(Received for publication, August 7, 1995)
From the
Glucagon receptor mutants were characterized with the aim of
elucidating minimal structural requirements for proper biosynthesis,
ligand binding, and adenylyl cyclase coupling. One N-terminal deletion
mutant and five truncation mutants with progressively shorter C termini
were expressed in transiently transfected monkey kidney (COS-1) cells.
Each truncation mutant was designed so that the truncated C-terminal
tail would remain on the cytoplasmic surface of the receptor. In order
to characterize the cellular location of the expressed receptor
mutants, a highly specific, high affinity antipeptide antibody was
prepared against the extracellular, N-terminal tail of the receptor.
Immunoblot analysis and immunofluorescence microscopy showed that the
presence of all seven putative transmembrane segments, but not an
intact N-terminal tail, was required for cell surface expression of the
receptor. Membranes from cells expressing receptor mutants lacking a
large portion of the N-terminal tail or any of the seven putative
transmembrane segments failed to bind glucagon. Membranes from cells
expressing the C-terminal tail truncation mutants, which retained all
seven transmembrane segments, bound glucagon with affinities similar to
that of the native receptor and activated cellular adenylyl cyclase in
response to glucagon. These results indicate that all seven helices are
necessary for the proper folding and processing of the glucagon
receptor. Glycosylation is not required for the receptor to reach the
cell surface, and it may not be required for ligand binding. However,
the N-terminal extracellular portion of the receptor is required for
ligand binding. Most of the distal C-terminal tail is not necessary for
ligand binding, and the absence of the tail may increase slightly the
receptor binding affinity for glucagon. The C-terminal tail is also not
necessary for adenylyl cyclase coupling and therefore does not play a
direct role in G protein (G) activation by the glucagon
receptor.
The glucagon receptor belongs to the superfamily of putative
seven-helical, transmembrane, G protein-coupled receptors. The most
immediate relatives to the glucagon receptor include receptors for
glucagon-like peptide-1 (GLP-1) ()(Thorens, 1992), secretin
(Ishihara et al., 1991), vasoactive intestinal peptide
(Ishihara et al., 1992), vasoactive intestinal peptide-2 (Lutz et al., 1993), calcitonin (Lin et al., 1991), growth
hormone-releasing hormone (Mayo, 1992), parathyroid hormone (PTH)
(Jüppner et al., 1991), PTH-related
peptide (Abou-Samra et al., 1992), pituitary adenylyl
cyclase-activating peptide (Pisegna and Wank, 1993), gastric inhibitory
peptide (Usdin et al., 1993), and corticotrophin-releasing
factor (Chen et al., 1993). This branch of receptors makes up
a unique subfamily within the larger classification of G
protein-coupled receptors. Although fairly homologous to each other,
these peptide hormone receptors share very few of the conserved
sequence motifs found in many of the more extensively studied receptors
(Probst et al., 1992; Sprengel et al., 1994).
The
receptors for the glycoprotein hormones luteinizing hormone,
follicle-stimulating hormone, thyroid-stimulating hormone, and
chorionic gonadotropin contain very large extracellular N-terminal
domains of over 300 amino acids (Salesse et al., 1991). The
specificity of gonadotropin receptors is determined by high affinity
hormone binding to N-terminal leucine-rich repeats. Activation of the
receptor may then proceed after the bound ligand interacts with the
transmembrane segments of the receptor (Braun et al., 1991).
Thus, the glycoprotein hormone receptors use the N-terminal portion of
the receptor as well as other extracellular loops and the transmembrane
helices to bind their ligands. In contrast, the photopigment rhodopsin
(Zhukovsky et al., 1991) and the -adrenergic receptors
(Strader et al., 1989) use only the transmembrane helices to
bind chromophore or agonist ligands, respectively. These receptors have
considerably smaller N-terminal extensions compared with those of the
glycoprotein hormone receptors (Probst et al., 1992).
According to tentative structural models, the hormone-binding site of the glucagon receptor probably consists of a contribution from the large extracellular domain of the receptor (Fig. 1), which includes the N-terminal tail and loops connecting transmembrane helices. However, transmembrane signaling must involve ligand-mediated communication between the extracellular domain and the intracellular domain where heterotrimeric G proteins are activated. To investigate the molecular mechanism of hormone-receptor interaction and of receptor activation, we previously designed and synthesized a gene for the rat glucagon receptor. COS cell membranes expressing the synthetic receptor gene bound glucagon with high affinity and displayed the appropriate peptide hormone specificity. The transfected COS cells also showed increased intracellular cAMP levels in response to glucagon (Carruthers et al., 1994).
Figure 1:
Schematic representation of the rat
glucagon receptor primary and secondary structure. Seven putative
transmembrane helices (helix A through helix G) based on previous
models of G protein-coupled receptors are shown. The N terminus and
extracellular surface is toward the top, and the C terminus
and cytoplasmic surface is toward the bottom of the figure.
The four sites of potential N-linked glycosylation on the N
terminus are labeled with asterisks. The 12 amino acids in the
N-terminal tail, which were used to design a peptide DK-12 for antibody
production, are boxed. Asp, which was previously
studied by site-specific mutagenesis, is numbered and labeled
with an arrow (Carruthers et al., 1994). Mutant D1
contained a deletion of 96 amino acid residues from the N-terminal tail
as indicated by arrows. Mutants T1, T3, T5, T7a, and T7b were
truncated as shown. In addition, mutant T3 included the tetrapeptide
RKLH derived from the first intracellular loop after Thr
because the intracellular boundary of transmembrane helix 3 was
not well defined.
In order to evaluate the minimal structural requirements for glucagon binding and for adenylyl cyclase activation, a series of six site-directed glucagon receptor mutants was prepared (Fig. 1). Mutants T7b and T7a had portions of the C-terminal tail removed. Mutants T5, T3, and T1 consisted of the N terminus followed by five, three, or one transmembrane helix, respectively. In addition to the truncated receptors, mutant D1 contained a 96-residue deletion from the N terminus, such that the seven helices were intact, but none of the N-linked glycosylation sites remained. The mutant receptor genes were expressed in COS cells and studied by immunoblot analysis, glycosidase treatment, immunofluorescence microscopy, competitive displacement ligand-binding assays, and adenylyl cyclase activation assays. The results showed that to reach the plasma membrane, the receptor did not need to be glycosylated but did require all seven helices. The N terminus was shown to be important for ligand binding, but the detailed role of the transmembrane helices for ligand binding remains to be elucidated. In contrast to the N terminus, most of the C terminus was not necessary either for the binding of glucagon or for activating adenylyl cyclase.
Figure 5:
Competition for I-glucagon
binding to COS cell membranes expressing native or mutant glucagon
receptor genes. A, competitive displacement of
I-labeled glucagon bound to transfected cell membranes
was determined by incubation with
I-glucagon alone and
with the indicated concentrations of unlabeled glucagon. Data are
presented as percentage of total binding of the radiolabeled hormone versus the log of glucagon concentration. Maximum binding
(100% on the y axis) was less than 10% of total added
radioactivity. Each symbol represents the mean of duplicate
determinations and was curve-fitted where appropriate based on a single
ligand-binding site model as described under ``Experimental
Procedures.'' Cells transfected with control vector pMT3 showed
insignificant binding of
I-glucagon that was considered
to be nonspecific. The concentration of unlabeled glucagon required to
displace 50% of receptor-bound
I-glucagon, the
IC
value, was calculated to be 10.4 nM for the
native receptor (pMT5), 4.7 nM for mutant receptor T7a, and
4.0 nM for mutant receptor T7b. B, membranes from COS
cells transiently transfected with the glucagon receptor gene (pMT5),
T1, T3, T5, or D1 were incubated with radiolabeled glucagon and
increasing concentrations of unlabeled glucagon. Total radioactivity
bound (cpm) is plotted versus log of glucagon concentration,
where each symbol represents the mean of triplicate
measurements. Each of the four mutations resulted in the complete
inability of the mutant receptor to bind
glucagon.
Figure 6:
Adenylyl cyclase activity of COS cells
expressing the glucagon receptor gene and C-terminal mutants. COS cells
were transfected with vector containing the synthetic glucagon receptor
gene (pMT5), mutant receptor T7a, or mutant receptor T7b. The increase
in intracellular cAMP level when cells were incubated with increasing
concentrations of glucagon was determined. cAMP was quantitated using
an assay method that measured the ability of cAMP in each sample to
displace [8-H]cAMP from a cAMP-binding protein.
Each symbol represents the mean of duplicate determinations
and is plotted as the percentage of total cAMP accumulation versus the log of glucagon concentration. The effective concentrations at
50% stimulation of adenylyl cyclase (EC
values) for cells
expressing the glucagon receptor or the two mutants are given in Table 1.
Figure 2: Immunoblot analysis of the rat glucagon receptor mutants expressed in transiently transfected COS-1 cells. Membrane preparations were separated by 10% SDS-polyacrylamide gel electrophoresis, transferred to membranes, and probed with DK-12 anti-peptide glucagon receptor antibody. Immunoreactive bands were visualized by chemiluminescence (ECL). Control cells were transfected with vector alone (pMT3), or with vector containing the synthetic glucagon receptor gene (pMT5). Samples were divided, and one fraction was treated with N-glycosidase F to remove N-linked carbohydrates. Lanes labeled (+) were treated with N-glycosidase F and lanes labeled(-) were untreated. Molecular mass (kDa) indicators are shown to the right of each immunoblot. Each lane contains 10 µg of total protein. A, mutant receptors T1, T3, D1, and controls undigested(-) and digested (+) with N-glycosidase F. B, mutant receptors T5, T7a, T7b, and controls undigested (-) and digested (+) with N-glycosidase F.
Immunoblot analysis of the synthetic rat glucagon receptor mutants expressed in transfected COS cells is shown in Fig. 2. Membrane preparations of cells transfected with the expression vector (pMT3) without the glucagon receptor gene did not react with the antibody upon immunoblot analysis. Membrane preparations of cells transfected with vector containing the synthetic glucagon receptor gene (pMT5) showed a faint broad band migrating with an apparent molecular mass of 55-75 kDa. A potential receptor dimer band migrated at about 100 kDa. An additional weak band migrating at about 35 kDa not seen in the pMT3 lane was also apparent as previously discussed (Carruthers et al., 1994).
Membrane preparations from cells transfected with the N-terminal deletion mutant (D1) and the five truncation mutants (T1, T3, T5, T7a, and T7b) were immunoreactive with the DK-12 antibody since the peptide epitope was not disrupted by the deletion or the truncations (Fig. 2). Each of the mutant receptors was present in membrane preparations of transfected cells. The immunoblot band patterns and levels of expression of the individual mutants are described below. The first step in evaluating whether the mutant receptors were capable of glucagon binding and signal transduction was to demonstrate that they had been properly inserted into the plasma membrane of the cell. Membrane localization was measured by a series of deglycosylation experiments and by immunofluorescence microscopy described below.
The pattern of N-linked glycosylation differed among the truncated mutant receptors and fell into two groups. The first group consisted of mutants T1, T3, and T5, where each displayed a doublet band pattern. Upon deglycosylation, each of the doublet band patterns collapsed to a single band with lower apparent molecular weight. Mutant receptors T3 and especially T5 also displayed bands corresponding to receptor dimer and higher order multimers as described previously for the native receptor (Carruthers et al., 1994). Dimer bands, and multimer bands in the case of mutant T5, were also noted after deglycosylation.
The second group of mutant receptors consisted of T7a and T7b. These two mutants displayed behavior similar to that of the native receptor. Each of these receptors was visualized as a faint broad band corresponding to the receptor monomer. Deglycosylation yielded one band for these receptors, again with a lower apparent molecular weight. With each of these receptors, the DK-12 anti-peptide antibody seemed to bind more avidly to the N-glycosidase F-treated form compared with the untreated form. In addition to the major bands, mutant T7b and native pMT5 each displayed a faint band migrating faster than the principal bands. The positions of these bands did not change upon N-glycosidase F treatment. The significance of this 35-kDa band in the case of pMT5 was been discussed previously (Carruthers et al., 1994). Mutant T7a had a consistently lower expression level compared with the other receptors as judged by immunoblot analysis.
Deletion mutant D1 showed no difference in immunoblot band pattern between untreated and treated membranes. This result was anticipated since mutant D1 contained none of the four potential N-linked glycosylation sites on the N-terminal tail (Fig. 1). Also, the immunoreactive band intensity was also not affected by the N-glycosidase F treatment of cells transfected with mutant D1.
Figure 3: Endo H sensitivity of glucagon receptor mutants in membrane preparations. Membrane preparations were separated by 10% SDS-polyacrylamide gel electrophoresis, transferred to a membrane, and probed with DK-12 anti-peptide glucagon receptor antibody. Each lane contains 10 µg of total protein. Immunoreactive bands were visualized by chemiluminescence (ECL). Samples (T1, T3, T5, T7a, and T7b) were divided, and one fraction was untreated(-), one fraction was treated with N-glycosidase F (N) to remove N-linked carbohydrates, and one fraction was treated with Endo H (E) to remove only high mannose carbohydrates. Mutant receptor D1 was not tested since it lacks glycosylation sites. The native receptor pMT5 (not shown) displayed the same pattern as that of mutants T7a and T7b. It was resistant to Endo H cleavage, but sensitive to N-glycosidase F cleavage (see Fig. 2).
Figure 4: Immunofluorescence microscopy of COS cells transfected with mutant glucagon receptor genes. Transfected cells were prepared as described under ``Experimental Procedures.'' For each mutant receptor (T1, T3, T5, T7a, T7b, and D1) and control (pMT3 and pMT5), permeabilized cells (+) are compared with nonpermeabilized cells(-). Anti-peptide receptor antibody DK-12, which recognizes the extracellular N-terminal tail of the receptor, was used as the primary antibody. A rhodamine-conjugated antibody (lissamine rhodamine B sulfonyl chloride-conjugated goat anti-rabbit IgG) was used as the secondary antibody.
A series of six glucagon receptor mutants was prepared to attempt to define the minimum size required for proper biosynthesis and ligand binding. A schematic representation of the rat glucagon receptor primary and secondary structure is shown in Fig. 1. Seven putative transmembrane helices (helix A through helix G) based on previous models of G protein-coupled receptors are shown (Dratz and Hargrave, 1983; Sakmar et al., 1989; Baldwin, 1993). Four sites of potential N-linked glycosylation on the N terminus are labeled with asterisks (*). The N-terminal amino acid residues 126-137 were used to design a peptide, DK-12, for anti-peptide antibody production. A highly specific, high affinity antibody was obtained, which was used for immunoblot analysis of the expression of the glucagon receptor gene and site-directed mutant genes. The immunoblots in Fig. 2show that the antibody reacted with the products of expression of the vector containing the synthetic glucagon receptor gene. The antibody showed affinity for the monomeric and oligomeric forms of the receptor.
Three of the six mutant receptors,
T1, T3, and T5, were not expressed on the cell surface of transfected
COS cells. The Endo H susceptibility analysis (Fig. 3) combined
with immunofluorescence microscopy (Fig. 4) showed that these
receptors could have been transported only as far as the medial Golgi
(Kornfeld and Kornfeld, 1985). However, most of the population of these
mutant receptors probably were located in the endoplasmic reticulum
(ER). Abnormally processed membrane proteins often are specifically
retained in the rough ER (Lodish, 1988). Fig. 2and Fig. 3show that mutants T1, T3, and T5 all were glycosylated to
a high mannose form, which is the degree of glycosylation expected to
occur in the ER. However, mutants T1 and T3 both lack the highly
conserved pair of Cys-Cys
, which are likely
to form an extracellular disulfide bond analogous to the essential
Cys
-Cys
linkage in rhodopsin (Karnik et
al., 1988; Ridge et al., 1995). Therefore, there is at
least one key structural element lacking in mutants T1 and T3 that
might prevent proper folding. Mutant T5 has the cysteine residues for
the putative disulfide bond, but lacks the last two transmembrane
helices. It is likely that these two helices are essential for assuming
a native-like conformation in the ER membrane. In the case of
rhodopsin, it was shown that a structure involving all transmembrane
helices, the N-terminal tail, and the three intradiscal loops were all
essential for the correct folding and assembly of a functional receptor
(Anukanth and Khorana, 1994). Thus, each of these three mutants lacks
at least one key structural element.
In spite of the demonstrated importance of glycosylation for proper folding for some receptors, the glucagon receptor clearly does not require glycosylation to reach the plasma membrane. The immunofluorescence data in Fig. 4show surface expression of mutant D1, which lacks all four potential N-linked glycosylation sites. A lack of mutant D1 receptor glycosylation is shown experimentally by a failure of electrophoretic mobility change following N-glycosidase F treatment (Fig. 2A). Glycosylation cannot be a required signal for intracellular trafficking and targeting of this receptor to the plasma membrane in COS cells. In addition, a considerable portion of the N terminus can be deleted without affecting the global structure responsible for ER and Golgi processing. The glucagon receptor may therefore differ from other membrane glycoproteins, for which it has been argued that glycosylation is required for protein folding and for routing to the cell membrane (Kusui et al., 1994). In addition, receptor glycosylation also may not be necessary for glucagon binding, since preliminary results show that treating pMT5 membranes with N-glycosidase F does not alter glucagon-binding affinity (data not shown).
A tentative model for receptor-hormone interaction
suggests the existence of at least three distinct functional regions:
Region 1, the proximal N-terminal loop, which contains all four
putative N-linked glycosylation sites; Region 2, the distal
portion of the N-terminal loop, the seven transmembrane helices, and
the three extracellular loops; and Region 3, the intracellular region
consisting of the three interhelical loops and the C-terminal tail.
Region 1 is likely to contain structural elements necessary for proper
receptor structure, as suggested by the finding that substitution of a
conserved aspartic acid residue (Asp) resulted in a
receptor mutant that failed to bind glucagon (Carruthers et
al., 1994). In another recent analysis, a series of chimeras in
which various domains of the human glucagon receptor were replaced by
homologous regions from the GLP-1 receptor was generated (Buggy et
al., 1995). These two receptors share 47% amino acid identity, and
glucagon binds to the GLP-1 receptor with an affinity 1000-fold less
than for its own receptor. With respect to glucagon binding, the
chimeras were classified as being more similar to either the glucagon
receptor or to the GLP-1 receptor. For those receptors that did not
bind well to glucagon, the cell surface expression was confirmed by
immunofluorescence. The dichotomous nature of binding specificities was
used to identify certain segments of Region 2 as important for glucagon
binding: the distal portion of the N-terminal domain, the first
extracellular loop, and the third, fourth, and sixth transmembrane
domains (Buggy et al., 1995). The importance for biological
activity of the N-terminal domain and the first extracellular loop was
also demonstrated in the secretin-vasoactive intestinal peptide
receptor family (Holtmann et al., 1995). These findings are
generally consistent with the present results. Mutants T7a and T7b both
lack part or most of the intracellular C-terminal tail, and yet both
bind glucagon with an affinity comparable with that of the native
receptor (Fig. 5, Table 1). All other truncation mutants
and the deletion mutant D1 lack at least one of the domains implicated
to be important for glucagon binding.
A direct role for Region 1 in
ligand binding cannot be ruled out. The chimeric receptor analysis was
inherently incapable of distinguishing a structural from a functional
role for Region 1, since the proximal N-terminal region of the GLP-1
receptor shares many of the same residues as the glucagon receptor,
including Asp (Buggy et al., 1995). Four glucagon
receptor mutants (D64E, D64G, D64K, and D64N), were previously shown to
be incapable of binding glucagon (Carruthers et al., 1994).
Each of these mutant receptors is resistant to Endo H cleavage (data
not shown) and is present on the cell surface. The strict conservation
of this residue throughout the glucagon receptor family suggests a
generalized role for Asp
. Whether this Asp is involved
directly in ligand-binding is still open to question.
The
intracellular portions of the receptor, Region 3, are directly
responsible for signal transduction. Previous studies have shown the
second and third intracellular loops of G protein-coupled receptors to
mediate G protein activation (Ernst et al., 1995). The role
for the C-terminal tail, however, is less well defined. The present
work demonstrates that most of the tail is not required for binding
glucagon. In fact, mutants T7a and T7b have affinities for the hormone
that are slightly stronger than normal (Table 1). This
improvement may be explained by a receptor-ligand interaction where the
receptor is in equilibrium between an active and inactive state. An
agonist for the receptor, such as glucagon, would stabilize the active
state, and lead to activation of the signal transduction pathways (Bond et al., 1995; Samama et al., 1993). The presence of
the native C-terminal tail destabilizes the active state and pushes the
equilibrium toward an inactive state that has less affinity for a pure
agonist such as glucagon. Such a response already has been observed
with the rat PTH/PTH-related peptide receptor, where the removal of
wild-type C-terminal sequences increased the mutant receptor affinity
for agonist and increased the efficacy with which the receptor
interacted with G (Iida-Klein et al., 1995).
In
an analogous study, mutant avian -adrenergic receptors with
progressively truncated C-terminal tails were prepared (Parker and
Ross, 1991). Membranes from cells that expressed the truncated
receptors displayed elevated basal and agonist-stimulated adenylyl
cyclase activities. Counterintuitively, the truncation mutants also
demonstrated adenylyl cyclase activation upon exposure to alprenolol
and propranolol, molecules classically considered as pure
-adrenergic receptor antagonists. It would appear that the
C-terminal tail does modulate the receptor's affinity for various
ligands. While it is not directly required for G
activation, the C-terminal tail does affect the overall coupling
efficiency between receptor and G protein.
Accumulation of cAMP was
measured as an assessment of the ability of a receptor to couple to
G. A large body of work has demonstrated a clear connection
between glucagon binding to its receptor and the generation of cAMP via
G
(Iyengar et al., 1988). However, it is possible
that this ligand-receptor complex can activate additional effector
pathways. Glucagon-induced elevations in inositol 1,4,5-trisphosphate
(Wakelam et al., 1986) and
[Ca
]
have been reported
(Jelinek et al., 1993). The key question remains as to whether
this change occurs through G
, (Christophe, 1995) as has
been postulated for the glucagon receptor. G
has been
implicated as the G protein for other peptide hormone receptors such as
that for thyroid-stimulating hormone (Allgeier et al., 1994)
and PTH/PTH-related peptide, (Iida-Klein et al., 1995) but
recent evidence has shown that G
(Sternweis, 1994)
and cAMP (Cooper et al., 1995) also can elevate intracellular
calcium concentrations. However, the source for calcium may not
necessarily be the ER stores released by inositol 1,4,5-trisphosphate
(Berridge and Irvine, 1985). Rather, a
G
-stimulated adenylyl cyclase or cAMP directly may
open channels at the plasma membrane to increase calcium influx (Cooper et al., 1995). Investigations are under way to distinguish the
possible roles for each of these components.
In conclusion, the present research provides biochemical and cell biological information about the glucagon receptor, which may be relevant to related G protein-coupled peptide hormone receptors. It is clear that all seven helices are necessary for the proper folding, processing, and cell surface expression of the glucagon receptor. Truncated receptors may be glycosylated, but they are not transported to the Golgi and subsequently to the plasma membrane. At least for the glucagon receptor, glycosylation is not required to reach the cell surface, and it may not be required for ligand binding. The N-terminal extracellular portion of the receptor is required for ligand binding, but it is unclear from the present study which particular transmembrane helices interact directly with the ligand glucagon. In contrast, most of the C-terminal region of the receptor is not necessary for ligand binding, and the presence of an intracellular tail may in fact decrease the receptor's binding affinity for glucagon. Finally, the distal C-terminal region was shown to be unnecessary for coupling to adenylyl cyclase. We are currently investigating which portions of the intracellular region are responsible for activating adenylyl cyclase, increasing intracellular calcium concentrations, and regulating receptor activity at the cell surface.