The ternary complex model suggests that G-protein-coupled receptors resonate between inactive (R) and active (R*) forms. Physiologically, R sites ordinarily predominate with a few R* sites giving rise to basal activity. Agonists recognize, stabilize and increase the R* population, thus altering intracellular activity. There is evidence to suggest the possibility of a spectrum of conformations between R and R*. Our aim is to study the consequences of putative GR (glucagon receptor)-activating mutations using glucagon and partial agonist des-His1-[Glu9]glucagon amide (glucagon-NH2). Alanine substitution in TM (transmembrane) helix 2 of Arg173 or of His177 detrimentally affected glucagon and glucagon-NH2 response maxima. TM2 receptor mutant, Phe181-Ala, displayed reduced maximum cAMP accumulation in response to glucagon-NH2. Thr353-Cys (TM6) and Glu406-Ala (TM7) receptors demonstrated constitutive activity and enhanced EC50 values for glucagon-NH2; Arg346-Ala (TM6) and Asn404-Ala (TM7) receptors were activated by sub-fmol glucagon concentrations, yet were not constitutively active and demonstrated wild-type receptor-like EC50 values for glucagon-NH2. Unlike Arg346-Ala receptors, Thr353-Cys, Asn404-Ala and Glu406-Ala receptors demonstrated improved EC50 values for glucagon, whereas their maximal responses to and their affinity for glucagon were comparable with the wild-type receptor. In contrast, despite slightly reduced glucagon-NH2 affinity, Arg346-Ala, Thr353-Cys, Asn404-Ala and Glu406-Ala receptors displayed glucagon-NH2 response maxima that exceeded those seen for wild-type receptors. Interestingly, we observed biphasic glucagon-mediated signalling responses. Our results are consistent with the concept of different agonists promoting the formation of distinct active states from partially active R*low to fully active R*high forms.

Introduction

The extended ternary complex model for GPCR (G-protein-coupled receptor) activation suggests that they interchange between inactive (R) and active (R*) states [1,2]. Classical antagonists bind indiscriminately to R and R* without physiological activity. Inverse agonists bind preferentially to R, shifting the equilibrium towards R, thus decreasing basal levels of activity. In contrast, agonists recognize, stabilize and promote R* formation. Agonists may occupy a low proportion of receptor sites, yet elicit a maximal cellular response or exhibit low-level activity despite high receptor occupancy. Classically, the former are full agonists and the latter, partial agonists. Since occupancy equates to affinity and signalling to efficacy it seems these are independent variables [3].

Agonist-independent (or constitutive) activity may be increased by receptor overexpression, causing a proportionate increase in R and R* sites or, alternatively, by mutations that shift the R–R* equilibrium towards R*. There is evidence for ligand acting both as agonist and inverse agonist at a GPCR, depending on the level of constitutive activity [4]. The ability to discriminate experimentally between different activation pathways is of potential therapeutic value.

The glucagon receptor (GR)

Glucagon, through GR activation, up-regulates blood sugar levels through stimulatory G-protein-linked cAMP release [5]. The GR is a secretin-like GPCR, which characteristically possess large extracellular N-termini with which their respective protein ligands make extensive contacts. Although the commensurate intracellular signalling events remain ill defined, there is little doubt that the TM (transmembrane) bundle transmits the agonist's signal through a series of conformational changes to the intracellularly associated G-protein [6,7].

Predictive modelling of the GR

There is little sequence identity between Family-B secretin-like receptors and Family-A rhodopsin-like receptors. However, there are some conserved features. TM3 has a conserved disulphide bond, TM4 a tryptophan, TM6 a proline and TM7 an asparagine residue. TM3 in Family B also has an EXXY motif that corresponds to the Family A D/ERY motif, with the arginine being provided by the intracellular region of an adjacent TM helix. After sequence alignment of these four helices with bovine rhodopsin, a partial TM bundle was constructed using the crystal structure of rhodopsin as a template [8]. The remaining TM helices were built manually, keeping alignments similar to bovine rhodopsin. On the basis of this model, Arg173, His177, Phe181 (TM2), Arg346, Thr353 (TM6), Asn404 and Glu406 (TM7) had predicted involvement in receptor activation. These residues were singularly mutated in the cDNA encoding the human GR.

Evaluation of receptor mutants

Receptor cDNAs were expressed in HEK-293 cells (human embryonic kidney 293 cells) stably transformed with a vector containing CRE (cAMP-response element) 5′ to DNA-encoding luciferase [9]. Adenylate cyclase stimulation increases cAMP levels and this promotes phosphorylation of the CRE-binding protein and ultimately luciferase production. Luciferase catalyses the conversion of luciferin into luminescent adenyl-oxyluciferin. Therefore the levels of luminescence theoretically correlate with intracellular cAMP levels. [125I]Glucagon competition analyses were used to determine affinities of glucagon and glucagon-NH2 (a partial agonist des-His1-[Glu9]glucagon amide at the rat GR [10]) for the transiently expressed GRs.

Evidence in support of a continuum of GR active states

Our results indicate that agonist affinity is a poor marker of mutation-induced constitutive activity. All the mutant receptors exhibited approximately wild-type [125I]glucagon-binding profiles, with the exception of the Arg173-Ala and His177-Ala GRs, which displayed negligible binding. The rat GR equivalent of His177 has been previously implicated in receptor activation [10]. Intriguingly, all receptor mutants (except Phe181-Ala) exhibited biphasic glucagon-mediated luciferase activity. EC50 values for the low concentration glucagon responses (EC501) were affected for Arg346-Ala, Thr353-Cys, Asn404-Ala and Glu406-Ala receptors (Figure 1 and Table 1).

Glucagon (•) and glucagon-NH2 (○) stimulated luciferase activity in receptor-expressing cells

Figure 1
Glucagon (•) and glucagon-NH2 (○) stimulated luciferase activity in receptor-expressing cells

Luciferase activity is expressed as a percentage of the response to 10 μM forskolin (a direct adenylate cyclase stimulator). Accumulated data from eight or more experiments performed in triplicate.

Figure 1
Glucagon (•) and glucagon-NH2 (○) stimulated luciferase activity in receptor-expressing cells

Luciferase activity is expressed as a percentage of the response to 10 μM forskolin (a direct adenylate cyclase stimulator). Accumulated data from eight or more experiments performed in triplicate.

Table 1
Data summary

IC50 values were determined by [125I]glucagon competition analysis and EC50 values by luciferase activity. NDB, no detectable binding; NDLA, no detectable luciferase activity. Glucagon-NH2 activity is quoted as the glucagon-NH2/glucagon activity ratio to determine intrinsic activity. Means±S.E.M. and basal activity determinations are given. Results are from three or more experiments performed in triplicate.

 Glucagon Glucagon-NH2   
Receptor IC50 (nM) EC501 (pM) EC502 (nM) IC50 (nM) EC50 (nM) Glucagon-NH2/Glucagon Basal activity 
Wild-type 20.0±7.3 8.2±0.4 49.2±24.3 14.5±5.4 288.1±87.4 0.21 3.1 
Arg173-Ala NDB NDLA NDLA NDB NDLA NDB/NDLA − 
His177-Ala NDB NDLA 12.0±3.4 NDB NDLA NDB/NDLA − 
Phe181-Ala 15.0±4.0 Not calculated 48.4±11.0 51.4±1.7 438.6±189.8 0.14 3.9 
Arg346-Ala 68.8±14.0 74.1±5.1 24.7±9.3 30.0±8.1 185±73.4 0.60 3.4 
Thr353-Cys 11.4±4.4 0.79±0.1 17.6±5.8 24.5±4.5 26.2±1.3 1.03 21.3 
Asn404-Ala 31.1±9.7 1.5±0.1 27.9±8.3 58.0±17 158.3±14.2 0.80 4.9 
Glu406-Ala 13.0±0.9 1.9±0.1 26.2±9.1 52.7±9.7 35.7±9.6 1.18 10.5 
 Glucagon Glucagon-NH2   
Receptor IC50 (nM) EC501 (pM) EC502 (nM) IC50 (nM) EC50 (nM) Glucagon-NH2/Glucagon Basal activity 
Wild-type 20.0±7.3 8.2±0.4 49.2±24.3 14.5±5.4 288.1±87.4 0.21 3.1 
Arg173-Ala NDB NDLA NDLA NDB NDLA NDB/NDLA − 
His177-Ala NDB NDLA 12.0±3.4 NDB NDLA NDB/NDLA − 
Phe181-Ala 15.0±4.0 Not calculated 48.4±11.0 51.4±1.7 438.6±189.8 0.14 3.9 
Arg346-Ala 68.8±14.0 74.1±5.1 24.7±9.3 30.0±8.1 185±73.4 0.60 3.4 
Thr353-Cys 11.4±4.4 0.79±0.1 17.6±5.8 24.5±4.5 26.2±1.3 1.03 21.3 
Asn404-Ala 31.1±9.7 1.5±0.1 27.9±8.3 58.0±17 158.3±14.2 0.80 4.9 
Glu406-Ala 13.0±0.9 1.9±0.1 26.2±9.1 52.7±9.7 35.7±9.6 1.18 10.5 

Glucagon-NH2 affinity (IC50) for the wild-type GR was approximately equimolar to that of glucagon. Glucagon-NH2 demonstrated a marginally reduced affinity for all the GR mutants when compared with the wild-type receptor (Table 1). Despite this, the response maxima to glucagon-NH2 were increased for Arg346-Ala, Thr353-Cys, Asn404-Ala and Glu406-Ala receptors. At Thr353-Cys and Glu406-Ala receptors, glucagon-NH2 acted as a full agonist (Figure 1). Thr353-Cys and Glu406-Ala receptors displayed increased EC50 values for glucagon-NH2 and evidence of constitutive activity as indicated by increased basal luminescence (Table 1). Although constitutive activity was not seen for the Arg346-Ala or Asn404-Ala receptors, sub-fmol glucagon concentrations elicited signalling to approx. 18 and 10% of the forskolin maxima respectively (see Figure 1). Phe181-Ala receptors demonstrated reduced glucagon-NH2 response maxima but not decreased EC50 values for this agonist. Negligible and substantially reduced glucagon-mediated luciferase activities were seen for Arg173-Ala and His177-Ala respectively. No luciferase activity was observed for either of these mutants after exposure to glucagon-NH2 (results not shown).

Conclusions

Our results support the view that the existing ternary complex models do not fully accommodate all possible receptor recognition and activation events. The discrepancy between the increase in glucagon-NH2 response maxima and apparently reduced glucagon-NH2 affinity (as seen for Thr353-Cys and Glu406-Ala receptors) suggests glucagon-NH2 may simply be unable to recognize the same R* conformation as [125I]glucagon. This points to the existence of at least two R* populations. We propose that glucagon-NH2 exerts its effects through binding to a subset of receptor sites that are distinct from, or overlapping with, the subset highlighted by [125I]glucagon. For instance, there may be R*glucagon-NH2 and R*glucagon sites, consistent with the proposal that there are ligand-specific receptor conformations [11].

When compared with wild-type receptors, the increased activity of glucagon-NH2 suggests that Thr353-Cys and Glu406-Ala mutant receptors are more readily capable of adopting the fully activated R* conformations necessary for G-protein activation. This is further evidence for the existence of a spectrum of R* states from partially active R*low to fully active R*high. Agonists may preferentially recognize a range of active receptor conformations. A proportion of these sites may be R*high sites. At the other extreme, sites may approximate states from which the agonist would dissociate, having triggered events culminating in signalling (R*low). This accommodates the observed increased basal levels of activity exhibited by some mutant receptors. Hence, basal activity and agonist affinity may change independently, in contrast with the two-state theory [12], providing further evidence that activation occurs through a series of conformational events [13]. The existence of a spectrum of receptor active states has potential consequences for rational drug design, particularly since different receptor sites may exhibit variable activation thresholds.

Research Colloquia: Research Colloquia at BioScience2004, held at SECC Glasgow, U.K., 18–22 July 2004. Edited by M. Bouvier (Montreal, Canada), G. Milligan (Glasgow, U.K.), V. O'Donnell (Cardiff, U.K.), M. Brand (MRC-Dunn Human Nutrition Unit, Cambridge, U.K.), M. Schweizer (Heriot-Watt University, Edinburgh, U.K.), R. Insall (Birmingham, U.K.), A. Ridley (Ludwig Institute for Cancer Research, London, U.K.) and M. Sutcliffe (Leicester, U.K.). The first eight papers featured in this Section were presented as a part of the GPCR Regulation and Signalling Research Colloquium, incorporating the GPCR–Ion Channel Interactions Pfizer-Sponsored Research Colloquium.

Abbreviations

     
  • CRE

    cAMP-response element

  •  
  • GPCR

    G-protein-coupled receptor

  •  
  • GR

    glucagon receptor

  •  
  • TM

    transmembrane

This project was funded by a BBSRC CASE studentship in collaboration with GlaxoSmithKline (U.K.). We acknowledge GlaxoSmithKline for providing materials including the GR cDNA and the HEK-293 CRE luciferase cell line.

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