EC50 values. Together, these results emphasize receptor
binding and receptor activation as separate entities.
The observation that the [H178R] substitution, when present in a construct physically deleted for essentially the entire extracellular domain, is capable of conferring constitutive activity provides further evidence for the separation of binding and activation, as the TM domain of the receptor by itself is sufficient to establish an active conformation of the receptor. This notion further agrees with a series of chimeric studies employing pairs of receptors from this family. In these studies substitution and/or deletion of parts of the N-terminal domain disrupt high-affinity binding of the peptide (7, 8, 9, 10), as seen also after the mutation of a single residue, e.g. Asp64 in the glucagon receptor (29), or either of the six highly conserved cysteine residues that have been presumed to play at least an important structural role for this receptor domain (29, 30).
Distinct Activation Mechanisms for 7TM Receptor Subfamilies
In the PTH receptor, substitution of a series of residues, which
together could form a polar surface on the TM-II helix, suggested this
domain as being important for signaling (31). Substitution in the TM-II
domain in all instances impaired the ability of the receptors to
stimulate adenylyl cyclase. Notably, substitution of the highly
conserved histidine at the cytoplasmic pole of TM-II into either
aspartate or alanine was accompanied by an improved affinity for the
PTH peptide ligand, similar to the observation of the present study for
the His178 mutants. In the secretin receptor, similar
results were obtained after the exchange of the corresponding histidine
residue (32), i.e. improved ligand binding affinity.
However, for none of these receptor mutants was constitutive adenylyl
cyclase activity discerned. The strict requirement for exchange into a
particular side chain (e.g. His Arg) is in contrast to
observations in the adrenergic 1B-receptor, in which the
alteration of an aspartate at the bottom of TM-III (31a) or an alanine
at the bottom of TM-VI (20), respectively, led to enhanced receptor
activity irrespective of the choice for substitution. In the glucagon
and PTH receptors, the specific choice for substitution is seemingly
critical in determining whether or not basal receptor activity is
affected.
As summarized in Table 3, even identical
substitutions at homologous positions (here His‘178’
Arg) affect signaling properties dissimilarly among members of the
glucagon/VIP/calcitonin receptor family. In the PTH, the VIP, and the
glucagon receptors, substitution of histidine into argine was
accompanied by enhanced cAMP signaling (23, 32), yet in the GLP-1, the
GIP, and the calcitonin receptors this same histidine into arginine
substitution was neutral or even impaired the ability to signal through
adenylyl cyclase (33, 34). Furthermore, while a naturally occurring
TM-VI substitution in the PTH receptor enhanced the basal cAMP level,
signaling was barely affected upon introduction of the identical
substitution in the glucagon receptor in the present study. Rather, the
glucagon receptor substitution into an alternate residue,
i.e. alanine, provided the largest enhancement of signaling,
exemplifying once again the importance of the actual amino acid
substitutent present at these highly conserved positions, as recently
seen also for the PTH receptor (27). The distinctly different signaling
phenotypes arising from even identical exchanges in homologous
receptors was recently pursued in the rhodopsin-like gonadotropin
receptors. Here the exchange of a conserved residue in IC loop 3 had no
effect on the basal activity of the FSH receptor, whereas in the
homologous LH receptor this represents a naturally occurring mutation
associated with constitutive activation. Interestingly, when this
substitution was instead presented also in the context of the two
surrounding LH receptor segments, TM-V and TM-VI, activation of the
chimeric FSH receptor was indeed observed (35).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Expression of Receptor Constructs in COS-7 Cells
Receptor cDNAs were transiently expressed and introduced into
COS-7 cells by the calcium phosphate precipitation method using 40 µg
of plasmid DNA per 6 x 106 cells (39) except in the
gene dosage experiment in which variable amounts of cDNA were used for
transfection as indicated in the legend to Fig. 4. After addition of
the DNA-CaPO4 precipitate, cells were incubated in the
presence of chloroquine (100 µM) for 5 h. One day
after transfection the cells were harvested and seeded in 6-, 12-, or
24-well culture plates (1.0–7.5 x 104 cells per
well) for whole-cell analysis by radioligand competition binding.
Analysis of basal and ligand-stimulated cAMP synthesis was performed in
parallel on cells seeded in six-well plates at a density of
3.5–5.0 x 105 cells per well.
Radioligand Binding Analysis of Transfected COS-7 Cells
The transfected COS-7 cells were analyzed by radioligand binding
analysis on intact cells on the second day after transfection. The
cells were incubated for 16 h at 4 C in 1 ml of buffer consisting
of 25 mM Tris-HCl, pH 7.4, 5 mM
MgCl2, using 35 pM [125I]glucagon
as radioligand. Unlabeled peptide, used as a competitor, was present at
concentrations ranging from 10-11 to 10-5
M. The cells were lysed by the addition of 1 ml lysis
buffer (8 M carbamide, 3 M acetic acid, 2%
NP40), and specifically bound radioligand was calculated as the
difference between total counts of radioligand bound and counts bound
in the presence of 1 µM glucagon. Monoiodinated
[125I]glucagon, at a specific activity of 15 MBq/µg,
was kindly provided by Dr. Ulla Dahl Larsen (Novo Nordisk A/S,
Copenhagen, Denmark). The binding data were analyzed and
IC50 values determined by nonlinear regression analysis
using Inplot 4.0 (GraphPad Software, San Diego, CA). Kd,
inhibition constant (Ki) and Bmax values were
calculated from competition binding experiments using the equations:
Kd = IC50 - L (L is the concentration of free
radioligand), Ki = IC50/[1 + (L/Kd)], and
Bmax = Bo(IC50/L).
Peptides
The peptide ligands, glucagon and
des-His1-[Glu9]glucagon amide were kindly
provided by L. B. Knudsen (Novo Nordisk A/S). Peptides were
dissolved in 10-3 M acetic acid, 0.1% BSA for
use in competition binding analysis and cAMP determination.
cAMP Production
The assay was performed as a slightly modified version of the
method of Solomon et al. (40). Cells were cultured in
six-well plates (5 x 105 cells per well) and
incubated overnight with medium containing 2 µCi/ml of
[3H]adenine (Amersham TRK311). After two washes in HBS
buffer (25 mM HEPES pH = 7.2, 0.75 mM
NaH2PO4, 140 mM NaCl) 1 ml of HBS
containing 1 mM of the phosphodiesterase inhibitor
3-isobutyl-1-methylxanthine was added per well, and the cells were then
stimulated with peptide agonist for 15 min at 37 C. The cells were
chilled on ice, medium was removed, and the incubation was terminated
using 1 ml of ice-cold 5% trichloroacetic acid containing 0.1
mM of unlabeled cAMP and ATP. After 30 min incubation on
ice, the supernatants were applied first to a Bio-Rad 50W-X4 resin
(Bio-Rad, Richmond, CA) and subsequently to an alumina column (SIGMA
A9003; Sigma Chemical Co., St. Louis, MO). The [3H]cAMP
generated was eluted into scintillation tubes using 6 ml of 0.1
M imidazole, after which 15 ml of scintillation liquid
(HighSafe 3) were added and the samples counted. The levels of cAMP
production attained for each construct, agonist-induced as well as
basal levels, were normalized relative to that obtained for the
wild-type construct in the presence of 50 µM forskolin.
The maximum response for the wild-type construct during glucagon
stimulation (attained at 10-8 M) was
48 ± 5% of that obtained in the presence of 50 µM
forskolin.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This study was supported by grants from the Danish Medical Research Council, The Biotechnology Research Unit for Molecular Recognition, and the Novo Nordisk Foundation.
Received for publication June 19, 1997. Revision received September 16, 1997. Accepted for publication October 3, 1997.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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1B-adrenergic
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1B-adrenergic receptor:
Potential role of protonation and hydrophobicity of a highly conserved
aspartate. Proc Natl Acad Sci USA 94:808–813This article has been cited by other articles:
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