Constitutive Activity of Glucagon Receptor Mutants

Siv A. Hjorth, Cathrine Ørskov and Thue W. Schwartz

Laboratory for Molecular Pharmacology (S.A.H., C.Ø., T.W.S.) Department of Pharmacology
Department of Anatomy (C.Ø.) The Panum Institute DK-2200 Copenhagen N, Denmark


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Increased constitutive activity has been observed in the PTH receptor in association with naturally occurring mutations of two residues that are conserved between members of the glucagon/vasoactive intestinal peptide/calcitonin 7TM receptor family. Here, the corresponding residues of the glucagon receptor, His178 and Thr352, were probed by mutagenesis. An elevated level of basal cAMP production was observed after the exchange of His178 into Arg, but not for the exchange into Lys, Ala, or Glu. However, for all of these His178 substitutions, an increased binding affinity for glucagon was observed [dissociation constant (Kd) ranging from 1.1–6.4 nM, wild type: Kd = 12.0 nM]. A further increase in cAMP production was observed for the [H178R] construct upon stimulation with glucagon, albeit the EC50 surprisingly was increased approximately 10-fold relative to the wild-type receptor. Substitution of Thr352, located at the intracellular end of transmembrane segment VI, with Ala led to a slightly elevated basal cAMP level, while the introduction of Pro or Ser at this position affected rather the binding affinity of glucagon or the EC50 for stimulation of cAMP production. The large extracellular segment, which is essential for glucagon binding, was not required for constitutive activation of the glucagon receptor as the introduction of the [H178R] mutation into an N-terminally truncated construct exhibited an elevated basal level of cAMP production. The analog des-His1-[Glu9]glucagon amide, which in vivo is a glucagon antagonist, was an agonist on both the wild-type and the [H178R] receptor and did not display any activity as an inverse agonist. It is concluded that the various phenotypes displayed by the constitutively active glucagon receptor mutants reflect the existence of multiple agonist-preferring receptor conformers, which include functionally active as well as inactive states. This view agrees with a recent multi-state extension of the ternary complex model for 7TM receptor activation.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The glucagon peptide is an important up-regulator of blood glucose levels and in this way counteracts the insulinotropic activities of the structurally closely related peptide glucagon-like-peptide 1 (GLP-1) (1). The receptors that recognize glucagon and GLP-1 with a high degree of selectivity are similarly homologous and comprise, together with receptors for a number of other peptide hormones and neuropeptides such as vasoactive intestinal peptide (VIP), pituitary adenylate cyclase activating peptide (PACAP), secretin, glucose-dependent insulinotropic peptide (GIP), calcitonin, PTH, CRF, calcitonin gene-related peptide, and GRHR, a structurally distinct family of 7TM G protein-coupled receptors (2, 3, 4). Distinct structural features of this glucagon/VIP/calcitonin receptor family include the presence of a large N-terminal extracellular domain containing six fully conserved cysteine residues that are presumed to serve an important structural role via the formation of disulfide bridges. Functionally, all members of the glucagon/VIP/calcitonin receptor family convey hormone-dependent stimulation of cAMP production through Gs-mediated activation of adenylyl cyclase.

Structure-functional analysis of the glucagon/VIP/calcitonin receptor family is still at a relatively early stage as compared with the quantitatively much larger family of rhodopsin-like 7TM peptide receptors for which a more detailed picture of important ligand-receptor interactions has emerged. For instance, for the rhodopsin-like receptors binding of small agonist ligands such as monoamines appears to involve distinct mechanisms from those involved in binding of the larger peptide ligands (5). Thus, while binding of the small ligands is believed to occur through high-affinity interaction with ‘binding pockets’ buried relatively deeply in the transmembrane domain, the peptide ligands seem to acquire their binding energy predominantly through interactions with residues present in the extracellular domain of the receptor. No common mechanism therefore seems to account for the initial recognition events between individual agonist ligands and the corresponding 7TM receptors, although the signaling mechanism appears to be shared (6). For the glucagon/VIP/calcitonin receptors, binding of the peptide ligands appears to be critically dependent on major parts of the extracellular N-terminal extension, although this receptor domain by itself does not suffice for high-affinity peptide binding and/or receptor activation (7, 8, 9, 10, 11, 12, 13, 14, 15).

Constitutive receptor activity, i.e. signaling by receptors in the absence of ligand binding, has been described extensively for the rhodopsin-like 7TM receptor family. Here, constitutive activity has been observed in the form of naturally occurring mutants associated with human diseases (reviewed in Ref.16) and in some wild-type/unmutated monoamine receptors (17, 18), as well as for engineered substitutions (19, 20). For several of these mutant forms the substitutions are located in the intracellular (IC) loop segments, in particular the IC loop 3, although examples of mutations located in essentially any subdomain of the rhodopsin-like receptor structure have been demonstrated to cause increased signaling activity; these include exchanges at positions located in the extracellular loops, e.g. in the MSH receptor (21), and the thrombin receptor (22). Recently the first examples of constitutively active receptor mutants in the glucagon/VIP/calcitonin family were reported (23, 24). The mutation of a highly conserved histidine residue present at the presumed junction between TM-II and IC loop 1 was identified in the PTH receptor gene of patients suffering from a rare form of dwarfism, Janssen-type chondrodysplasia (23). This mutation [H223R], which appears to form (part of) the molecular basis for the disease, was shown by expression studies to be accompanied by elevated levels of ligand-independent levels of adenylyl cyclase activity. Subsequently, the exchange of a another highly conserved residue located at the cytoplasmic end of TM-VI (T410P) was shown also to be associated with this syndrome (24).

In the present study we have exchanged the corresponding conserved residues in the rat glucagon receptor, His178 and Thr352, respectively (Fig. 1Go), and characterized receptor constructs carrying different substitutions at each of these positions. The highly diverse phenotypes exhibited by these mutants shed further light on the molecular mechanisms that cause and accompany constitutive activity of the glucagon receptor and underlines similarities as well as differences between the glucagon/VIP/calcitonin receptor family and the quantitatively dominating 7TM family, the rhodopsin-like receptors.



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Figure 1. A Serpentine Diagram of the Glucagon Receptor

Residues conserved between all members of the glucagon/VIP/calcitonin 7TM peptide receptor family are indicated in white on black. Three putative disulfide bridges are indicated in the large N-terminal extracellular domain. The indicated pairing of (consecutive pairs of) cysteine residues is purely speculative.

 

    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Receptor Constructs
The presumed membrane topology of the glucagon receptor is shown in Fig. 1Go, indicating the substituted conserved residues His178 and Thr352, located at the membrane-cytosol interphase of TM-II and TM-VI, respectively. The receptor constructs ([H178R], [H178K], [H178A], [H178E], [T352P], [T352S], and [T352A]) were expressed transiently in COS-7 cells and characterized by competition binding analysis using [125I]glucagon and with regard to basal and stimulated levels of cAMP accumulation.

Radioligand Binding Experiments with His178
In homologous competition binding analysis, each of the four mutant His178 receptors bound glucagon with an improved affinity compared with the wild-type receptor (Table 1Go), up to a 10-fold increase for the [H178E] construct. The receptor density (Bmax) estimated on the basis of [125I]glucagon competition binding experiments indicated a reduced level of receptor expression for all mutant constructs, the [H178E] receptor in particular (Table 1Go). The glucagon analog des-His1-[Glu9]glucagon amide, which both in vivo and in vitro has been characterized as a glucagon receptor antagonist (25, 26), bound the two His178-substituted glucagon receptors that were analyzed with affinities similar to that seen for the wild-type receptor (Table 1Go).


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Table 1. Ligand Binding Affinities for the Glucagon Wild-Type and His178-Substituted Receptors

 
Adenylyl Cyclase Stimulation by His178-Substituted Receptors
Of the four His178-substituted constructs, only one [H178R] exhibited an elevated basal level of cAMP accumulation (Fig. 2AGo), while for each of the other His178 constructs ligand-independent adenylyl cyclase activity was indistinguishable from the wild-type response. The basal, i.e. ligand- independent level of adenylyl cyclase activity observed for the [H178R] construct was elevated 4.9 ± 1.2-fold relative to that observed for the wild-type receptor (Figs. 2Go and 3Go) and further stimulated, in response to glucagon, with an increment of approximately half that obtained for the wild-type receptor. Adenylyl cyclase activity reached a maximal level at {approx}10-7 M glucagon, with a slight decrease in activity occurring at even higher concentrations (Fig. 3Go). This biphasic response, which has been also reported previously in liver membranes (25), was seen both for the wild-type receptor and for the [H178R] mutant (Fig. 3Go). Surprisingly, the potency of glucagon was reduced 12-fold for the constitutively active [H178R] receptor construct (from 0.30 ± 0.04 nM to 3.6 ± 1.2 nM, Fig. 3Go and Table 2Go). Thus for this mutant the IC50 for glucagon binding and the EC50 for glucagon-mediated stimulation of adenylyl cyclase were shifted in opposite directions.



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Figure 2. Basal and Stimulated Levels of cAMP Production in COS-7 Cells Expressing Wild-Type or Mutated Glucagon Receptors

A, Normalized accumulation of cAMP in COS-7 cells transfected with the wild type glucagon receptor and the His178 mutant forms. The level of cAMP accumulation is expressed in fold relative to the level obtained in unstimulated cells expressing the wild-type glucagon receptor. Black bars represent the constitutive, basal activity, and shaded bars represent the maximal activity obtained after glucagon stimulation (at {approx} 10-7 M). B, Activity of glucagon and des-His1-[Glu9]glucagon amide on the wild-type and the [H178R] glucagon receptor. Transfected COS-7 cells expressing either the wild-type or the constitutively active receptor construct [H178R] were stimulated with glucagon and (des-His1-[Glu9]glucagon amide, respectively, and cAMP production was determined as described in Materials and Methods. The intrinsic activity indicates the ratio between the maximal cAMP production obtained relative to that obtained with the full agonist glucagon on the identical construct.

 


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Figure 3. cAMP Production in COS-7 Cells Expressing the Wild-Type and the [H178R] Glucagon Receptor

Top panel, Dose-response curve for cAMP accumulation in cells stimulated with glucagon. Bottom panel, Dose-response curve for des-His1-[Glu9]glucagon amide. The level of cAMP accumulation is expressed as percentage of the maximal induction obtained for the wild-type receptor in response to glucagon and des-His1-[Glu9]glucagon amide, respectively.

 

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Table 2. EC50 Values for Stimulation of cAMP Production in Response to Glucagon

 
The glucagon analog des-His1-[Glu9]glucagon amide has been reported to act as a potent peptide antagonist for glucagon (25, 26). Yet, when tested alone on COS-7 cells expressing either the wild-type or the [H178R] construct, the glucagon analog mediated stimulation of the production of cAMP, albeit with reduced potency compared with glucagon (Fig. 3Go). Moreover, and in contrast to glucagon, des-His1-[Glu9]glucagon amide showed a similar potency for adenylyl cyclase stimulation on the wild-type and the [H178R] receptor, respectively [EC50 (wild-type) = 52.8 ± 6.8 nM; EC50 [H178R] = 35.3 ± 9.2 nM]. The maximal stimulation attained in response to des-His1-[Glu9]glucagon amide was reduced in comparison with the response exerted by glucagon on the wild-type receptor (Fig. 2BGo). Consequently the analog would be classified as a partial agonist. However, when acting on the constitutively active glucagon receptor [H178R] maximal activity exerted by the analog exceeded that obtained with the full agonist, glucagon. Thus on this receptor the glucagon analog would be characterized rather as a full agonist (or even a superagonist).

All four constructs mutated at the His178 position were expressed at reduced levels compared with the wild-type receptor (Bmax values in Table 1Go) and exhibited, accordingly, a lower maximal level of stimulation (Fig. 2AGo). To further examine the basal levels of cAMP production in relation to expression levels we performed a gene dosage experiment (Fig. 4Go). For the wild-type and the [H178A] receptors there was no detectable change (increase) in the basal level of cAMP activity even at high expression levels, thus indicating a very low inherent signaling activity of these receptors. This is in contrast to the steep increment in basal cAMP level observed for the constitutively active [H178R] receptor construct. Only the lower range of expression levels could be assessed for the two other constructs substituted at the His178 position, [H178K] and [H178E], but no indication of constitutive activity was observed for these receptors (data not shown).



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Figure 4. The Effect of Expression Level on Basal Level of Receptor Activation

COS-7 cells were transfected with increasing amounts of DNA encoding wild-type and mutant glucagon receptors. Basal levels of cAMP accumulation are expressed in femtomoles/well after 15 min of glucagon stimulation. The data for each of the constructs represent results from at least two independent experiments each performed in triplicate.

 
Substitution of the Thr352 Residue
Three substitutions (Pro, Ser, and Ala) were introduced at the Thr352 position in the glucagon receptor (Fig. 1Go). This resulted in rather pleiotropic effects, as reflected in distinctly different phenotypes with regard to 1) ligand binding affinity, 2) basal signaling activity, and 3) potency of glucagon for adenylyl cyclase stimulation. Thus, for each of the three constructs, at least one parameter differed distinctly from the corresponding wild-type value. For the [T352S] construct, ligand binding affinity was reduced to 45 nM (Table 1Go). For the [T352P] construct, the potency of glucagon for cAMP signaling was decreased from 0.3 nM to 1.6 nM (Table 2Go), while the [T352A] substitution was accompanied by 3.7-fold enhancement of the basal level of adenylyl cycase activity (Fig. 2AGo). Analysis of these constructs at increasing levels of expression affirmed the constitutive activity of the [T352A] construct, albeit less so than that seen for the [H178R] mutant (Figs. 2Go and 4Go). However, the basal level of cAMP production similar to the wild-type for the two constructs [T352S) (Figs. 2Go and 4Go) or [T352P] (Fig. 2Go).

Effect of [H178R] Substitution in N-Terminally Truncated Receptors
To assess further the mechanism responsible for the constitutive activity conferred by the [H178R] substitution, a set of receptors was constructed in which the N-terminal extracellular domain had been deleted. These constructs, {Delta}N-term and {Delta}N-term-[H178R], were, as expected, deficient in peptide binding (Fig. 5Go, top panel) and showed no glucagon-inducible accumulation of cAMP. However, the construct that included the [H178R] mutation exhibited a constant and elevated basal level of cAMP (Figs. 2Go and 5Go), suggesting that the receptor domain consisting of the TM segment and the intervening loops can mediate signaling by itself, and that the His178 -> Arg substitution promotes this activity even in the absence of the presumed main ligand-binding receptor domain of the receptor.



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Figure 5. Truncated Glucagon Receptors Expressed in COS-7 Cells

Competition binding analysis using [125I]glucagon (top panel) and dose-response curves for stimulation of cAMP accumulation (bottom panel) is shown for COS-7 cells expressing N-terminally truncated receptor constructs (for position of deletion endpoint see Fig. 1Go). The {Delta}N-term receptor (•), and the {Delta}N-term [H178R] glucagon receptor (O). The averaged response obtained for the full-length wild-type receptor is indicated as a stippled line.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Different ‘Phenotypes’ of Constitutively Active Receptors
In the present study we have characterized the pharmacological properties of a series of glucagon receptor mutants substituted at either of two highly conserved positions located at the IC side of TM-II and TM-VI. Both of these positions have been associated with altered signaling properties in other receptors of the glucagon/VIP/calcitonin 7TM receptor family (23, 24, 27). In the glucagon receptor the mutational exchange at either of these positions affected peptide binding as well as receptor activation. Thus, constitutive activation of adenylyl cyclase signaling was observed although only in a single instance at either of the two positions, His178 and Thr352. All of the mutants did, however, differ from the wild-type receptor with regard to other pharmacological parameters: either 1) the binding affinities for the peptides, glucagon and des-His1-[Glu9]glucagon amide; 2) the potency of glucagon for stimulation of adenylyl cyclase activity; or 3) the basal and stimulated cAMP levels.

According to the allosteric ternary complex model (19), 7TM receptors exist in equilibrium between inactive (R) and active (R*) conformational states, and the pharmacological property of a given ligand may be viewed simply as the ability of that ligand, upon binding to the receptor, to stabilize either the R or the R* state. Agonists (A) thus act by virtue of stabilizing the receptor R* state. Importantly, in this model active receptor complexes include bound (R*-A) as well as unbound (R*) species, the latter representing the constitutively active forms. In this two-state model, an increased prevalence of active receptor states (as in constitutive active mutants) is inseparably linked to an enhanced agonist affinity as was indeed originally observed for the adrenergic receptors (19, 20). However, as pointed out by Cotecchia, Costa and co-workers (28) in a recent analysis of a series of {alpha}1B-adrenergic receptor mutants, the two parameters, basal activity and agonist affinity, may in fact change independently upon mutagenesis. In that study, enhanced agonist affinity was observed in a number of mutants, but not necessarily accompanied by constitutive activation. To fully account for these observations, a multi-state extension of the two-state model was proposed. Central to this new model is the assumption that the (macroscopic) functional states that are discerned experimentally could be composed of ensembles of multiple different (microscopic) receptor conformations, and importantly that the subsets of agonist-preferring and active conformations, respectively, within this ensemble need not be identical although they must clearly overlap. In the multi-state model the preferential binding of an agonist therefore does not explicitly imply the formation of an active state as reflected in, for example, stimulation of adenylyl cyclase. The multi-state model thus, in contrast to the two-state model, accommodates independent changes in agonist affinity and basal and ligand-stimulated activity. In the present analysis we have observed not only a lack of correlation between agonist affinity and basal (constitutive) activity but, in addition, disparate changes in agonist affinity and the potency for receptor stimulation. The data therefore, when viewed in the context of the multi-state model, suggest the existence of receptor conformations that, although characterized by an enhanced agonist affinity, exhibit no increase in basal activity and even a decreased ability to interact productively with the G-protein as reflected in the rightward shift in EC50 values. Together, these results emphasize receptor binding and receptor activation as separate entities.

The observation that the [H178R] substitution, when present in a construct physically deleted for essentially the entire extracellular domain, is capable of conferring constitutive activity provides further evidence for the separation of binding and activation, as the TM domain of the receptor by itself is sufficient to establish an active conformation of the receptor. This notion further agrees with a series of chimeric studies employing pairs of receptors from this family. In these studies substitution and/or deletion of parts of the N-terminal domain disrupt high-affinity binding of the peptide (7, 8, 9, 10), as seen also after the mutation of a single residue, e.g. Asp64 in the glucagon receptor (29), or either of the six highly conserved cysteine residues that have been presumed to play at least an important structural role for this receptor domain (29, 30).

Distinct Activation Mechanisms for 7TM Receptor Subfamilies
In the PTH receptor, substitution of a series of residues, which together could form a polar surface on the TM-II helix, suggested this domain as being important for signaling (31). Substitution in the TM-II domain in all instances impaired the ability of the receptors to stimulate adenylyl cyclase. Notably, substitution of the highly conserved histidine at the cytoplasmic pole of TM-II into either aspartate or alanine was accompanied by an improved affinity for the PTH peptide ligand, similar to the observation of the present study for the His178 mutants. In the secretin receptor, similar results were obtained after the exchange of the corresponding histidine residue (32), i.e. improved ligand binding affinity. However, for none of these receptor mutants was constitutive adenylyl cyclase activity discerned. The strict requirement for exchange into a particular side chain (e.g. His -> Arg) is in contrast to observations in the adrenergic {alpha}1B-receptor, in which the alteration of an aspartate at the bottom of TM-III (31a) or an alanine at the bottom of TM-VI (20), respectively, led to enhanced receptor activity irrespective of the choice for substitution. In the glucagon and PTH receptors, the specific choice for substitution is seemingly critical in determining whether or not basal receptor activity is affected.

As summarized in Table 3Go, even identical substitutions at homologous positions (here His‘178’ -> Arg) affect signaling properties dissimilarly among members of the glucagon/VIP/calcitonin receptor family. In the PTH, the VIP, and the glucagon receptors, substitution of histidine into argine was accompanied by enhanced cAMP signaling (23, 32), yet in the GLP-1, the GIP, and the calcitonin receptors this same histidine into arginine substitution was neutral or even impaired the ability to signal through adenylyl cyclase (33, 34). Furthermore, while a naturally occurring TM-VI substitution in the PTH receptor enhanced the basal cAMP level, signaling was barely affected upon introduction of the identical substitution in the glucagon receptor in the present study. Rather, the glucagon receptor substitution into an alternate residue, i.e. alanine, provided the largest enhancement of signaling, exemplifying once again the importance of the actual amino acid substitutent present at these highly conserved positions, as recently seen also for the PTH receptor (27). The distinctly different signaling phenotypes arising from even identical exchanges in homologous receptors was recently pursued in the rhodopsin-like gonadotropin receptors. Here the exchange of a conserved residue in IC loop 3 had no effect on the basal activity of the FSH receptor, whereas in the homologous LH receptor this represents a naturally occurring mutation associated with constitutive activation. Interestingly, when this substitution was instead presented also in the context of the two surrounding LH receptor segments, TM-V and TM-VI, activation of the chimeric FSH receptor was indeed observed (35).


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Table 3. Constitutive Activity Associated with a Highly Conserved Histidine Residue in Receptors from the Glucagon/VIP/Calcitonin Receptor Family

 
Inverse Agonism
The presence of spontaneous receptor activity affords the possibility to observe inverse agonism, a phenomenon that had until recently been reported only for the rhodopsin-like class of 7TM receptors (17). When the naturally occurring constitutively active PTH receptors were used, it became feasible to test a number of PTH peptide analogs with regard to inverse agonism and thus to identify two peptides as being inverse agonists, i.e. capable of suppressing the basal level of activity (36). Similar compounds, peptidic as well as nonpeptidic, would therefore be expected to exist also for other members of the glucagon/VIP/calcitonin receptor family.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Construction of Mutant Receptor cDNAs
Point mutagenesis was performed by the PCR overlap extension technique using the wild-type glucagon cDNA as a template (37). Mutant constructs were identified initially through the presence of diagnostic restriction sites that were introduced via the mutated oligonucleotides, and mutations were subsequently verified by sequencing of the PCR-generated segment of the cDNA using either USB Sequenase or Thermo Sequenase (Amersham, Arlington Heights, IL) with the ALFexpress DNA Sequencer (Pharmacia, Piscataway, NJ). Pfu Polymerase (Stratagene, La Jolla, CA) was employed as PCR enzyme. All mutant and wild-type receptor cDNAs were present as inserts in the expression vector pTEJ8 (38).

Expression of Receptor Constructs in COS-7 Cells
Receptor cDNAs were transiently expressed and introduced into COS-7 cells by the calcium phosphate precipitation method using 40 µg of plasmid DNA per 6 x 106 cells (39) except in the gene dosage experiment in which variable amounts of cDNA were used for transfection as indicated in the legend to Fig. 4Go. After addition of the DNA-CaPO4 precipitate, cells were incubated in the presence of chloroquine (100 µM) for 5 h. One day after transfection the cells were harvested and seeded in 6-, 12-, or 24-well culture plates (1.0–7.5 x 104 cells per well) for whole-cell analysis by radioligand competition binding. Analysis of basal and ligand-stimulated cAMP synthesis was performed in parallel on cells seeded in six-well plates at a density of 3.5–5.0 x 105 cells per well.

Radioligand Binding Analysis of Transfected COS-7 Cells
The transfected COS-7 cells were analyzed by radioligand binding analysis on intact cells on the second day after transfection. The cells were incubated for 16 h at 4 C in 1 ml of buffer consisting of 25 mM Tris-HCl, pH 7.4, 5 mM MgCl2, using 35 pM [125I]glucagon as radioligand. Unlabeled peptide, used as a competitor, was present at concentrations ranging from 10-11 to 10-5 M. The cells were lysed by the addition of 1 ml lysis buffer (8 M carbamide, 3 M acetic acid, 2% NP40), and specifically bound radioligand was calculated as the difference between total counts of radioligand bound and counts bound in the presence of 1 µM glucagon. Monoiodinated [125I]glucagon, at a specific activity of 15 MBq/µg, was kindly provided by Dr. Ulla Dahl Larsen (Novo Nordisk A/S, Copenhagen, Denmark). The binding data were analyzed and IC50 values determined by nonlinear regression analysis using Inplot 4.0 (GraphPad Software, San Diego, CA). Kd, inhibition constant (Ki) and Bmax values were calculated from competition binding experiments using the equations: Kd = IC50 - L (L is the concentration of free radioligand), Ki = IC50/[1 + (L/Kd)], and Bmax = Bo(IC50/L).

Peptides
The peptide ligands, glucagon and des-His1-[Glu9]glucagon amide were kindly provided by L. B. Knudsen (Novo Nordisk A/S). Peptides were dissolved in 10-3 M acetic acid, 0.1% BSA for use in competition binding analysis and cAMP determination.

cAMP Production
The assay was performed as a slightly modified version of the method of Solomon et al. (40). Cells were cultured in six-well plates (5 x 105 cells per well) and incubated overnight with medium containing 2 µCi/ml of [3H]adenine (Amersham TRK311). After two washes in HBS buffer (25 mM HEPES pH = 7.2, 0.75 mM NaH2PO4, 140 mM NaCl) 1 ml of HBS containing 1 mM of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine was added per well, and the cells were then stimulated with peptide agonist for 15 min at 37 C. The cells were chilled on ice, medium was removed, and the incubation was terminated using 1 ml of ice-cold 5% trichloroacetic acid containing 0.1 mM of unlabeled cAMP and ATP. After 30 min incubation on ice, the supernatants were applied first to a Bio-Rad 50W-X4 resin (Bio-Rad, Richmond, CA) and subsequently to an alumina column (SIGMA A9003; Sigma Chemical Co., St. Louis, MO). The [3H]cAMP generated was eluted into scintillation tubes using 6 ml of 0.1 M imidazole, after which 15 ml of scintillation liquid (HighSafe 3) were added and the samples counted. The levels of cAMP production attained for each construct, agonist-induced as well as basal levels, were normalized relative to that obtained for the wild-type construct in the presence of 50 µM forskolin. The maximum response for the wild-type construct during glucagon stimulation (attained at {approx} 10-8 M) was 48 ± 5% of that obtained in the presence of 50 µM forskolin.


    ACKNOWLEDGMENTS
 
We thank Susanne Hummelgaard for enthusiastic and highly skilled technical assistance.


    FOOTNOTES
 
Address requests for reprints to: Siv A. Hjorth, Laboratory for Molecular Pharmacology, Department of Pharmacology, The Panum Institute, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark. Email: siv@molpharm.dk

This study was supported by grants from the Danish Medical Research Council, The Biotechnology Research Unit for Molecular Recognition, and the Novo Nordisk Foundation.

Received for publication June 19, 1997. Revision received September 16, 1997. Accepted for publication October 3, 1997.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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