Cecilia G.

Unson
The Rockefeller University,
1230 York Avenue, Molecular Determinants of
Box 294,
New York, NY 10021
Glucagon Receptor Signaling

Abstract: A 29-amino acid polypeptide hormone, glucagon has been one of the most prolific
models in the study of hormone action. The key biologic function of glucagon is to counterbalance
the actions of insulin and maintain a normal level of serum glucose. Diabetes mellitus can thus be
considered a bihormonal disorder with an excess of glucagon contributing to the hyperglycemic
state. The effects of glucagon are mediated by the glucagon receptor, which is itself a prototypical
member of a distinct category called family B receptors within the G protein-coupled superfamily
of seven-helical transmembrane receptors (GPCRs). At the structural level, the peptide ligands of
family B receptors are highly homologous, in particular in the N-terminal region of the molecules.
The mechanism by which highly homologous peptide ligands selectively recognize their receptors
involves distinct molecular interactions that are gradually being elucidated. This review focuses on
structural determinants of the glucagon receptor that are important for its activity with respect to
interaction with its ligand and G proteins. Information about the glucagon receptor is presented
within the context of what is known about other members of the family B GPCRs. © 2002 Wiley
Periodicals, Inc. Biopolymers (Pept Sci) 66: 218 –235, 2002

Keywords: G protein-coupled receptors; glucagon receptor; peptide ligand binding; G protein;

family B GPCR; structure–activity; receptor chimeras; receptor binding model

INTRODUCTION ogous hormones: glucagon, glucagon-like peptide 1

The principal role of glucagon is to maintain normal
serum glucose concentration. Glucagon is a 29-amino
acid peptide hormone that is produced and secreted in
the A cells of the islets of Langerhans when glucose
levels fall below the physiological range. Glucagon
exerts its effects mostly in the liver, where it coun-
terbalances the action of insulin by stimulating gly-
cogenolysis, gluconeogenesis, and ketogenesis, a se-
ries of events that ultimately results in a rise in hepatic
glucose output.1 Biosynthetic studies have shown that
glucagon is synthesized as the 180-amino acid pre-
cursor molecule proglucagon, which is proteolytically
processed in specific tissues to produce three homol-
(GLP-1), and glucagon-like peptide 2 (GLP-2).2– 4
Glucagon is the major product of proglucagon pro-
cessing by prohormone convertase 2 (PC2) in the islet
A cells and GLP-1 and GLP-2 are produced in the
intestinal L-cells.5 In addition to a major role in glu-
cose homeostasis, glucagon is known to have a wide
range of effects in tissues other than the liver. Gluca-
gon receptor mRNA expression was also found to be
relatively abundant in adipose tissue, kidney, heart,
spleen, pancreatic islets, ovary, and thymus.6 In the
pancreas, glucagon potentiates glucose-induced insu-
lin release from B cells.7,8 Glucagon also has a role in
free fatty acid metabolism and induces lipolytic re-
sponse in adipocytes.9 Glucagon has been shown to

Correspondence to: C.G. Unson; email: unsonc@mail.

rockefeller.edu
Biopolymers (Peptide Science), Vol. 66, 218 –235 (2002)
© 2002 Wiley Periodicals, Inc.

218
 Molecular Determinants of Glucagon Receptor Signaling
exert a positive inotropic effect on the heart and
regulate renal function.10,11 Lower expression levels
of receptor mRNA were detectable in skeletal muscle,
adrenal glands, stomach, small intestine, and brain,
signifying that novel activities of glucagon have yet to
be determined.
The driving force for continued efforts to study
glucagon lies primarily in the still unresolved problem
of diabetes mellitus, which ranks among the most
widespread diseases of our time, afflicting 1–2% of
the population of the Western world. The careful
regulation of plasma glucagon and insulin levels is
seriously impaired in patients with either insulin-de-
pendent (IDDM) or non-insulin-dependent (NIDDM)
diabetes mellitus. A two-hormone abnormality hy-
pothesis has been enlisted to account for the observa-
tion that the major metabolic complications of diabe-
tes, impaired glucose tolerance and ketoacidosis, are
often accompanied by an abnormal increase in the
level of plasma glucagon relative to insulin.12,13 Thus,
glucose underutilization due to insulin deficiency and
overproduction of glucose by elevated physiological
concentrations of glucagon both contribute to hyper-
glycemia and implicate a role for glucagon in the
pathogenesis of diabetes.
Consistent with these observations, a structural
analog of glucagon that inhibits glucagon action by
competing effectively for receptor binding sites is a
reasonable target for drug design as an alternative or
adjunct to insulin therapy in the management of dia-
betic complications. In addition, genetic evidence has
led to the proposal that a defect in the glucagon
receptor that mediates the actions of glucagon might
constitute a genetic basis for the disease. A single
heterozygous missense mutation in exon 2 of the
glucagon receptor gene that changes a glycine to a
serine (Gly40Ser) was reported to be associated with
late-onset NIDDM in some populations.14,15 Subse-
quent expression of a cDNA containing a Gly40Ser
single-point mutation in the extracellular region of
GR produced a mutant receptor with decreased ligand
binding affinity and attenuated signaling response to
glucagon. Thus, a full understanding of the interaction
between glucagon and its receptor would confirm
their involvement in the pathogenesis of diabetes and
might lead to effective therapeutics.
GLUCAGON RECEPTOR
The glucagon receptor (Figure 1) is a member of the
superfamily of G protein-coupled receptors (GPCRs),
219
which constitutes approximately 1–2% of the total
number of genes in the human genome. A cDNA
encoding the rat glucagon receptor was isolated from
rat liver mRNA by an expression cloning strategy.16,17
The human glucagon receptor (hGR) cDNA obtained
from a liver library by a combination of polymerase
chain reaction (PCR) and colony hybridization en-
codes a 477-residue protein that is 80% identical to rat
GR in its primary structure.18 Fluorescence in situ
hybridization to metaphase chromosome preparations
mapped the gene encoding the human glucagon re-
ceptor to chromosome band 17q25.19,20 Analysis of
the genomic sequence shows that the coding region
spans over 5.5 kb and is interrupted by 12 introns,
allowing for alternative splicing and receptor hetero-
geneity.21 However, because no alternate products
were detected by RT-PCR analysis, the glucagon re-
ceptor expressed in all tissues appears to exist as one
form and derives from the same gene.22,23
The biologic effects of glucagon are initiated by
high-affinity binding to its membrane-bound receptor.
Upon activation of a GPCR, a heterotrimeric G pro-
tein transduces the signal from the interior of the cell
membrane to activate cellular signaling pathways.
The activated receptor induces a conformation change
in the associated G protein, and, following GTP ex-
change for GDP, the GTP-bound ␣-subunit of the G
protein, G␣GTP, dissociates from the receptor and
from the ␤␥ subunits. Both G␣GTP and the released
␤␥ dimer can modulate several downstream effector
pathways. Glucagon receptors activate adenylyl cy-
clase via the heterotrimeric G protein Gs to produce
cAMP, which mediates most of glucagon’s cellular
effects. However, it is now known that glucagon
activates alternative signaling pathways, leading to
intracellular accumulation of additional second mes-
sengers, inositol triphosphates (IP3) and free Ca⫹2.24,25
It has been suggested but not proven that the glucagon
receptor is coupled to the phosphoinositide signaling
pathway via the G protein Gq or G11. Subsequent
work has shown that glucagon-induced increase in
intracellular [Ca⫹2] results from activation of PKA
and thus is mediated by an increase in cyclic AMP
concentration.26 The generation of these second mes-
sengers triggers other physiological effects via a com-
plex synergism among effector pathways that remain
to be sorted out. Emerging evidence show that GPCR
signaling previously thought to be sequential but uni-
directional involves cross-activation and concurrent
regulation between different GPCRs and other path-
ways.
Recently, a stable cell line expressing glucagon
receptors activated the mitogen-activated protein ki-
nase (MAPK) pathway upon glucagon treatment,
220 Unson

FIGURE 1 Schematic representation of the rat glucagon receptor primary and secondary struc-
tures. Amino acid residues are depicted in single-letter code. The extracellular domain consisting of
the amino terminal tail and extracellular loops e1, e2, and e3 is at the top, and the cytoplasmic
domain consisting of the carboxy terminal tail and intracellular loops i1, i2, and i3 is toward the
bottom. The red arrow indicates a putative signal sequence proteolytic site. The four sites of
potential N-linked glycosylation on the N terminus are labeled with asterisks. Six of the cysteines
in the N terminus are conserved in family B GPCRs and are involved in a pattern of three disulfide
bonds shown in orange to link Cys44 and Cys68, Cys59 and Cys101, and Cys82 and Cys122. An
essential disulfide bridge between extracellular loops e1 and e2 is a conserved feature in all GPCRs,
and is depicted here between Cys225 and Cys295. The seven TM ␣-helical segments I to VII are
shown in blue cylinders.

causing a dose-dependent phosphorylation of extra- to be Gs-mediated and involved a concerted action of

cellular signal-regulated protein kinases (ERK1/2).27
While G protein-coupled receptors lack intrinsic ty-
rosine kinase activity, G proteins can couple GPCRs
to MAP kinases, which are key components of a
signaling pathway that controls cell proliferation.28,29
The transactivation of MAPK by glucagon appeared
glucagon-induced intracellular [Ca2⫹] elevation and
cAMP-dependent protein kinase A. The specific bio-
chemical routes that switch a glucagon-dependent sig-
nal from a G protein-dependent pathway to a signal-
ing cascade used by receptor tyrosine kinases have not
been determined.27
 Molecular Determinants of Glucagon Receptor Signaling
GLUCAGON RECEPTOR STRUCTURE
GPCRs do not share overall sequence homology, but
significant sequence homology is conserved within
each of the five GPCR subfamilies.30,31 The isolation
of the cDNA of rat and human glucagon receptors
confirmed that it is a prototype of family B, a unique
class of receptors within the GPCR superfam-
ily16,18,32,33 (Figure 1). Members of family B GPCRs
include receptors for structurally related peptides glu-
cagon-like peptide 134; glucagon-like peptide 235; se-
cretin36; vasoactive intestinal peptide37,38; gastric inhib-
itory peptide39; growth hormone releasing hormone40;
pituitary adenylyl cyclase-activating peptide41; as well
as receptors for peptides unrelated to glucagon such as
calcitonin42; calcitonin gene-related peptide43; PTH/
PTH-related peptide44,45; and corticotropin-releasing
factor.46 The molecular basis by which these receptors
recognize their peptide ligands and convey the signal
across the membrane to G protein-coupled effectors
inside the cell is still largely unknown.
GPCRs have in common a central core consisting
of seven membrane-spanning helices connected by
alternating extracellular and intracellular loops, along
with an amino-terminal extracellular domain and an
intracellular carboxyl terminal extension. The extra-
cellular domains typically contain consensus sites for
asparagine-linked glycosylation. The transmembrane
(TM) domains form a seven-helical bundle oriented
roughly orthogonal to the plane of the membrane in a
counterclockwise configuration. A characteristic di-
sulfide bridge that connects extracellular loops 1 and
2 is found in all GPCRs and is probably important for
packing and stabilization of the central core. This
basic arrangement was validated in the 2.8-Å resolu-
tion structure of rhodopsin, the first 3-D structure of a
GPCR.47,48 The most conserved residues among
GPCRs are located within the TM domains and rep-
resent essential structural determinants of receptor
structure and function. For all GPCRs, receptor acti-
vation is associated with a change in conformation
within this core domain. Other domains are not as
conserved and sequence variations in the extracellular
and intracelullar domains bestow specific properties
to each receptor to regulate molecular events leading
to this change.
The most conserved family B receptors do not
contain any of the hallmarks that characterize family
A receptors. Notably the Asp-Arg-Tyr (DRY) motif is
absent and the prolines conserved among family A
receptors are distinct from those conserved in family
B receptors. The most prominent feature of family B
GPCRs is a long N-terminal domain of 100 –200
amino acids, which contains six conserved cysteines
221
that are presumed to form a complex network of three
disulfide bridges. The human glucagon receptor
cDNA encodes a 477-aa protein with a putative 27-
amino acid signal peptide that is presumably cleaved
between A27 and Q28 to yield a mature 450-aa recep-
tor, consisting of a 121-aa extracellular amino-termi-
nal domain, a 404-aa seven-helical core, and a 73-aa
intracellular carboxyl-terminal domain. The conflu-
ence of the extended N-terminus and the three extra-
cellular loops is believed to embody the hormone
binding site.
RECEPTOR–G PROTEIN INTERACTION
As for all GPCRs, the binding of glucagon to GR is
thought to trigger conformational changes in the re-
ceptor that constitute the common activation mecha-
nism. These changes might first involve reorganiza-
tion of the extracellular loops to accommodate the
ligand, followed by movement of the TM domains at
the central core of the receptor. It is the modification
of interhelical constraints comprised of electrostatic
and hydrophobic interactions that subsequently affect
the cytoplasmic side of the receptor and enable spe-
cific regions of the intracellular loops to interact with
G proteins. Among family A GPCRs, one major in-
tramolecular constraint was found to be interactions
surrounding the highly conserved E(D)RY(W) motif.
In the crystal structure of rhodopsin, E134R135Y136 are
involved in hydrogen bonds and a salt bridge and a
reorientation of interactions in this region occurs dur-
ing activation.47 A closer look at family B GPCRs
reveals that the DRY counterpart could be furnished
by a conserved R (R174 in GR) in the intracellular end
of TM II predicted to be at the same level in the
membrane as the R in DRY.49 The E(D) function is
predicted to align with an ExxY (E246 in GR) motif in
the intracellular end of TM III.49 The conserved res-
idues Y239 and L240 of human VPAC1 receptor are
predicted to be at the same location as the asparagine
and arginine in the “DRY” motif in the rhodopsin
family of GPCRs. The VPAC1 receptor mutants
Y239A had a moderate and L240A a pronounced
impaired ability to produce cAMP, suggesting that the
effect of these mutants is a perturbation of the G
protein interaction.50 Thus, the different chemical
properties of the hydrophobic YL motif and charged
DR(Y) motif could be a crucial difference between
the family B GPCRs and the rhodopsin family with
respect to receptor activation and G protein coupling.
The general topography of GPCRs necessitates
that receptor G protein-coupling domains lie within
222 Unson
sequences of the cytoplasmic domains, specifically
regions close to the plasma membrane at the ends of
the TM helices. It is well known that the second (i2)
and third (i3) intracellular loops of GPCRs are closely
involved in coupling to G proteins and in determining
specificity toward a particular G protein class. The
change in conformation of the helical bundle in gen-
eral affects the disposition of the i2 and i3 loops, both
of which are directly connected to TMIII and TMVI.
It has been proposed that the C-terminus of the G
protein ␣-subunit binds in a pocket formed by these
intracellular loops. The residues in the first intracel-
lular loop of family B receptors are highly conserved,
which may indicate an essential role for these residues
in protein folding and in surface expression of the
receptors. In GR, the long C-terminal tail does not
contribute significantly to ligand binding or G protein
coupling. Truncation of variable portions of the
COOH terminal of GR had no effect on binding and
glucagon-stimulated cAMP accumulation, or Ca2⫹
mobilization.51,52 Indeed, the absence of the tail
slightly increased receptor affinity for glucagon, per-
haps by making the intracellular loops more accessi-
ble for coupling to G proteins.51 However, the C-
terminal tail appears to be involved in mechanisms
that regulate the functional level and activity of GR.
Glucagon-induced phosphorylation of serine residues
in the cytoplasmic C-terminal tail is crucial for recep-
tor internalization.53
All members of the glucagon family of receptors
couple to Gs to activate adenylyl cyclase and elevate
intracellular cAMP concentration. At high nonphysi-
ological concentration (⬎10⫺5), glucagon inhibits the
accumulation of cAMP induced by isoproterenol, an
effect that is attenuated by pertussis toxin, suggesting
involvement of Gi.54 Glucagon-stimulated GR also
results in mobilization of intracellular Ca⫹2 stores and
the breakdown of inositol lipids to produce inositol
phosphates and diacylglycerol. Recently, it was
shown that glucagon activates the MAP kinase path-
way via a cAMP-dependent pathway in a cell line
stably expressing GR.27 In most GPCRs, activation of
phospholipse C and the activation of the MAP kinases
are mediated by Gq or G11. While it is well established
that GR couples to Gs to generate cAMP, the involve-
ment of Gq with activated GR is less clear. In earlier
studies, a glucagon antagonist, (1-N-alpha- trinitro-
phenylhistidine,12-homoarginine)glucagon (TH-glu-
cagon), which does not activate adenylyl cyclase, was
shown to stimulate the production of inositol phos-
phates.24,55 These observations initially suggested ei-
ther that hepatocytes possessed two distinct receptors
for glucagon, one coupled to adenylyl cyclase activa-
tion and another coupled to the phospholipase C path-
way, or, more likely, that glucagon receptors were
activating two signaling pathways via different G
protein species.24 Subsequent mutagenesis of the cy-
toplasmic domains of GR demonstrated that the sec-
ond and third intracellular loops, i2 and i3, synergis-
tically contribute determinants required for activation
of Gs.56 Deletion or replacement of residues from each
of either the i2 or i3 loops resulted in a marked
attenuation of the ability of glucagon to activate ad-
enylyl cyclase.56,57 Replacement of both the i2 and i3
loops of GR with a sequence from the i1 loop of the
unrelated D4 dopamine receptor in the receptor mu-
tant G23D1R resulted in the abrogation of all gluca-
gon-dependent activities.27,56 Consequently, GR ap-
pears to regulate its primary signal transduction path-
ways by faithfully coupling only to Gs.
GR is known to exhibit both high and low binding
affinities for glucagons, which is thought to be due to
a heterogeneous population of receptors that are mod-
ulated by association with Gs, even prior to ligand
binding.58 – 60 Variations in the relative densities of
receptors and G proteins in turn influence effector
coupling and the efficacy of activation.21 Two pre-
dominant splice variants of G␣s, a short and a long
form (G␣s-S and G␣s-L) resulting from differential
splicing of a single precursor RNA, exist in most
cells, but their relative proportions vary.61– 63 To as-
sess functional differences in the interactions of the
GR with the two predominant splice variants of G␣s,
GR was covalently linked to the short and the long
forms, G␣s-S and G␣s-L, to produce the fusion pro-
teins GR-G␣s-S and GR-G␣s-L.64 GR-G␣s-S bound
glucagon with an affinity similar to that of GR, while
GR-G␣s-L showed a 10-fold higher affinity for glu-
cagon. Both GR-G␣s-L and GR-G␣s-S were consti-
tutively active, causing elevated basal levels of cAMP
even in the absence of glucagon. The mutant GR that
failed to activate Gs (G23D1R) was fused to G␣s-L.
G23D1R-G␣s-L bound glucagon with high affinity
but failed to elevate cAMP levels, suggesting that the
mechanisms of GR-mediated G␣s-L activation and
G␣s-L-induced high-affinity glucagon binding are in-
dependent.64 Both GR-G␣s-S and GR-G␣s-L bound
the glucagon antagonist desHis1[Nle9Ala11Ala16]-
glucagon amide with similar affinities as GR.64 The
antagonist displayed partial agonist activity with GR-
G␣s-L, but not with GR-G␣s-S, proving that the par-
tial agonist activity of the antagonist observed in
intact cells appears to be due to GRs coupled to
G␣s-L. That G␣s-S and G␣s-L isoforms appear to
interact differently with GR has physiological impli-
cations and emphasizes that the efficacy and potency
of ligands are regulated not only by the receptor but
also by the type and relative amounts of G proteins
 Molecular Determinants of Glucagon Receptor Signaling
FIGURE 2 Comparison of sequences of peptide ligands of the glucagon receptor family. The
most invariant residues are located in the N-terminal part of the peptides. Based on homology
between both receptors and ligands, the conformation of receptor-bound ligand may be similar.
accessible to the receptor molecules. Receptors asso-
ciating with different isoforms of G␣s or other G
proteins may vary in their ability to isomerize to the
activated state and thus differ in their affinity for
agonists and antagonists and provoke a graded range
of potencies. Some endogenous GPCRs exhibit de-
tectable activity even in the absence of agonist, an
occurrence frequently associated with pathogenic
states. This can be accounted for by mutations in the
cytoplasmic domains that facilitate isomerization of
the receptor to the active conformation. There are no
known constitutively active GR mutants except for
one report in which His178 in the i1 loop, which is
strictly conserved in family B receptors, was replaced
with an arginine.65 The corresponding mutation in the
PTH-PTHrP and VIP1 receptors exhibited constitu-
tive activation of Gs.66,67 In the PTH-PTHrP receptor,
this mutation was identified in a patient with Jansen-
type metaphyseal chondrodysplasia characterized by
the severe ligand-independent hypercalcemia and hy-
pophosphatemia and a rare form of dwarfism.66 Con-
stitutively active GPCRs allow the study of ligands
that elicit inverse agonism or lowering of the basal
activity. Thus, the constitutive activity of GR-G␣s-L
will be useful in assessing novel glucagon analogs for
inverse agonist activity.
LIGAND–RECEPTOR INTERACTION
The peptide ligands of family B receptors are highly
homologous to each other, yet each will activate a
specific receptor to elicit the appropriate physiological
response (Figure 2). Despite the initial suggestion that
the C-terminal part of the peptide is primarily in-
volved in receptor recognition and binding while the
N-terminal half is important for transduction, the gen-
eral picture that has emerged is that the active phar-
macophore is dispersed throughout the glucagon mol-
ecule. Numerous structure–function studies of gluca-
gon have singled out strategic positions throughout
the peptide ligand in which the amino acid residues
223
influence either binding affinity or activation and af-
forded some insight into the molecular requirements
of the receptor for optimum recognition and interac-
tion. The negatively charged side-chain of the aspartic
acid residue at position 9 is absolutely required for
receptor activation and less for binding, so that re-
placement with any other amino acid resulted in a
glucagon antagonist.68,69 The N-terminal histidine,
which is strictly conserved within the glucagon fam-
ily, furnishes determinants of both binding and activ-
ity of the hormone.70 –72 Aspartic acids at positions 15
and 21 also regulate the binding or activity function of
the hormone.69,73 The positively charged groups at
positions 12, 17, and 18 stabilize the binding interac-
tion to ensure maximum potency.74 –76 In addition, the
crystal structure of the peptide superagonist [Lys17,18,
Glu21]glucagon amide and cyclic analogs of glucagon
suggest that an intramolecular salt bridge between
side-chains of aspartic acid and lysine may be a fea-
ture of the active conformation of the hormone.75,77
These studies confirmed that even the flexible middle
portion of the peptide ligand between positions 12 to
21 is important for recognition and transduction. In
addition to electrostatic interactions, a topographical
requirement for the proper stacking of hydrophobic
residues Phe6 and Tyr10,13 in glucagon has also been
proposed.78 – 80 Collectively, structure–function stud-
ies of glucagon confirmed that virtually the entire
hormone is necessary for the expression of full hor-
monal activity.
HORMONE BINDING SITE
While extensive structure–activity studies of glucagon
have identified specific residues in the peptide ligand
that play a role in binding and transduction, the com-
plementary recognition site that is the “switch” on the
receptor has yet to be deduced. Members of the glu-
cagon family of peptide hormones are 50% identical
and more than 70% homologous (Figure 2). The
structural basis by which the receptors discriminate
224 Unson
among similar ligand structures most likely lies in
structural subtleties within the receptors themselves.
Structure–function studies of related family B recep-
tors indicate that the peptide ligand binding site is
discontinuous and consists of contact points from the
extended N-terminus and the three extracellular loops
connecting the TM helices.81– 85 This observation was
further corroborated by cross-linking experiments that
have mapped the peptide ligand binding site to the
extracellular domain of the receptors.86 –90
The putative 121-residue N-terminal domain of
GR contains six cysteine residues, which are strictly
conserved within family B members and are pre-
sumed to form three specific disulfide bonds. The
precise disulfide connectivity was determined for the
N-terminal fragment of human parathyroid hormone
receptor (PTHR), for the corticotropin releasing factor
(CRF) type 1 receptor, and, most recently, for GLP-
1R,91–93 suggesting a conserved structural pattern in
the N-terminal domain of family B receptors. Treat-
ment with cell non-permeabilizing reducing reagents
or mutagenesis of the cysteine residues to alanine had
adverse effects on receptor binding, validating the
critical importance of the complex, highly folded,
disulfide arrangement to family B receptor func-
tion.94,95 Based on the known disulfide-linking pattern
of these family B receptors, the analogous disulfide
bonds in rat GR can be predicted to occur between
cysteines 44 and 68, 59 and 101, and 82 and 122
(Figure 1).
Mutagenesis studies of family B GPCRs have
shown direct involvement of the N-terminal domain
in peptide ligand binding. A GR mutant with a 96-
residue deletion from the N-terminal tail, including 5
of the 6 conserved cysteine residues, was expressed
on the cell surface but failed to bind glucagon, em-
phasizing that the integrity of the tertiary structure
was crucial for high-affinity glucagon binding.51 Sin-
gle point mutations throughout the N-terminal tail of
family B receptors have resulted in either complete
loss of binding or an attenuation of the activity func-
tion. Photoaffinity cross-linking and mutational data
showed that residues at the extreme amino-terminus
of the PTH receptor participate in ligand binding.96 In
GR, single amino acid replacements of D64 resulted in
GR mutants that failed to bind glucagon.97 This site in
the related growth hormone releasing factor receptor
was shown to be responsible for the little mouse (lit)
genetic defect that results in mice of small size with
hypoplastic pituitary glands.98 A G40S GR mutant
exhibited attenuated adenylyl cyclase activity and has
been linked to late-onset NIDDM in some popula-
tions.14,15 Five out of six tryptophan residues in the
N-terminal domain of GLP-1 receptor are essential for
its ability to bind GLP-1. In particular, the replace-
ment of W39 in GLP-1R led to loss of receptor func-
tion.85,99 More recently, a nonpeptide ligand was
shown to bind to the human GLP-1R and inhibit
GLP-1-induced cAMP production by targetting
W.33,100 Most of these tryptophan residues are con-
served in both rat and human GR and undoubtedly
play a similar role in the binding of glucagon to GR.
The N-terminal domains of the human parathyroid
hormone, PACAP, GLP-1, and CRF type 1 receptors
have been expressed, correctly refolded, and puri-
fied.84,91–93,101 The soluble proteins corresponding to
the receptor N-terminus manifested significant ligand
binding activity. In particular, the CRF-R1 N-terminal
fragment bound a CRF agonist and antagonist just as
avidly as intact wild-type CRF-R1, indicating that the
major determinants for high-affinity binding of CRF
are found in this domain.92,102 Nonetheless, the bind-
ing affinities of the isolated N-terminal fragments of
PTHR, PACAPR, and GLP-1R for their ligands were
sufficiently weak to infer that the N-terminal domain
alone cannot account for the binding affinity of an
intact receptor.84,91,93,101 In addition, when the entire
N-terminal domains of the glucagon and the GLP-1
receptors were exchanged, glucagon or GLP-1 bound
to neither the GR-GLP-1R nor the GLP-1R-GR chi-
mera.103 The accepted view that has emerged from
these studies is that the N-terminus is an important
part of the peptide binding pocket but is not adequate,
requiring supplementary contributions from residues
located in the extracellular loops and in the upper
portion of the TM helices close to the intradiscal
membrane bilayer interface.
The partial boundaries of the glucagon binding site
on the glucagon receptor were demarcated using anti-
GR antibodies (Figure 3). Antibodies DK-12 and
KD-14 generated against synthetic peptides with
amino acid sequences corresponding to residues 126 –
137 of the N-terminal tail and 206 –219 of the first
extracellular loop, respectively, competed with gluca-
gon for receptor binding binding sites.104 In contrast,
ST-18 antibody raised against an 18-residue peptide
from the C-terminal end of GR located in the cyto-
plasmic domains had no effect on the binding of
glucagon. DK-12 and KD-14 also behaved like glu-
cagon antagonists, preventing glucagon-dependent
activation of adenylyl cyclase.104 Although the results
indicate that the antibodies bind to the glucagon bind-
ing site in GR, the exact nature of the competition
cannot be specified. It is possible that antibody bind-
ing causes an effect from a distance and sterically
displaces bound glucagon. The peptide epitope may
actually be part of the binding interface or may be a
sequence spatially close to the binding site. Nonethe-
 Molecular Determinants of Glucagon Receptor Signaling 225

FIGURE 3 Schematic representation of the GR showing the locations of the peptide segments that
were used to generate polyclonal antibodies to GR. PR-15 and DK-12 were raised against amino
acid residues 103–117 and 126 –137 of the N terminus, respectively. KD-14 was raised against
residues 206 –219 of the e1 loop. ST-18 was raised against C-terminal residues 468 – 485. Antibod-
ies DK-12 and KD-14 competed with glucagon for GR binding and inhibited glucagon-dependent
receptor activation. A series of chimeric receptors in which the corresponding peptide epitopes were
each replaced with their counterparts from GLP-1R and SR indicated that the sequences played a
role in GR binding and activation.

less, the antibody studies were consistent with the
idea that the glucagon binding domain involves part
of the N-terminal tail close to the junction at which
the first TM domain begins and includes the first
extracellular loop.
Chimeric constructs of structurally related recep-
tors have been used to correlate a specific structural
domain with function and identify determinants of
ligand binding affinity and selectivity. A chimeric
receptor in which residues 29 –32 of the N-terminus of
the GLP-1 receptor were replaced with the corre-
sponding amino acids from GR showed a 10-fold
decrease in affinity for GLP-1 and an increase in
affinity for glucagon, representing a 50-fold decrease
in the selectivity of this receptor for its natural li-
gand.105 A composite of findings utilizing glucagon/
GLP-1 receptor chimeras demonstrated that the glu-
cagon binding pocket is a discontinuous domain that
encompasses the membrane-proximal region of the
amino-terminal extension and the first extracellular
loop, further corroborating the GR antibody studies.81
In addition, the chimeric receptor studies revealed that
glucagon binding was sensitive to replacement of TM
helices III, IV, and VI with the corresponding se-
quences from GLP-1R, indicating that amino acid
residues within these TM domains, in particular those
located close to the lipid and aqueous interphase, are
in contact with ligand during the binding event.81
A receptor chimeric study was conducted to exam-
ine the role of these domains in greater detail. Resi-
dues 103–117 and 126 –137 of the N-terminus and
206 –219 and 220 –231 of the first extracellular loop
were each replaced with the corresponding sequences
from the GLP-1 receptor or secretin receptor (SR).106
Evidence that polyclonal antibodies against peptides
representing sequences 126 –137 and 206 –219 effec-
tively blocked glucagon binding strongly indicated
that these domains were involved in ligand bind-
ing.104 Receptors for GLP-1 and secretin are the clos-
est relatives of GR within family B GPCRs, sharing
50% overall identity in their primary structures. In-
terestingly, the substituted sequences represented
nonconserved areas in these receptors and implied
that these domains may also embody determinants of
ligand selectivity. The chimeric receptor study
showed that these segments indeed played a role in
glucagon binding.106 Segment substitutions within the
first extracellular loop led to a greater detrimental
effect on both binding and activity than either replace-
ment of residues 103–117 or 126 –137 in the mem-
brane proximal region of the N-terminal extension,
supporting the notion that the N-terminal domain of
GR is not sufficient for glucagon binding. Replace-
ment with e1 sequence from SR had a more detrimen-
tal effect than a sequence from GLP-1R, which sug-
gested that glucagon recognized structural patterns in
the sequence from the e1 loop of GLP-1R that were
not contained in the analogous SR sequence.
A cross-chimeric analysis was employed to ad-
dress the possibility that the homologous peptide hor-
226 Unson
mones GLP-1 and glucagon may interact with the
same regions in their respective receptors. A peptide
hybrid comprising the N-terminal 1–14 residues of
glucagon linked to the C-terminal 15–31 residues of
GLP-1 was shown to possess the chemical features
necessary for selective recognition by both recep-
tors.107 The e1 loop chimera bearing a sequence from
GLP-1R exhibited greater potency toward the GLP-
1/glucagon chimera relative to glucagons, suggesting
both GLP-1 and glucagon associate with amino acid
residues in the e1 loop of their respective receptors
during binding and activation.106 This observation is
consistent with the finding that residues located at the
N-terminal half of glucagon predominantly determine
selective recognition of GR. Conversely, the selective
binding epitope of GLP-1 is primarily located near the
C-terminus.
Residues 206 –219 of the first extracellular loop are
most likely situated in the central portion of the e1
loop. Single substitution mutagenesis of the charged
residues within the 206 –219 stretch failed to mimic
the phenotype of the entire 206 –219 amino acid sub-
stitution, suggesting that the effect may be conforma-
tional or hydrophobic in nature.106 In the human
PTH1 receptor (hPTHR), a leucine (L261) located in
the central helical portion of the e1 loop has been
identified as the contact site for K27 of PTH(1-
34).108,109 Replacement of the adjacent 220 –231 res-
idues that extend to the border of TM helix III of GR
did not seriously impair high-affinity binding to glu-
cagon. The central portion of the e1 loop is important
in glucagon binding but residues 220 –231 near helix
III are not. A conserved cysteine in this segment
(C225) is likely to be involved in a disulfide bridge
with C295 of the second extracellular loop (e2 loop). A
disulfide bond joining the e1 and e2 loops is a struc-
tural feature that is strictly conserved in both family A
and family B GPCRs.30 In the crystal structure of
rhodopsin, a disulfide bridge between C110 and C187
draws the e2 loop away from the surface and extends
it over a crevice in the seven-helix bundle.47,110,111
The analogous disulfide bridge in GR that occurs
between C225 of the e1 loop and C295 of the e2 loop
may restrain the 220 –231 segment of the e2 loop and
pull it toward the closely packed region at the top of
the helices, an area not easily accessible to a peptide
ligand the size of glucagon. Indeed, a receptor chi-
mera in which residues 220 –231 were replaced re-
tained glucagon binding affinities similar to that of
wild-type GR but exhibited a disproportionate loss in
the ability to transduce the signal. A disproportionate
change in agonist potency vs. binding affinity indi-
cates that the 220 –231 region contributes less directly
to ligand binding affinity but may instead provide
molecular contacts that trigger activation.106
Single substitution mutagenesis of the e1 loop fur-
ther revealed a critical requirement for a positive
charge at the extracellular border of TM helix II at the
junction with the e1 loop. Replacement of R202 with a
negatively charged aspartic acid in R202D resulted in
a complete loss of receptor function.106 Although a
positively charged group is required to define the
topology of a TM helix within the bilayer, it is also
possible that by replacing R202 an important associa-
tion with specific residues of the ligand was lost. The
importance of amino acid residues H1, S8, D9, D15,
S16, and D21 of glucagon for either binding or activa-
tion has been established.69,70,72,112,113 Any of these
amino acids residues could contribute to glucagon
binding energy through ionic or hydrogen bond for-
mation with R202. In vasoactive intestinal peptide
(VIP), D3 was shown to contact an arginine and a
lysine residue at analogous positions at the extracel-
lular end of TM helix II of the VIP receptor.114,115
Alternatively, arginines can interact with aromatic
side-chains of phenylalanine, tyrosine, or tryptophan
through cation ␲-orbital interaction. This potent non-
covalent binding force occurs at or near the surface of
proteins and contributes to ligand–receptor interac-
tion.116 –118
BINDING MODEL FOR FAMILY B
GPCRS
The ultimate goal of the different approaches to study
the molecular mechanism of ligand-specific recogni-
tion, binding, and receptor activation is to determine
the structure and conformation of the peptide ligand
while bound within a ligand–receptor complex and
generate a template for rational drug design. Difficul-
ties in the expression and solubilization of family B
receptors have not provided practical amounts of pu-
rified receptors required for structural studies. Re-
cently, the conformation of an analog pituitary adeny-
lyl cyclase activating polypeptide PACAP(1-21),
bound to a PACAP–specific receptor, was determined
by nuclear magnetic resonance (NMR) spectroscopy.119
This is the first structure determination of a ligand
peptide bound to a GPCR, which provided a unique
insight on the structural basis for how a receptor
recognizes its ligand. The PACAP receptor was ex-
pressed in Sf9 insect cells, solubilized in digitonin,
and purified by affinity chromatography.120 The struc-
ture revealed a unique ␤-coil structure formed by
residues 3–7 next to an N-terminal extended tail. The
␤-coil creates a hydrophobic cluster made up of the
 Molecular Determinants of Glucagon Receptor Signaling 227

FIGURE 4 Working model for the binding and activation of family B GPCRs. The mechanism
is proposed to involve initial association of the peptide with the membrane, followed by recognition
and capture of the C terminus of the ligand by the N-terminus of the receptor, which reins in the
N-terminus of the peptide toward the extracellular loops and the helical bundle. Both ligand and
receptor undergo conformational changes that subsequently lead to activation.

side-chains of I5, F6, and Y10, together with an R14. A
G4 is necessary for the formation of a type II’ ␤-turn,
which is important in maintaining the relative orien-
tation of the hydrophobic patch and the extreme N-
terminus. In contrast, the C-terminal region of recep-
tor-bound PCAP21 forms an ␣-helix similar to its
structure in micelles.119 At the structural level, pep-
tide ligands of family B GPCRs are highly homolo-
gous, in particular in the N-terminal domain of the
molecules where the characteristic ␤-coil structure is
induced upon receptor binding. Based on the homol-
ogy between both receptors and ligands, the structural
features that were found in receptor-bound PACAP21
may parallel those found in ligands belonging to the
glucagon branch of family B GPCRs. Indeed, the
NMR structure of glucagon in a micelle–water inter-
face showed that residues 17–29 of glucagon form an
amphiphilic helix.121 Synthetic analogs containing
amino acid replacements that increased the ␣-helical
character of the C-terminus displayed improved bind-
ing affinities.122,123 Structure–activity studies have
proposed a role for a hydrophobic patch in glucagon
and emphasized that proper stacking of F6, Y10, and
Y13 was important for binding and transduction.78 – 80
More importantly, the fact that H1, G4, F6, T7, and Y10
are invariant in the N-terminal part of both PACAP
and glucagon strongly implies that glucagon may
behave similarly in the presence of its receptor.
Based on a composite of structure–function anal-
yses of single replacement and chimeric receptor mu-
tants as well as analogs of the peptide ligands, tenta-
tive models have been proposed for the binding of
family B receptors to their peptide ligands.103,108 One
model proposes a sequential process that involves
low-affinity association and initial capture of the pep-
tide by the extracellular N-terminus of the receptor
(Figure 4). Results from structure–function studies of
C-terminally truncated secretin and VIP molecules
suggest that the C-terminal end of secretin and VIP
interacts with the N-terminal domains of their recep-
tors.124 Thus, the receptor N-terminus may initially
associate with the C-terminus of the peptides. Mem-
bers of the glucagon family are most divergent at the
C-terminus. In addition, while the three disulfide
bonds establish a common overall tertiary fold for all
family B GPCRs, many of the intervening residues
are not conserved and may function as the first line of
selectivity filters that restrict access of inappropriate
ligands to an activation site. The ligand now brought
within the receptor molecule interacts more avidly
with additional residues in the central core of the
receptor, leading to formation of a high-affinity com-
plex and ultimately to activation (Figure 4).103 The
working model is bolstered further by the NMR struc-
ture of PACAP, which advocates that binding of the
peptide ligand to its receptor is kinetically favored
when it occurs in two steps. The ligand first associates
nonspecifically with the membrane surface, establish-
ing an ␣-helical structure at the peptide C-terminus,
then diffuses two-dimensionally until it finds its re-
ceptor. The characteristic N-terminal ␤-coil is in-
duced upon binding, which enables interaction with
residues that drives the receptor activation process.
Extensive studies of the PTH receptor system have
also corroborated the two-site hypothesis of ligand–
receptor interaction.125–127 Moreover, the combined
functional and photoaffinity labeling data have estab-
lished the relative spatial orientation of PTH(1-34)
when bound to the PTH receptor. The N-terminal
residue in PTH(1-34) cross-links to M425 located in
228 Unson

FIGURE 5 Schematic of a model for the interaction of glucagon with GR. Glucagon forms an
␣-helix at the C-terminal region upon association with the membrane bilayer, and initially engages
sequences involving residues 103–117 and 126 –137 of the N-terminal domain of GR. The N-
terminus of the peptide subsequently interacts with a predominantly ionic binding pocket involving
acidic residues from 206 –219 of the e1 loop and is positioned for additional interaction with sites
that lead to receptor activation.

TMVI of PTH receptor.128 K13 contacts a site close to
R186 at the junction of the N-terminal extracellular
domain and TMI of PTHR, and K27 cross-links to
L261 in the e1 loop of PTHR.90,108,129 These findings
further support a model in which the C-terminal por-
tion of PTH(1-34) initially interacts with the large
extracellular N-terminal domain close to the junction
with TMI. This facilitates a second interaction be-
tween the N-terminal region of the ligand with the
juxtamembrane domain at the top of the seven-helix
bundle forming an interface with the e1 loop and
making contact with the e3 loop. Computer modeling
of this topology shows the active conformation to be
a closed U-shaped hairpin structure with the N-termi-
nus of the ligand facing the extracellular loops.126,130
The PTH receptor belongs to family B GPCRs but is
not a close relative of GR. Interestingly, receptor
binding assays demonstrated that bovine parathyroid
hormone (PTH) and human PTH(1-34) can displace
[125I]iodoglucagon from GR in rat liver plasma mem-
branes.131 The displacement of [125I]iodoglucagon re-
quired several thousand-fold more bovine PTH or
human PTH(1-34) than glucagon. However, the PTH
peptides were more effective than secretin, which up
to a concentration of 10⫺5M displayed no ability to
displace [125I]iodoglucagon, despite the fact that se-
cretin is homologous with glucagon while PTH is not.
Circular dichroism studies showed that in the pres-
ence of 3 mM SDS glucagon and hPTH(1-34) have
similar secondary structure, which indicated that re-
ceptors can recognize gross conformational features
of a peptide hormone as well as a specific amino acid
sequence.131
Unlike in the PTH system, specific contact sites
between glucagon and GR have not been identified. A
recent report of a mutational analysis of GR suggested
that Q3 of glucagon binds close to a region at the top
of TM II, consistent with the findings on the R202D
GR mutant.106,132 Nevertheless, structure–activity
studies of glucagon analogs and GR mutants are also
consistent with the two-site models for the binding of
peptide ligands like PTH(1-34) to family B receptors.
 Molecular Determinants of Glucagon Receptor Signaling 229

FIGURE 6 The organization of the genes that have been sequenced from vertebrates are shown
for the glucagon superfamily members. The structural organization of the genes is similar and
ecodes a signal peptide (SP), one to three bioactive peptides, and a C-terminal peptide. Exons are
depicted as boxes and the introns as lines. Glucagon, GRF, GIP, and secretin genes evolved from
the same line of evolution because all lack an exon between the exon coding for the signal peptide
and bioactive peptide. The glucagon gene is unique in that it encodes three peptides— glucagon,
GLP-1, and GLP-2—suggesting that there were exon duplications (adapted from Ref. 135).

It is in general accepted that the N-terminus of glu-
cagon, which contains both H1 and D9 residues, is the
principal region responsible for receptor activa-
tion.69,70 Indeed, a receptor mutant in which the N-
terminal domain of GR is replaced by the first 15
residues of glucagon displayed a significant level of
constitutive activity (Unson, unpublished results).
Constitutive activation by a similarly tethered ligand–
receptor chimera has recently been demonstrated for
the PTH and corticotropin-releasing factor recep-
tors.133,134 This observation further supports a model
in which the C-terminus of the ligand initially engages
the N-terminus of the receptor and subsequently
draws the N-terminus of the peptide toward the e1
loop and the body of the receptor, where additional
favorable interactions provide the stabilization energy
that leads to activation (Figure 5).106 Whether the bio-
active conformation of receptor-bound glucagon is ex-
tended (Figure 5) or folded into a U-hairpin (Figure 4) as
has been postulated for PTH ultimately will have to be
determined by NMR analysis or X-ray crystallography.
MOLECULAR EVOLUTION OF THE
GLUCAGON RECEPTOR
The glucagon subfamily includes nine homologous
polypeptide ligands. The most ancient and tightly
conserved members are PACAP and glucagon, which
may be the ancestral molecules.135 A gene encoding
PACAP was isolated from a protochordate, suggest-
ing that the origin of the ancestral family goes as far
back as the invertebrates.136 The organization of the
known genes for the glucagon family is similar (Fig-
ure 6). Each gene encodes a signal peptide, an N-
terminal peptide, bioactive peptides, and a C-terminal
peptide. Glucagon, GRF, GIP, and secretin genes lack
an exon between the exons encoding the signal pep-
tide and the bioactive peptide. Evidence to date indi-
cates that the glucagon gene family arose from exon
duplication of a single ancestral gene that continued to
diverge. These gene duplications are likely to have
occurred around 400 –500 million years ago prior to
the evolution of vertebrates.137 The receptors for these
ligands are structurally related and similarly origi-
nated by gene duplication events. It is reasonable to
speculate that the glucagon family of receptors may
also have evolved from a common ancestral gene in
invertebrates, although no receptors have been iso-
lated. One theory surmises that the genes for PACAP
and glucagon receptors are derived from protochor-
date (invertebrate) ancestral genes that led to the
vertebrate forms of the receptors. Early in chordate
evolution, two gene duplications might have led to
four copies of each gene that encoded a family B
230 Unson
receptor, followed by structural alterations that might
have resulted in eight receptor types.135
The mammalian proglucagon gene is unique in that
it encodes three biologically active peptides: gluca-
gon, glucagon-like pepide 1 (GLP-1), and glucagon-
like pepide 2 (GLP-2). Each of these functionally
distinct peptides exerts its actions through a specific
and unique but structurally related receptor.16,34,35
Glucagon is functionally and structurally conserved
across vertebrate species, although some of its activ-
ities have diversified during evolution. The tight con-
servation of the primary structure of glucagon
throughout most vertebrates suggests a strong evolu-
tionary pressure to preserve the molecule, consistent
with its pivotal role in the regulation of metabo-
lism.138 Two proglucagon cDNAs were characterized
from the intestine of the sea lamprey Petromyzon
marinus.139 As in other vertebrates, sea lamprey pro-
glucagon genes encode three related sequences— glu-
cagon and GLP-1 and 2—which is an indication that
all three glucagon-like sequences existed prior to the
divergence of jawed and jawless vertebrates. Esti-
mates of the rates of evolution for the glucagon-like
sequences suggest that glucagon had the most ancient
origin and diverged nearly 1 billion years ago, while
GLP-1 and GLP-2 diverged from each other by an
exon duplication event about 700 million years ago.
Whether the origin of the receptors for these pep-
tides parallel their ligands is not known. While the
amino acid sequence of many mammalian glucagons,
bird, several species of frogs, bony fish, cartilaginous
fish, and jawless fish are known, to date, only a few
nonmammalian glucagon receptors have been charac-
terized.3,138,140 –144 However, based on the sequences
known, a phylogenetic analysis using both parsimony
and distance methods reveals that the evolutionary
history of the receptors for proglucagon-derived pep-
tides differs from that of the ligands. These observa-
tions suggest that the diversification of the peptides
encoded by the proglucagon gene and the receptors
for these peptides occurred independently and that the
hormones or their receptors have been recruited for
new functions.144 The difference in the phylogenies
for receptors and ligands may indicate that the spec-
ificity of the receptor–ligand pair evolved much later
than the peptides or receptors themselves. The pep-
tides and receptors originally may have had a gluca-
gon-like function and the different physiological func-
tions evolved only much later. For example, while the
physiological function of glucagon remained un-
changed throughout vertebrate evolution, the function
of GLP-1 has changed.145 In fish, glucagon and
GLP-1 have overlapping glucagon-like activity that
counteracts insulin action, which deviated to an insu-
linotropic function in mammals.144,146 Recent isola-
tion and structure function studies of a GLP-1 recep-
tor from goldfish Carassius auratus demonstrated that
it responded to goldfish glucagon, human glucagon,
and human GLP-1, which are structurally different
peptides (S. Mojsov, personal communication). This
was evidence that the goldfish receptor was more
accommodating than its mammalian counterpart. Ear-
lier characterization of frog GRs showed varying se-
lectivities toward mammalian and fish gluca-
gons.138,144 Deviations in glucagon sequences are
found in teleost fishes, suggesting that GRs in species
that existed before frogs would have different ligand
recognition specificities (Figure 7). The observations
led to the speculation that evolutionary pressures
manifested at the time of the emergence of the frogs
may have necessitated mutations in the sequences of
the ligands and the recognition domains of the recep-
tors.138 These mutations directed more stringent rec-
ognition requirements that were maintained through-
out the evolution of the mammalian species and re-
sulted in highly specific receptors. Functional
characterization of GRs from other species is needed
to fully understand the molecular evolution of verte-
brate glucagons and their receptors and the structural
determinants that dictate specificity within the gluca-
gon receptor family.
CONCLUSION AND PERSPECTIVES
The glucagon signaling system has been one of the
most prolific models in the study of peptide hormone
action. About 30 years ago, Rodbell first characterized
receptors for glucagon in hepatocytes and demon-
strated that glucagon-dependent stimulation of adeny-
lyl cylcase had a GTP requirement, an observation
that gave rise to the field of signal transduction me-
diated by guanine nucleotide binding proteins (G pro-
teins) and the classification of a superfamily of recep-
tors coupled to G proteins. Considerable structure and
activity studies of GPCRs have since been carried out
that have provided a general picture of GPCR struc-
ture and improved the understanding of receptor func-
tion. To date, only the photoreceptor rhodopsin, a
prototype of family A GPCRs, has its structure deter-
mined to atomic resolution. Because there is consid-
erable divergence in the way GPCR ligands interact
with their receptors, the rhodopsin crystal structure
may not be an adequate homology template on which
to base predictions of how peptide ligands of family B
interact with their receptors. Nevertheless, it is a good
vantage point from which information from structure–
function studies of family B GPCRs can be reexam-
 Molecular Determinants of Glucagon Receptor Signaling 231

FIGURE 7 Comparison of amino acid sequences of glucagon in vertebrate species. Shaded areas
represent invariant residues (adapted from Ref. 138).

ined and molecular models refined and validated.
Structural and functional relationships among mem-
bers of the glucagon subfamily of GPCRs have gen-
erated a model for receptor binding and activation.
The experimental data has been supported by molec-
ular modeling, computational simulations, and, in the
case of the receptor for PACAP, an NMR-determined
structure of receptor-bound PACAP. It is now gener-
ally accepted that the large amino terminus of family
B GPCRs plays a key role in the binding of peptide
ligands with additional interactions from the extracel-
lular loops. At present, there is no clear evidence that
any of the peptides penetrate deep into a TM binding
crevice. The situation will most likely be different in
the case of nonpeptide ligands of family B GPCRs
and will require a modification of the existing models.
Small-molecule nonpeptide compounds would diffuse
more easily into the TM bundle and occupy a binding
pocket distinct from the natural peptide ligands.
The mechanism by which different ligands activate
receptors believed to share similar overall structure is
still poorly understood, especially within the GR sub-
family where ligands themselves are highly homolo-
gous. Thus, ligand structure–activity studies and re-
ceptor structure–function relationships will continue
to provide important information. The amino acid
residues that serve as selectivity filters that screen for
appropriate ligands and permit entry into the receptor
binding pocket have to be delineated. No doubt, as
GR family receptor sequences from other species
become available, differences in receptor structure
might provide additional insight into how highly spe-
cific receptors evolved in vertebrates.
The nature of the conformational changes that link
agonist binding to receptor activation and subsequent
transmission of the initial signal to G proteins remains
to be addressed. The counterpart for the D(E)-R-Y
sequence that is a key player in the activation of all
family A receptors has not been identified in GR and
members of family B GPCRs. Biophysical studies
similar to those that have been carried out with rho-
dopsin and ␤2-adrenergic receptors will allow direct
structural observation of movements of TM domains
and other conformational changes accompanying re-
232 Unson
ceptor activation. Methods of expression and isolation
of detergent-stabilized receptor protein will have to be
developed for these studies. This is currently an oner-
ous undertaking for the GR subfamily of GPCRs.
Adequate amounts of receptor protein will enable
determination of high-resolution structure of receptor-
bound glucagon by NMR and eventually atomic res-
olution structure by X-ray analysis. Understanding
GR function at a molecular level will advance the
development of new molecules for the management of
diabetes mellitus.
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