Alterations in Receptor Activation and Divalent Cation Activation of Agonist Binding by Deletion of Intracellular Domains of the Glucagon Receptor*

(Received for publication, October 29, 1997)

Gary G. Chicchi , Michael P. Graziano , Greg Koch , Patricia Hey , Kathleen Sullivan , Pasquale P. Vicario and Margaret A. Cascieri §

From the Department of Molecular Pharmacology and Biochemistry, Merck Research Laboratories, Rahway, New Jersey 07065

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

Deletion of residues 252-259 within the putative second intracellular loop of the human glucagon receptor results in a protein with high affinity for glucagon but with attenuated agonist activation of adenylyl cyclase. The Delta 252-259 mutant has 4-fold higher affinity for glucagon than does the wild type receptor. The nonhydrolyzable GTP analog, guanosine 5'-(beta ,gamma -imido)triphosphate (Gpp(NH)p), inhibits binding of 125I-glucagon to the wild type receptor but not to the Delta 252-259 mutant. Divalent cations such as MgCl2 and CaCl2 stimulate the binding of 125I-glucagon to the wild type receptor by increasing glucagon affinity. The rate of dissociation of 125I-glucagon is decreased 4-fold by MgCl2 and increased 6-fold by Gpp(NH)p. However, divalent cations do not affect the binding of 125I-glucagon to the Delta 252-259 mutant. The rate of dissociation of 125I-glucagon from the Delta 252-259 mutant protein is equivalent to the rate of dissociation from the wild type receptor in the presence of MgCl2. These data suggest that at least three conformations of the glucagon receptor can exist in the membrane based on their differing affinities for 125I-glucagon. Deletion of residues 252-259 appears to lock the protein in the conformation promoted by divalent cations and prevents the protein from normal coupling to Gs.


INTRODUCTION

Glucagon is a major counterregulatory hormone that attenuates the inhibition of liver gluconeogenesis and glycogenolysis by insulin. Glucagon action is mediated by a G protein-coupled receptor (1) that stimulates cyclic AMP accumulation via activation of Gs. G protein-coupled receptors are characterized by the ability of agonists to promote the formation of a high affinity ternary complex between the agonist, the receptor, and the G protein (2). The affinity of the agonist for the receptor-G protein complex is higher than its affinity for the uncomplexed receptor, so that disruption of the ternary complex reduces the affinity of the receptor for the agonist. Thus, the affinity of agonists for G protein-coupled receptors is a function of the affinity of the receptor for G protein. The changes in affinity of agonist and G protein for receptor are thought to reflect changes in the conformation of the receptor. In contrast, most antagonists bind with the same affinity to the receptor in the presence or absence of G protein coupling.

G protein-coupled receptors such as the glucagon receptor are predicted to have seven transmembrane domains linked by hydrophilic loops (1, 2). Mutagenesis and modeling studies predict that the binding domain for small molecules is within the transmembrane helical domains, although peptide agonist binding also involves the hydrophilic extracellular domains (3). Coupling of the beta -adrenergic receptors to Gs is disrupted by deletion of either the amino-terminal or carboxyl-terminal sections of the third intracellular loop (4). These mutant proteins maintained high affinity for agonist. A similar phenotype has been observed in the receptor for glucagon-like peptide 1, a receptor that is 50% homologous to the glucagon receptor, when three residues predicted to be at the amino-terminal of the third intracellular loop are deleted (5). The second intracellular loop has also been shown to be involved in G protein coupling (2). In the current work we have investigated the properties of glucagon receptors with deletions within the second and third intracellular loops.


MATERIALS AND METHODS

Preparation and Expression of Mutants

Mutagenesis of a cloned human glucagon receptor (6) was performed using the Bio-Rad Mutagene Phagemid in vitro mutagenesis kit, version 2. The putative second intracellular loop was deleted by modification of the sequence of the wild type receptor cDNA to produce the Delta 252-259 mutant protein using the oligonucleotide primer 5' CTG TAC CTG CAC AAC GAG AGG AGC TTC TTC 3'. This primer sequence corresponds to nucleotides 739 through 753 and 778 through 792 of the coding sequence of the human glucagon receptor. The deletion (i.e. nucleotides 754 through 777 of the coding sequence of the human glucagon receptor) was confirmed by sequencing. The cDNA was ligated into the vector PCI/neo (Promega) for expression studies. Deletion of the amino-terminal end of the putative third intracellular loop (Delta  334-340) was prepared using oligonucleotide primer 5'CTC GTG GCC AAG CTG ACA GAC TAC AAG TTC 3', and the deletion was confirmed by sequencing. Deletion of the carboxyl-terminal end of the putative third intracellular loop (Delta 341-347) was prepared using oligonucleotide primer 5' CGG CAG ATG CAC CAC GCC AAG TCC ACG CTG 3', and the deletion was confirmed by sequencing. Deletion of amino acid residues 332-334 was accomplished by recombinant polymerase chain reaction using the oligonucleotide primer 5'-ATC GTT CAG CTG CTC GTG GCC GCA CGG CAG ATG CAC CAC-3' and its complement. The cDNA was ligated into the vector PCI/neo-wild type glucagon receptor for expression studies. The deletion of amino acid residues 332-334 was confirmed by DNA sequencing.

COS cells were transfected in T175 flask monolayers using 10 µg of cDNA and 8 mg of DEAE-dextran in Dulbecco's modified Eagle's medium containing 10% Nu-Serum (Collaborative Biomedical Products) and 100 µM chloroquine for 2 h at 37 °C in 5% CO2 incubation followed by 2-min shock in 10% Me2SO. Cells were maintained in fresh growth medium overnight then were harvested and seeded into T175 flasks for binding and 24-well dishes for cyclase activity measurement. Stable cell lines were isolated after transfection into CHO1 cells and selection with G418.

Characterization of the Binding and Functional Activity

After 2 days, COS cells were harvested from flasks by scraping in 1 mM Tris, pH 7.4, homogenized, and the crude membrane pellet was recovered by centrifugation. 125I-Glucagon (58 pM) binding to the membrane preparation was measured in 20 mM Tris, pH 7.4, containing 2.5 mM MgCl2, 1 mM dithiothreitol, 5 µg/ml leupeptin, 10 µg/ml benzamidine, 40 µg/ml bacitracin, 5 µg/ml soybean trypsin inhibitor, and 3 µM o-phenanthroline ± 1 µM glucagon for 1 h. Bound counts/min were recovered by filtration using a Tomtec harvester and quantified in a gamma -scintillation counter. Functional responses in transfectants were measured by exposing the cell monolayers to Dulbecco's modified Eagle's medium (basal), Dulbecco's modified Eagle's medium with increasing concentrations of glucagon or Dulbecco's modified Eagle's medium with 10 µM forskolin for 30 min at 22 °C to generate intracellular cAMP. Cells were lysed in HCl, and cAMP in lysates was acetylated and quantified by radioimmunoassay. In stable CHO cell lines expressing the receptors, cells were harvested from monolayers with enzyme-free cell dissociation solution (Specialty Media, Inc.) and were pelleted at 500 × g. The cells were resuspended at 100,000 cells/100 µl in 75 mM Tris-HCl, pH 7.4, containing 250 mM sucrose, 12.5 mM MgCl2, 1.5 mM EDTA, 0.1 mM Ro-20-1724 (Biomol, Inc.), leupeptin (5 µg/ml), benzamidine (10 µg/ml), bacitracin (40 µg/ml), soybean trypsin inhibitor (5 µg/ml), and 0.02% bovine serum albumin. Cells were incubated for 30 min at 22 °C with increasing peptide concentrations followed by lysis by boiling. Lysates were analyzed for cAMP content versus a nonacetylated cAMP standard curve using the Amersham cAMP radioimmunoassay SPA kit. Data were analyzed using Packard TopCount with RIASmart and GraphPad Inplot4 software. Transient expression studies in COS cells were repeated at least twice with each mutant protein with similar results. In addition, stable CHO cell lines expressing the Delta 252-259 mutant protein were isolated and analyzed.

Quantitation of Expressed Protein by Western Analysis

The amount of receptor protein in COS membranes was quantitated by binding to an antibody to the COOH terminus of the glucagon receptor (7) on a dot blot using various concentrations of membrane protein. Membranes (0.06-2 µg of protein) were applied to nitrocellulose, incubated with primary antibody in the presence or absence of 100 µg/ml of the antigenic peptide, incubated with 125I-protein A, and then exposed to a PhosphorImager screen overnight before quantitation.


RESULTS

Deletion mutations were made in the human glucagon receptor within putative intracellular loops 2 (Delta 252-259) and 3 (Delta 334-340, Delta 341-347, and Delta 332-334). The positions of these mutations are shown schematically in Fig. 1. After transfection into COS cells, binding of 125I-glucagon to the Delta 252-259, Delta 341-347, and Delta 332-334 mutant proteins is comparable (40-60%) with binding to the wild type glucagon receptor. However, binding to the Delta 334-340 mutant protein is <5% of that observed to the wild type receptor, suggesting that this mutant protein is not well expressed.


Fig. 1. Schematic diagram of the human glucagon receptor showing the putative topological location of the deletion mutants.
[View Larger Version of this Image (31K GIF file)]


After transfection into COS cells, deletion of residues 252-259 results in marked attenuation of the ability of glucagon to activate adenylyl cyclase (Fig. 2). The EC50 for glucagon activation of the wild type receptor in transiently transfected COS cells is 84 ± 67 pM (Table I). Under conditions where glucagon stimulates adenylyl cyclase activity of the wild type receptor 6-fold, glucagon (100 nM) stimulates adenylyl cyclase activity of the Delta 252-259 mutant protein 47% (Fig. 2). In several independent transfections into COS cells, glucagon stimulates adenylyl cyclase activity of the wild type glucagon receptor 819 ± 743% (n = 5). In contrast, glucagon stimulation of activity of the Delta 252-259 mutant protein is 40 ± 20% (n = 3). This is 12 ± 3% of the glucagon activation of wild type receptors in these experiments (Table I). Basal and forskolin-stimulated adenylyl cyclase activities are comparable for the COS cells transfected with wild type receptor and the Delta 252-259 mutant protein (data not shown). A similar phenotype is observed in three independent CHO cell lines expressing the Delta 252-259 mutant protein. Maximal glucagon activation of the Delta 252-259 mutant protein is 56 ± 40% (n = 3) over basal activity. This is 7 ± 5% of the activation observed with wild type glucagon receptor expressed at similar levels (approximately 500 fmol/mg of protein).


Fig. 2. Glucagon-activated adenylyl cyclase activity of the wild type human glucagon receptor (solid circles) and the Delta 252-259 mutant protein (open circles). Proteins were expressed transiently in COS cells, and the cells were grown in monolayer culture. Cells were stimulated with glucagon or forskolin (10 µM) for 15 min before lysis and measurement of cAMP levels by radioimmunoassay. Data are the mean ± S.D. of triplicate wells.
[View Larger Version of this Image (18K GIF file)]


Table I.

Characterization of the activity of intracellular loop deletion mutants of the human glucagon receptor


Mutant Cell type Glucagon-stimulated adenylyl cyclase activity (mean ± S.D. (n))
EC50 % of wild type activation

pM
Wild type COS 84  ± 67 (5) 100
 Delta 252-259 COS 12  ± 3 (3)
 Delta 252-259 CHO 7  ± 5 (3)
 Delta 332-334 COS 3500  ± 2500 (2) 61  ± 10 (2)
 Delta 334-340 COS 10350  ± 13647 (2) 29  ± 18 (2)
 Delta 341-347 COS 2000  ± 200 (2) 67  ± 12 (2)

Deletion of residues 332-334 from the amino-terminal end of putative intracellular domain 3 results in a 42-fold increase in the EC50 for glucagon activation and a decrease of 39% in the maximal activation observed (Table I). Even though the Delta 334-340 mutant protein is poorly expressed, glucagon (100 nM) stimulates adenylyl cyclase activity to 29 ± 18% of the maximal stimulation observed in cells expressing the wild type glucagon receptor (Table I). However, glucagon is >100-fold less active at this mutant compared with the wild type receptor. Deletion of residues 341-347 from the carboxyl-terminal end of the third intracellular loop increases the EC50 for glucagon activation 24-fold, and maximal glucagon-stimulated adenylyl cyclase activity was 67% of the stimulation observed with the wild type receptor (Table I). Little effect on basal or forskolin-stimulated adenylyl cyclase activity is observed with these mutations (data not shown).

Membranes prepared from COS cells transfected with the wild type human glucagon receptor, the Delta 252-259 mutant protein or the beta 3 adrenergic receptor were applied to nitrocellulose paper by dot blot and incubated with a polyclonal antibody raised against a peptide corresponding to the carboxyl-terminal of the receptor. This antibody has been previously demonstrated to specifically interact with the glucagon receptor (7), and no bands are observed by Western analysis of gels loaded with untransfected COS cell membranes (10 µg of protein). Similarly, no spots are observed on the dot blots loaded with membranes expressing beta 3-adrenergic receptors (4 µg of protein). Densitometric analysis of the conjugates after incubation with 125I-protein A is linear versus membrane protein added (Fig. 3). Coincubation with the antigenic peptide attenuates the signal (data not shown). Analysis of the slopes of the lines in Fig. 3 indicate that the membranes expressing wild type glucagon receptor express 60% higher levels of protein than membranes expressing the Delta 252-259 mutant protein.


Fig. 3. Quantitation of the levels of expressed wild type glucagon receptor or Delta 252-259 mutant protein by Western analysis. Membranes were prepared from COS cells from the experiment shown in Fig. 2 and applied to nitrocellulose using a dot blot apparatus. After incubation with an antibody to the carboxyl terminus of the glucagon receptor and 125I-protein A, the nitrocellulose was exposed to a PhosphorImager screen for quantitation. Data shown are the mean ± S.E. for two applications at each protein concentration. The slopes of the lines are 4.99 × 104 and 3.11 × 104 for the wild type glucagon receptor (solid circles) and the Delta 252-259 mutant protein (open circles), respectively, suggesting that the membranes expressing the wild type receptor have 60% more protein than the membranes expressing the Delta 252-259 mutant protein.
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The nonhydrolyzable GTP analog Gpp(NH)p inhibits binding of 125I-glucagon to the wild type human glucagon receptor with an IC50 = 22 nM (Fig. 4). The maximal inhibition of binding at excess levels of Gpp(NH)p is 90% of the specific binding of 125I-glucagon observed in the absence of nucleotide. These data suggest that 90% of the observed binding of 125I-glucagon is to receptor coupled to Gs. However, Gpp(NH)p does not inhibit the binding of 125I-glucagon to the Delta 252-259 mutant protein, suggesting that this protein is not coupled to Gs (Fig. 4).


Fig. 4. Inhibition of 125I-glucagon binding to the wild type glucagon receptor (closed circles) and the Delta 252-259 mutant protein (open circles) by Gpp(NH)p. Data shown are specific binding to membranes prepared from CHO cells stably expressing either the wild type glucagon receptor or the Delta 252-259 mutant protein.
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Glucagon inhibits binding of 125I-glucagon to the Delta 252-259 mutant protein with 4-fold higher affinity than to the wild type protein in the absence of added divalent cations. The IC50 (mean ± S.D.) for three experiments in COS cells and three experiments in CHO cells (i.e. n = 6) is 3.5 ± 2.0 and 0.76 ± 0.39 for the wild type receptor and the Delta 252-259 mutant protein, respectively.

Binding of 125I-glucagon to the wild type human glucagon receptor expressed in CHO cells is stimulated 2-3-fold by divalent cations such as MgCl2, MgSO4, and CaCl2 (Fig. 5, top). The EC50 for this stimulation is ~0.3 mM. The primary effect of divalent cation is to increase the affinity for glucagon 4-fold. Unlabeled glucagon inhibits binding of 125I-glucagon to the wild type glucagon receptor with an IC50 = 2.6 ± 1.5 nM (n = 3). Inclusion of 5 mM MgCl2 in the incubation decreases the IC50 for glucagon to 0.65 ± 0.13 nM (n = 3). In contrast, divalent cations have no effect on binding of 125I-glucagon to the Delta 252-259 mutant protein (Fig. 5, bottom). Glucagon inhibits the binding with IC50 values of 0.76 ± 0.39 nM (n = 3) and 0.53 ± 0.15 nM (n = 3) in the absence or presence of 5 mM MgCl2, respectively.


Fig. 5. Inhibition of 125I-glucagon binding to the wild type glucagon receptor (top) or the Delta 252-259 mutant protein (bottom) by unlabeled glucagon in the absence of divalent cation (squares), presence of 5 mM MgCl2 (triangles), or presence of 5 mM CaCl2 (inverted triangles). The experiment shown is representative of at least three replicate experiments.
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The rate of dissociation of 125I-glucagon for the wild type receptor is decreased in the presence of divalent cation (Fig. 6). The dissociation rate of 125I-glucagon in the absence or presence of divalent cation is 0.04 ± 0.01 min-1 (n = 2) or 0.013 ± 0.001 min-1 (n = 2), respectively. In the presence of excess Gpp(NH)p, the rate of dissociation is increased to 0.27 ± 0.04 min-1 (n = 2). In contrast, divalent cation has no effect on the dissociation rate from the Delta 252-259 mutant protein (Fig. 6). The rates of dissociation of 125I-glucagon from the Delta 252-259 mutant protein are 0.013 ± 0.002 min-1 (n = 3) and 0.010 ± 0.004 min-1 (n = 3) in the absence and presence of 5 mM MgCl2, respectively.


Fig. 6. Dissociation of 125I-glucagon from the wild type glucagon receptor (closed symbols) or the Delta 252-259 mutant protein (open symbols) in the absence (circles) or presence of 5 mM MgCl2 (diamonds) or 100 µM Gpp(NH)p (squares). Membranes were incubated for 40 min with 125I-glucagon in the presence or absence of divalent cation as indicated at room temperature before addition of an excess (2 µM) of unlabeled glucagon to initiate dissociation of labeled ligand. As indicated, Gpp(NH)p was added at the same time as unlabeled glucagon. Data shown are the average of two separate experiments.
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DISCUSSION

Mutagenesis experiments with the adrenergic receptor family have shown that deletions or replacements within the third intracellular loop attenuate agonist-mediated signal transduction and, in many cases, result in a protein that has high affinity for agonist ligands in the absence of G protein coupling (4). Similar data have subsequently been obtained for other G protein-coupled receptors (2). More recently, deletion of a Lys-Leu-Lys tripeptide sequence predicted to be adjacent to the fifth transmembrane domain of the glucagon-like peptide-1 receptor produces a similar phenotype, displaying high affinity binding of glucagon-like peptide (7-37) amide, but only 10% of the maximal activation of adenylyl cyclase as the wild type receptor (5). However, our data show that deletion of the homologous Lys-Leu-Arg sequence of the related glucagon receptor (Delta 332-334 mutant protein) increases the EC50 for glucagon 42-fold, but does not completely attenuate the maximal activation observed. Deletion of residues 334-340 results in a protein that is poorly expressed as determined by its low binding of labeled glucagon and low potential for glucagon activation. Deletion of the seven amino acids predicted to form the carboxyl-terminal end of the third intracellular loop of the human glucagon receptor also results in a 24-fold increase in EC50 with only a 33% decrease in the maximal receptor activation. These data suggest that the third intracellular loop of the glucagon receptor is involved in receptor activation and that impairment of coupling efficiency results from deletions in this region.

In contrast, deletion of residues 252-259 within the putative second intracellular loop of the human glucagon receptor results in a protein that is well expressed and that has high affinity for glucagon with drastically attenuated glucagon-mediated signal activation. Its degree of activation is even lower than that for the poorly expressed Delta 334-340 protein. This phenotype more closely resembles that observed with intracellular loop 3 deletions in the adrenergic receptors, although reductions in coupling efficiency have been observed in both biogenic amine receptors and peptide receptors after second intracellular domain deletions (2).

The glucagon receptor and other members of this structural family (i.e. glucagon-like peptide-1, pituitary adenylyl cyclase-activating peptide, calcitonin, parathyroid hormone, and corticotropin-releasing factor receptors) lack the sequence markers that are conserved in other families of G protein-coupled receptors (8). Therefore, it has been more difficult to predict the beginning and ends of the transmembrane domain helices in this family of proteins. However, the similarity of the phenotypes of these putative second and third intracellular domain mutations in the glucagon receptor to the phenotypes of the analogous mutations in biogenic amine receptors is consistent with the assignment of the helices of the glucagon receptor as described by MacNeil et al. (6).

Analysis of binding isotherms shows that the Delta 252-259 mutant protein has 4-fold higher affinity for glucagon as compared with the wild type glucagon receptor. This increased affinity is reflected in a 4-fold slower rate of dissociation of 125I-glucagon from the Delta 252-259 mutant protein as compared with the wild type receptor in the absence of divalent cation.

Divalent cations have been shown to allosterically enhance the binding of agonists to many types of G protein-coupled receptors. Previous data suggest that divalent cations stimulate binding of 125I-glucagon to receptors in chicken liver membranes (9) but not from mammalian liver membranes (9, 10). Subsequently, Post et al. (11) reported that Mg2+ cations stimulated binding of 125I-glucagon to saponin-permeabilized canine hepatocytes 3-fold. In contrast to our data, calcium did not mimic the effects of magnesium in this system.

Both calcium and magnesium stimulate binding of 125I-glucagon to the human glucagon receptor. The increased binding is due to a 3-fold increase in affinity for glucagon in the presence of divalent cation. Inclusion of divalent cation decreases the rate of dissociation of 125I-glucagon from the receptor from 0.04 to 0.01 min-1 at room temperature. The receptor in the absence of magnesium does not simply reflect the affinity of agonist in the absence of G protein coupling, since inclusion of Gpp(NH)p in the incubation increases the rate of dissociation of 125I-glucagon to 0.29 min-1. This conclusion is also supported by the effects of calcium, a cation that does not alter the affinity of G proteins for receptors. These data suggest that the primary effect of divalent cation is not on G protein coupling, but on the conformation of the receptor. These data also suggest that at least three conformations of the glucagon receptor exist in the membrane, reflected in the differences in affinity observed in the absence or presence of divalent cation, and in the fully uncoupled state (i.e. in the presence of excess Gpp(NH)p).

Gpp(NH)p does not inhibit the binding of 125I-glucagon to the Delta 252-259 mutant protein, and these data together with the lack of glucagon-activated adenylyl cyclase activity are consistent with a lack of G protein coupling to this protein. In biogenic amine receptors, deletions within the second intracellular domain alter activation, and chimeric receptors in this region have altered specificity for G proteins, suggesting that this region is directly involved in coupling of receptor and G protein (4). However, divalent cation does not alter the affinity of 125I-glucagon for this mutant as measured by binding isotherms or dissociation kinetics. Since the affinity of the Delta 252-259 mutant protein for 125I-glucagon and the rate of dissociation of 125I-glucagon from this protein are identical to the affinity and rate of dissociation of 125I-glucagon from the wild type receptor in the presence of divalent cation, these data strongly suggest that this deletion locks the protein into the conformation that is normally promoted by the binding of divalent cation to the receptor. These data strongly suggest that the glucagon receptor contains a divalent cation binding site that allosterically regulates glucagon binding and receptor coupling. As such, the Delta 252-259 mutant protein will be a useful tool for assessing the differences in binding interactions among the different affinity states of the glucagon receptor.


FOOTNOTES

*   The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
   Current address: Schering-Plough Research Inst., Kenilworth, NJ 07033.
§   To whom correspondence should be addressed: Merck Research Laboratories, 80M-213, P. O. Box 2000, Rahway, NJ 07065. Tel.: 908-594-4609; Fax: 908-594-3337; E-mail: peggy_cascieri{at}merck.com.
1   The abbreviations used are: CHO, Chinese hamster ovary; Gpp(NH)p, guanosine 5'-(beta ,gamma -imido)triphosphate.

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