The glucagon-like peptide-1 receptor (GLP-1R) is a class B G protein–coupled receptor that has a critical role in the regulation of glucose homeostasis, principally through the regulation of insulin secretion. The receptor system is highly complex, able to be activated by both endogenous [GLP-1(1–36)NH2, GLP-1(1–37), GLP-1(7–36)NH2, GLP-1(7–37), oxyntomodulin], and exogenous (exendin-4) peptides in addition to small-molecule allosteric agonists (compound 2 [6,7-dichloro-2-methylsulfonyl-3-tert-butylaminoquinoxaline], BETP [4-(3-benzyloxy)phenyl)-2-ethylsulfinyl-6-(trifluoromethyl)pyrimidine]). Furthermore, the GLP-1R is subject to single-nucleotide polymorphic variance, resulting in amino acid changes in the receptor protein. In this study, we investigated two polymorphic variants previously reported to impact peptide-mediated receptor activity (M149) and small-molecule allostery (C333). These residues were mutated to a series of alternate amino acids, and their functionality was monitored across physiologically significant signaling pathways, including cAMP, extracellular signal-regulated kinase 1 and 2 phosphorylation, and intracellular Ca2+ mobilization, in addition to peptide binding and cell-surface expression. We observed that residue 149 is highly sensitive to mutation, with almost all peptide responses significantly attenuated at mutated receptors. However, most reductions in activity were able to be restored by the small-molecule allosteric agonist compound 2. Conversely, mutation of residue 333 had little impact on peptide-mediated receptor activation, but this activity could not be modulated by compound 2 to the same extent as that observed at the wild-type receptor. These results provide insight into the importance of residues 149 and 333 in peptide function and highlight the complexities of allosteric modulation within this receptor system.
The glucagon-like peptide 1 receptor (GLP-1R) is a class B peptide hormone G protein–coupled receptor (GPCR) with physiologically important actions, including increases in insulin biosynthesis and secretion from pancreatic β-cells and decreases in β-cell apoptosis, gastric emptying, and peripheral tissue resistance to insulin. For these reasons, the GLP-1R is one of the key targets in the development of therapeutics for type 2 diabetes mellitus (DM). However, with increasing interest in establishing novel, long-acting and orally available therapeutics that eliminate or at least significantly reduce detrimental side effects, the pharmacologic complexities of targeting this receptor system are becoming evident.
The most well documented consequence of GLP-1R activation is enhanced cAMP production, which, along with cell membrane depolarization and the influx of Ca2+, is critical in the biosynthesis and exocytosis of insulin from pancreatic β-cells (Fehmann and Habener, 1992; Lu et al., 1993). However, the GLP-1R can couple via other G protein–dependent mechanisms, including Gαi, Gαo, and Gαq/11 (Montrose-Rafizadeh et al., 1999; Hallbrink et al., 2001), as well as via β-arrestin recruitment and signaling (Jorgensen et al., 2005, 2007; Sonoda et al., 2008). Furthermore, with the ability to be activated by multiple endogenous agonists [GLP-1(1–36)NH2, GLP-1(1–37), GLP-1(7–36)NH2, GLP-1(7–37) and oxyntomodulin] as well as the exogenous peptide agonist exendin-4 (exenatide, Byetta; AstraZeneca, Wilmington, DE) that is currently used as a type 2 diabetic treatment and allosteric ligands such as compound 2 [6,7-dichloro-2-methylsulfonyl-3-tert-butylaminoquinoxaline], the phenomenon of biased agonism can be clearly observed at this receptor (Kenakin, 1995, 2011; Koole et al., 2010, 2012a,b). Adding to the complexity of this receptor system, recent pharmacologic analysis of GLP-1R single nucleotide polymorphisms (SNPs) (Beinborn et al., 2005; Fortin et al., 2010; Koole et al., 2011) identified two variants that significantly influence receptor function: M149 [transmembrane domain (TM) 1], which attenuates endogenous and exogenous peptide-mediated receptor function (Beinborn et al., 2005; Koole et al., 2011), and C333 [intracellular loop (ICL) 3], which reduces the allosteric agonism of the GLP-1R small-molecule compound 2, as well as significantly impacting its modulatory profile (Koole et al., 2011). Although population analysis of these receptor variants suggests a low heterozygous frequency and unknown homozygous frequency, for at least the M149 variant, there has been a direct implication in the onset of type 2 DM (Tokuyama et al., 2004). Moreover, the significant loss of peptide function at this receptor variant would also suggest that subjects administered peptide mimetics, such as exendin-4, would experience limited effectiveness in management of the condition (Koole et al., 2011).
In the absence of high-resolution crystal structures of the GLP-1R in its entirety, and, in fact, any class B GPCR as a whole entity, the structural role of these residues and their influence on the mechanistic function of the receptor are largely unclear. However, with the emergence of two class B TM crystal structures (Hollenstein et al., 2013; Siu et al., 2013) and a plethora of mutagenesis (Lopez de Maturana and Donnelly, 2002; Lopez de Maturana et al., 2004; Coopman et al., 2011; Koole et al., 2012a,b; Wootten et al., 2013) and photoaffinity labeling data (Al-Sabah and Donnelly, 2003; Dong et al., 2004, 2007, 2011; Chen et al., 2009, 2010; Miller et al., 2011; Coin et al., 2013), structurally and functionally important components of the GLP-1R can begin to be predicted and complementary molecular models can be further refined. In this study, we have created a series of mutations at two GLP-1R residues subject to polymorphic variance (amino acids 149 and 333; Fig. 1) at which receptor function is significantly affected, to examine more broadly their involvement in receptor structure and function. We observed that mutation of residue 149, at which Thr most frequently occurs, is poorly tolerated in the context of peptide-mediated receptor activation but not that of the allosteric agonist compound 2. In addition, mutants of this residue with significantly reduced peptide activity were able to have at least partial restoration of function through compound 2–mediated allosteric modulation of the receptor. Conversely, at residue 333, at which Ser most frequently occurs, receptor mutants exerted little influence on peptide function in any detected output; however, modulation of oxyntomodulin-induced cAMP formation by compound 2 was significantly attenuated despite compound 2 retaining agonism. Together, these results not only enhance our understanding of the role of SNPs in receptor activity but also facilitate the refinement of models in understanding the complex molecular mechanisms involved in GLP-1R activity.
Materials and Methods
Dulbecco’s modified Eagle’s medium, hygromycin-B, and Fluo-4 acetoxymethyl ester were purchased from Invitrogen (Carlsbad, CA). Fetal bovine serum (FBS) was purchased from Thermo Fisher Scientific (Melbourne, VIC, Australia). The QuikChange site-directed mutagenesis kit was purchased from Stratagene (La Jolla, CA). AlphaScreen reagents, Bolton-Hunter reagent ([125I]), and 384-well ProxiPlates were purchased from PerkinElmer Life and Analytical Sciences (Waltham, MA). SureFire ERK1/2 reagents were generously supplied by TGR Biosciences (Adelaide, SA, Australia). SigmaFast O-phenylenediamine dihydrochloride tablets and antibodies were purchased from Sigma-Aldrich (St. Louis, MO). Compound 2 was generated according to a method published previously (Teng et al., 2007) to a purity of >95%, and compound integrity was confirmed by NMR. GLP-1 and GLP-1 peptide analogs were purchased from American Peptide (Sunnyvale, CA). All other reagents were purchased from Sigma-Aldrich or BDH Merck (Melbourne, VIC, Australia) and were of an analytical grade.
SNPs of the GLP-1R with pharmacologic profiles deviating from wild-type, as determined from our previous study (Koole et al., 2011), were mutated to a selection of amino acids. Mutations were introduced to an N-terminally double c-myc labeled wild-type human GLP-1R in the pEF5/FRT/V5-DEST destination vector (Invitrogen); this receptor had equivalent pharmacology to the untagged human GLP-1R (data not shown). Mutagenesis was carried out using oligonucleotides for site-directed mutagenesis from GeneWorks (HindMarsh, SA, Australia) (Supplemental Table S1) and the QuikChange site-directed mutagenesis kit (Stratagene). Sequences of receptor clones were confirmed by cycle sequencing as previously described (May et al., 2007). In this study, wild-type GLP-1R is composed of T149 and S333.
Transfections and Cell Culture.
Wild-type and mutant human GLP-1R were isogenically integrated into FlpIn-Chinese hamster ovary (FlpInCHO) cells (Invitrogen), and selection of receptor-expressing cells was accomplished by treatment with 600 μg ml−1 hygromycin-B as previously described (May et al., 2007). Transfected and parental FlpInCHO cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated FBS and incubated in a humidified environment at 37°C in 5% CO2.
Radioligand Binding Assay.
FlpInCHO wild-type and mutant human GLP-1R cells were seeded at a density of 3 × 104 cells/well into 96-well culture plates and incubated overnight at 37°C in 5% CO2, and radioligand binding was carried out at 4°C as previously described (Koole et al., 2011). For each cell line in all experiments, total binding was defined by 0.5 nM [125I]exendin(9–39) alone, and nonspecific binding was defined by 1 μM exendin(9–39). For analysis, data are normalized to the B0 value for each individual experiment. Of note, the condition of assay for radioligand binding and functional experiments are different and as such cannot be directly compared.
cAMP Accumulation Assay.
FlpInCHO wild-type and mutant human GLP-1R cells were seeded at a density of 3 × 104 cells/well into 96-well culture plates and incubated overnight at 37°C in 5% CO2, and cAMP detection carried out as previously described (Koole et al., 2010). For interaction studies, increasing concentrations of peptide and 3 μM compound 2 were added simultaneously, and cAMP accumulation measured after 30 minutes of cell stimulation. All values were converted to concentration of cAMP using a cAMP standard curve performed in parallel, and data were subsequently normalized to the response of 100 μM forskolin in each cell line. Agonist stimulation and interaction studies were performed as two different series of experiments and on different cell passages.
Phosphorylated Extracellular Signal-Regulated Kinase 1 and 2 Assay.
FlpInCHO wild-type and mutant human GLP-1R cells were seeded at a density of 3 × 104 cells/well into 96-well culture plates and incubated overnight at 37°C in 5% CO2. Receptor-mediated phosphorylated extracellular signal-regulated kinase 1 and 2 (pERK1/2) was determined using the AlphaScreen ERK1/2 SureFire protocol as previously described (May et al., 2007). Initial pERK1/2 time course experiments were performed over 1 hour to determine the time at which agonist-mediated pERK1/2 was maximal. Subsequent experiments were then performed at the time required to generate a maximal pERK1/2 response (6 minutes). Data were normalized to the maximal response elicited by 10% FBS in each cell line, determined at 6 minutes (peak FBS response).
iCa2+ Mobilization Assay.
FlpInCHO wild-type and mutant human GLP-1R cells were seeded at a density of 3 × 104 cells/well into 96-well culture plates and incubated overnight at 37°C in 5% CO2, and receptor-mediated iCa2+ mobilization was determined as previously described (Werry et al., 2005). Fluorescence was determined immediately after peptide addition, with an excitation wavelength set to 485 nm and an emission wavelength set to 520 nm; readings were taken every 1.36 seconds for 120 seconds. Peak magnitude was calculated using five-point smoothing, followed by correction against basal fluorescence. The peak value was used to create concentration-response curves. Data were normalized to the maximal response elicited by 100 μM ATP.
Cell-Surface Receptor Expression.
FlpInCHO wild-type and mutant human GLP-1R cells, with receptor DNA previously incorporated with an N-terminal double c-myc epitope label, were seeded at a density of 25 × 104 cells/well into 24-well culture plates and incubated overnight at 37°C in 5% CO2, washed three times in 1× PBS, and fixed with 3.7% paraformaldehyde at 4°C for 15 minutes. Cell-surface receptor detection was then performed as previously described (Koole et al., 2011). Data were normalized to the basal fluorescence detected in FlpInCHO parental cells. Specific [125I]exendin(9–39) binding at each receptor mutant, as identification of functional receptors at the cell surface, was also determined [corrected for nonspecific binding using 1 μM exendin(9–39)].
All data were analyzed using Prism 5.04 (GraphPad Software Inc., San Diego, CA). For all analyses, the data were unweighted, and each y value (mean of replicates for each individual experiment) was considered an individual point. Concentration-response signaling data were analyzed using a three-parameter logistic equation (eq. 1) as previously described (May et al., 2007):(1)where bottom represents the E value in the absence of ligand(s), top represents the maximal stimulation in the presence of ligand(s), [A] is the molar concentration of ligand, and EC50 represents the molar concentration of ligand required to generate a response halfway between top and bottom. Similarly, this equation was used in the analysis of inhibition binding data, replacing EC50 with IC50. In this case, bottom defines the specific binding of the radioligand that is equivalent to nonspecific ligand binding, whereas top defines radioligand binding in the absence of a competing ligand, and the IC50 value represents the molar concentration of ligand required to generate a response halfway between top and bottom. IC50 values obtained were then corrected for radioligand occupancy as previously described (Cheng and Prusoff, 1973) using the radioligand affinity (Ki) experimentally determined for each mutant.(2)
where top represents the maximal stimulation in the system; KA is the agonist-receptor dissociation constant, in molar concentration; τ is the estimated measure of efficacy in the system, which incorporates both signaling efficacy and receptor density; and all other parameters are as defined for eq. 1. Constraints for this model were determined by fitting the most efficacious peptide with the eq. 3:(3)The value obtained for the system maximum (Em) was then globally constrained as the parameter, top, in the operational model (eq. 2) when applied at each mutant receptor. All estimated τ values were then corrected to cell surface expression (τc) as determined by percent specific [125I]exendin(9–39) binding and errors propagated from both τ and cell surface expression relative to wild-type receptor. Of note, differences in functional KA values derived from fitting the operational model may arise from the presence of noninterconverting states that are unique to ligand-receptor-effector complexes (Leff et al., 1997; Holst et al., 2001; Zheng et al., 2008; McPherson et al., 2010; Strachan et al., 2010; Nijmeijer et al., 2012), and this is observed for peptide agonists at the wild-type GLP-1R (Supplemental Table S2).
Changes in peptide affinity, potency, efficacy, and cell-surface expression of human GLP-1R mutants in comparison with wild-type human GLP-1R control were statistically analyzed with one-way analysis of variance and Dunnett’s post-test, and significance was accepted at P < 0.05.
Mutation of Residue 149 or 333 Has Minimal Impact on Functional Human GLP-1R Expression at the Cell Surface.
Wild-type N-terminally c-myc–tagged human GLP-1R or receptors incorporating the mutations of residues 149 or 333 were isogenically introduced into FlpInCHO host cells by recombination, and cell surface expression was determined through antibody detection of the c-myc epitope label (Table 1). In this study, mutation of residue 333 to either Ala or Val caused little deviation in cell surface expression in comparison with the wild-type human GLP-1R. In contrast, mutation of residue 149 to Val or Tyr significantly reduced receptor cell surface expression, whereas mutation of residue 149 to Cys significantly increased cell surface expression in comparison with wild-type control. No significant change in specific [125I]exendin(9–39) binding was observed, although the C149 mutant trended toward increased binding (P = 0.11) consistent with the cell surface expression as monitored by c-myc antibody binding (Table 1). In addition, there was no significant difference in the level of receptor expression as determined by [125I]exendin(9–39) binding. Wild-type receptor expression was 1.87 ± 0.33 million receptors/cell, and the mean level of expression across all mutants was 1.55 ± 0.32 million receptors/cell (data not shown).
Mutation of Residue 149 but Not 333 of the Human GLP-1R Significantly Affects Peptide Agonist Binding Affinity but Not Antagonist Exendin(9–39) Binding Affinity.
Binding affinity of orthosteric GLP-1R peptide ligands at each of the mutant receptors was determined through equilibrium binding in the presence of the radiolabeled antagonist, [125I]exendin(9–39) (Fig. 2; Table 1). Homologous competition identified no significant changes in antagonist exendin(9–39) binding affinity at any mutant receptor (Table 1). Consistent with results previously published (Koole et al., 2012a), complete inhibition curves were unable to be determined in the presence of GLP-1(1–36)NH2 at any receptor mutant; given the small window for competition within the concentration range tested, any deviations in binding affinity of this peptide at receptor mutants in comparison with wild-type control were difficult to interpret (Fig. 2A). For the remaining agonist peptides, no significant changes in binding affinity were observed for either Ala or Val substitutions at residue 333, whereas decreases in the binding affinity of GLP-1(7–36)NH2, exendin-4, and oxyntomodulin were observed at all residue 149 receptor mutants (Fig. 2, B–D; Table 1). However, the extent of affinity reduction was variable, depending on both the residue substituted as well as the peptide present, with an overall greater effect of mutation on the affinity of GLP-1(7–36)NH2 than exendin-4 or oxyntomodulin (Table 1). This is clearly evident at mutant receptors A149 and V149, with reductions of 79-fold for exendin-4 and oxyntomodulin at both mutants but 200- and 251-fold, respectively, for GLP-1(7–36)NH2. Similarly, mutation of residue 149 to Phe resulted in decreases in binding affinity of 158-, 316-, and 501-fold for exendin-4, oxyntomodulin, and GLP-1(7–36)NH2, respectively. Perhaps not surprisingly, mutation of residue 149 to Ser, which has properties of greatest similarity to that of the most frequently occurring residue, Thr, had the least effect on peptide binding affinity, albeit most reductions still reached statistical significance.
Most Mutations of Residue 149 but Not 333 of the Human GLP-1R Significantly Decrease Peptide-Mediated cAMP Accumulation.
Whereas the GLP-1R is recognized as a pleiotropically coupled receptor, it is most well characterized for its role in enhancing adenylate cyclase activity and promoting the formation of cAMP, which is directly linked to the secretion of insulin (Baggio and Drucker, 2007). Consequently, we assessed the ability of each mutant receptor to augment cAMP accumulation in the presence of each peptide agonist (Fig. 3; Table 2). Consistent with effects on binding affinity, no significant effect of mutation of residue 333 on peptide potency in cAMP accumulation was observed (Table 2). Taking into account cell-surface receptor expression and pathway coupling efficiency, application of the operational model to yield the operational measure of efficacy (τc) illustrated a general trend for decreased peptide-mediated cAMP coupling at both Ala and Val mutants of residue 333, although this trend did not reach statistical significance. In contrast, decreases in peptide potency in cAMP were observed for all mutant receptors of residue 149 (Fig. 3; Table 2). In agreement with binding data, peptide-mediated cAMP was least affected at S149 with respect to wild-type (T149), and this was also reflected by no significant differences in coupling efficacy (τc) when the operational model was applied (Table 2). However, there were trends for decreases in coupling efficacy in the presence of GLP-1(7–36)NH2 and oxyntomodulin but increases in the presence of GLP-1(1–36)NH2 and exendin-4, suggesting that the additional methyl group of Thr may contribute to discrimination of orthosteric peptide signaling profiles. Mutation of residue 149 to Cys resulted in decreased peptide potency but reached statistical significance only for oxyntomodulin. Analysis with the operational model revealed no significant deviations in pathway coupling efficacy (τc) at C149 in the presence of any peptide, although there was a trend toward decreases for all peptides in comparison with the wild-type (T149) receptor. Significant reductions in peptide-mediated cAMP were noted with mutation of T149 to Ala, Phe, Ile, Val, and Tyr, with only weak responses detected and potency values unable to be determined for GLP-1(1–36)NH2, GLP-1(7–36)NH2, and oxyntomodulin. Despite the inability to determine peptide potency values at these mutants, the rank order of reduction in peptide-mediated cAMP was consistent for all peptides: Ala > Val > Phe > Ile > Tyr (Fig. 3).
Most Mutations of Residue 149 but Not 333 of the Human GLP-1R Abolish iCa2+ Mobilization.
Consistent with work presented previously (Koole et al., 2010), iCa2+ was weakly coupled to GLP-1R activation in FlpInCHO cells, with no notable response for GLP-1(1–36)NH2 at either the wild-type (T149, S333) or any receptor mutant (data not shown). Similarly, complete concentration-response curves in the presence of oxyntomodulin could not be established over the concentration range tested in this study. Comparison of responses at the highest peptide concentration tested (1 μM) revealed that mutation of residue 333 caused small increases in oxyntomodulin-mediated iCa2+ mobilization; however, these increases were not statistically significant (Table 3). Increases in Emax were also observed in GLP-1(7–36)NH2- and exendin-4–mediated iCa2+ mobilization responses at both 333 mutants, despite little deviation in peptide potency. Although this increase in Emax reached statistical significance for GLP-1(7–36)NH2, these data did not translate into significantly enhanced pathway coupling efficacy (τc) as determined through operational modeling (Table 3). Almost all substitutions at residue 149 had a significant impact on peptide-mediated iCa2+ mobilization (Fig. 4). The exception was S149, which displayed similar activity to that of the wild-type (T149) GLP-1R in the presence of all measurable peptide responses, reflected in the coupling efficacy (Fig. 4; Table 3). Interestingly, C149 showed no significant changes in peptide potency for either GLP-1(7–36)NH2 or exendin-4, but it showed significant attenuation of maximal response for exendin-4 and oxyntomodulin (the latter a measure of response at 1 μM) and was also reflected in the estimated coupling efficacy (τc) whereby although both GLP-1(7–36)NH2 and exendin-4 had reduced efficiency in the Ca2+ pathway, only exendin-4 reached statistical significance, indicating possible ligand specific effects of this residue on peptide activity (Table 3).
Most Mutations of Residue 149 but Not 333 of the Human GLP-1R Significantly Decrease Peptide-Mediated pERK1/2.
Peptide-mediated pERK1/2 was measured at 6 minutes for each receptor mutant, the time at which maximal stimulation of pERK1/2 occurred at the wild-type GLP-1R (data not shown). For mutants with measurable peptide-mediated pERK1/2 responses, there was no significant alteration of peptide potency (Fig. 5; Table 4). This finding is consistent with what we have previously seen at GLP-1R mutants, indicating that receptor mutation generically has a lesser impact on coupling to ERK1/2 signaling (Koole et al., 2011). In accord with iCa2+ mobilization data, no significant alterations in peptide potency were noted at either mutant of residue 333, yet both mutants displayed notable increases in Emax that were in turn reflected in an increase in pathway coupling efficacy. However, these increases did not reach statistical significance for any peptides (Table 4). Receptor mutants F149, I149, V149, and Y149 profoundly affected pERK1/2 signaling, with little to no detectable responses for both GLP-1 peptides and significant effects on maximal responses for exendin-4 and oxyntomodulin (Fig. 5; Table 4). However, in most cases operational modeling revealed no statistically significant changes in coupling efficiency (τc) at these mutants. Mutation of 149 to either Ala or Cys significantly impacted the Emax of both GLP-1(7–36)NH2 and oxyntomodulin, but it had little effect on exendin-4. However, application of the operational model revealed that although all peptides had reduced coupling efficacy (τc) at these mutants, none reached statistical significance. As predicted, there was little deviation in peptide responses at the S149 mutant.
Effect of Residue 149 or 333 of the Human GLP-1R on the Signaling Profile of the Allosteric Agonist Compound 2.
Previously, we demonstrated that despite a loss of peptide agonist binding and signaling at the naturally occurring M149 receptor variant, the agonist profile of the small-molecule GLP-1R allosteric ligand compound 2 was retained (Koole et al., 2011). In the present study, mutation of residue 333 did not significantly affect the potency of compound 2 in either cAMP or pERK1/2 signaling profiles (Tables 2 and 4). Despite significant reductions observed at the polymorphic variant C333, A333 and V333 did not display any significant effects on compound 2 agonism. Mutation of 149 did not significantly affect the potency of compound 2 in either cAMP accumulation or pERK1/2 outputs (Tables 2 and 4). Coupling efficacy in pERK1/2 increased for A149, F149, S149, and Y149, although these increases were not statistically significant (Table 4). Consistent with previous studies, no measurable agonism was observed for compound 2 in iCa2+ mobilization at either the wild-type or any of the mutant receptors (data not shown).
Effect of Residue 149 or 333 of the Human GLP-1R on the Modulatory Profile of Compound 2.
We have previously shown that the loss of agonist peptide binding and cAMP signaling at the naturally occurring M149 receptor variant could be partially rescued in the presence of compound 2 (Koole et al., 2011). In addition, the C333 receptor variant was not modulated by compound 2 to the extent observed at the wild-type GLP-1R (S333) (Koole et al., 2011). In the present study, we therefore examined the role of compound 2 in modulating peptide-induced cAMP responses of residue 149 and 333 mutants. Similar to previous observations, significant positive modulation of oxyntomodulin-mediated cAMP occurred with the addition of compound 2 at the wild-type GLP-1R, but no significant effects were seen on GLP-1(1–36)NH2, GLP-1(7–36)NH2, or exendin-4 (Table 5; Supplemental Figs. S1–S4; Koole et al., 2010). Although no modulation of these last peptides was observed at either 333 mutants, compound 2 also failed to modulate significantly the oxyntomodulin responses at A333 and V333 (Table 5; Supplemental Fig. S4). Similar to wild-type GLP-1R, mutation of residue 149 to Ser had little effect on the modulatory profile of compound 2, with a substantial enhancement of oxyntomodulin potency but no significant effect on other peptides (Table 5; Supplemental Figs. S1–S4). At all other substitutions of residue 149, significant compound 2–mediated augmentation of oxyntomodulin potency was observed; however, the extent of modulation was somewhat variable among mutants; the greatest detectable recovery of potency was observed at the A149 mutant, whereas only modest recovery was seen at the F149 mutant (100- and 16-fold enhancement of oxyntomodulin potency, respectfully) (Table 5; Supplemental Fig. S4). As seen with the M149 receptor variant (Koole et al., 2011), in addition to modulation of oxyntomodulin, compound 2 positively modulated most other peptide responses of 149 mutant receptors (Table 5; Supplemental Fig. S1–S3). In several cases (F149, I149, V149, Y149), compound 2 restored the function of undetectable or undefined cAMP responses (Table 5; Supplemental Fig. S1–S4). This was particularly evident for Y149, with no detectable cAMP response for low-potency agonists GLP-1(1–36)NH2 and oxyntomodulin but recovery of a detectable peptide response in the presence of compound 2. Despite positively modulating most peptide responses at 149 mutants, the extent of modulation by compound 2 was not consistent across all mutants. Clear examples are A149, for which compound 2 modulated GLP-1(7–36)NH2 and oxyntomodulin potency to a greater extent than GLP-1(1–36)NH2 and exendin-4 (changes of 63-, 100-, 6-, and 6-fold, respectively), whereas at F149, exendin-4 potency was modulated to a greater extent than oxyntomodulin (changes of 200- and 16-fold, respectively) (Table 5). Interestingly, compound 2 modulated both GLP-1(7–36)NH2 and oxyntomodulin at C149, but it had little influence on GLP-1(1–36)NH2 and exendin-4 (changes of 8-, 32-, 1-, and 3-fold, respectively) (Table 5). These subtle changes in modulation profiles suggest an important role of residue 149 in directing probe-dependent effects on cooperativity.
Class B GPCRs are important regulators for a number of physiologic processes. Consequently, they have become valuable therapeutic targets for multiple disorders including neurodegenerative and inflammatory conditions (vasoactive intestinal peptide and pituitary adenylate cyclase–activating peptide receptors) (Abad et al., 2006; Brenneman, 2007), bowel disorders (GLP-2 receptors) (Hornby and Moore, 2011), chronic stress [corticotropin releasing factor (CRF) receptors] (Gilligan and Li, 2004), bone-related disorders (calcitonin and parathyroid hormone receptors) (Mulder et al., 2006), and type 2 DM (glucagon, amylin, and GLP-1Rs) (Estall and Drucker, 2006; Brubaker, 2007; Adeghate and Kalasz, 2011). Many of these GPCRs exhibit single-nucleotide polymorphic variance, with the variant proteins linked to development of multiple diseases, and there is additional potential to impact the effectiveness of treatments targeted to that receptor (Hager et al., 1995; Taboulet et al., 1998; Schipani et al., 1999; Sadee et al., 2001; Siani et al., 2001; Tang and Insel, 2005).
The naturally occurring GLP-1R variant, M149, markedly reduces peptide-mediated functional responses in vitro, suggesting that this would engender a pathophysiological phenotype. In accord, possession of this variant has been associated with poor glycemic control (Tokuyama et al., 2004). The mechanistic basis behind this loss of function receptor variant is largely unclear because of the paucity of structural information within this class of GPCRs. Consequently, we have substituted amino acids with different physicochemical properties to examine critically the functional profile of the 149, as well as the 333 polymorphic variant of the human GLP-1R to further elucidate the potential contribution of these residues to receptor structure and function.
We have also shown, in a previous study, that an SNP of the human GLP-1R that results in the conversion of Ser to Cys at amino acid residue 333 attenuated both the allosteric agonism as well as the modulatory properties of the Novo Nordisk small molecule, compound 2 (Koole et al., 2011). This effect was observed in the absence of any significant effects on peptide binding affinity or function as measured through cAMP accumulation, pERK1/2, and iCa2+ mobilization outputs. In the current study, mutation of residue 333 to either Ala or Val did not significantly impact on peptide binding affinity or functional activity. Homology modeling of the GLP-1R using the glucagon receptor structure as a template places residue 333 in ICL3, close to the TM5/ICL3 boundary (Fig. 1) (Siu et al., 2013). There is significant evidence to suggest that ICL3 is a major interaction interface for G proteins (Bavec et al., 2003), and this is illustrated in multiple mutagenesis studies (Heller et al., 1996; Takhar et al., 1996; Mathi et al., 1997; Salapatek et al., 1999), as well as in studies where G protein activation can be achieved with ICL3-corresponding peptides (Hallbrink et al., 2001). This work suggests an involvement of residues V327, I328 (Mathi et al., 1997), V331 (Mathi et al., 1997; Salapatek et al., 1999), I332, A333 (Salapatek et al., 1999), K334 (Takhar et al., 1996; Mathi et al., 1997; Salapatek et al., 1999), and R348 (Heller et al., 1996) in allowing effective G protein coupling to the GLP-1R. Notably, these mutagenesis data have been obtained using the rat GLP-1R. The human, mouse, and rat GLP-1Rs share almost identical amino acid sequence in ICL3, suggesting that conservation of this region is important in the G protein coupling profile of the receptor across species. However, although S333 is conserved in both the human and mouse GLP-1R proteins, this residue is an Ala in the rat GLP-1R, consistent with our current data that indicate that this is not a critical residue in peptide-mediated G protein coupling. These data may suggest that S333 lies outside the domain of critical importance in receptor–G protein interaction. In agreement, Mathi et al. (1997) proposed that the N-terminal region of ICL3 is a helical projection of TM5 with S333 facing away from the G protein binding pocket, and this is supported by the glucagon receptor and CRF1 receptor crystal structures and in our homology model (Fig. 1) (Hollenstein et al., 2013; Siu et al., 2013).
Despite little deviation in peptide function at the 333 residue in either the case of the polymorphic variant (C333; Koole et al., 2011) or the mutations introduced in this study (A333, V333), all these substitutions impact the cooperativity between compound 2 and oxyntomodulin, with no significant modulation of oxyntomodulin-mediated cAMP formation at C333 (Koole et al., 2011) and only weak positive modulation at the A333 and V333 mutants. However, unlike the C333 mutant, which shows compromised compound 2–induced cAMP production in comparison with the wild-type GLP-1R (Koole et al., 2011), no significant change in cAMP production was observed at either the A333 or the V333 mutants. Whereas the mechanistic basis of these observations is unclear, the evidence suggests that the potentially distinct receptor conformations stabilized by compound 2 drive agonism versus cooperativity.
Recently, it was revealed that compound 2 acts via covalent modification of the GLP-1R at C347 (located at the juxtaposition of ICL3/TM6; Fig. 1), in a mechanism similar to that of the modulator BETP [4-(3-benzyloxy)phenyl)-2-ethylsulfinyl-6-(trifluoromethyl)pyrimidine] (Nolte et al., 2014). Mutation of C347 to Ala resulted in loss of compound 2 agonism, in addition to loss of the positive allosteric modulator activity in the presence of GLP-1(9–36)NH2, whereas peptide-mediated signaling was unaltered. It is possible that mutation of residue 333 impacts the neighboring interactions required for compound 2 to attach covalently and/or induce conformations that favor sampling of receptor active states. Furthermore, the greater loss of compound 2 activity seen with the C333 mutant (Koole et al., 2011) may arise, at least in part, through alternate cross-linking through this site.
Unlike residue 333, most mutations of residue 149 resulted in significant attenuation of peptide binding and functional activity. The loss of binding affinity tracked through most functional assay systems assessed, manifesting as an attenuation of functional efficacy. Not surprisingly, mutation of 149 to Ser, which has similar chemical properties to that of the most frequently occurring residue, Thr, was relatively well tolerated and had the least influence on peptide binding and function in comparison with the other mutants. Nonetheless, a significant reduction in affinity was observed with most peptides, with variable effects on signaling that suggest that even minor modification at the 149 position impact on receptor function.
Removal of the hydroxyl functional group from T149, with maintenance of chain length, by mutation to Val significantly reduced peptide binding affinity and cAMP and iCa2+ responses. Similarly, replacement of the hydroxyl group with a thiol group by means of mutation to Cys significantly reduced peptide binding affinity, which generally corresponded with decreases in cAMP and iCa2+ outputs, albeit not always to significance. Interestingly, peptide-mediated pERK1/2 coupling efficacy (τc) at both V149 and C149 was not significantly altered. This result is consistent with distinct mechanisms of conformational transition driving pERK1/2 versus cAMP/iCa2+ signaling (Koole et al., 2011, 2012a; Wootten et al., 2013).
All other assessed mutations of residue 149 were poorly tolerated by the GLP-1R. Similar to the naturally occurring polymorphism M149, these residues are considerably more hydrophobic and bulky than the wild-type Thr; consistent with M149, they have large detrimental effects, which is perhaps not surprising given the recent structural information emerging for the GLP-1R. Recently, Miller and colleagues used photoaffinity labeling that detected close proximity between residues 16 and 12 of the GLP-1 peptide with residues 141 and 145 of the GLP-1R, respectively (Chen et al., 2010; Miller et al., 2011). Given that these residues precede, and would neighbor residue 149 in an α-helical tertiary structure, it indicates that 149 is near the endogenous peptide binding pocket. Although not necessarily a ligand interaction point itself, as suggested by homology assessment with CRF1 and glucagon receptor TM structures (Fig. 6) (Hollenstein et al., 2013; Siu et al., 2013), it may provide essential conformational restraints involving other TMs. This hypothesis is supported by recent alanine mutagenesis data of conserved polar residues in TM1 and TM2, where mutation of S155 (TM1) and S186 (TM2) differentially changed peptide-mediated signal bias at the receptor (Wootten et al., 2013). These residues are predicted to be involved in tight packing interactions with TM7 and TM3, respectively (Fig. 6), and the data are consistent with a proximal role of residue 149 in the activation transition of the receptor following peptide binding.
Despite significant attenuation of peptide binding and function at almost all mutants of 149, the ability of compound 2 to signal via the receptor was, in most cases, minimally affected. Similar to our previous work showing that compound 2 was able to partially restore the binding affinity and cAMP response of M149, in this study, we observed that compound 2 was able to modulate almost all peptide responses when measured in cAMP. The most striking data observed here included the restoration of functional responses at the F149 and Y149 mutants that had severely abrogated cAMP signaling, demonstrating that compound 2 is able to lower the energy required for activation at receptor mutants that either have no detectable cAMP or very weakly stimulate a cAMP response. Given that antagonist binding was unaffected at any of these receptor mutants, this finding provides evidence for our previous hypothesis that residue 149 is involved in activation transition instead of direct disruption to peptide binding interactions (Koole et al., 2011).
Collectively, these results provide us with insight into domains that are essential for receptor function, as well as the role that allosteric ligands play in receptor modulation.
The authors thank Dr. Michael Crouch (TGR Biosciences) for the generous donation of the Surefire ERK reagents.
Participated in research design: Koole, Wootten, Simms, Sexton.
Conducted experiments: Koole.
Performed data analysis: Koole.
Wrote or contributed to the writing of the manuscript: Koole, Wootten, Simms, Miller, Christopoulos, Sexton.
- Received October 20, 2014.
- Accepted January 26, 2015.
↵1 Current affiliation: Department of Pharmacology, Aston University, Birmingham, United Kingdom.
This work was funded by National Health and Medical Research Council of Australia (NHMRC) project [Grant 1061044, 1065410] and program [Grant 1055134] grants. P.M.S. and A.C. are Principal Research Fellows of the NHMRC.
- Chinese hamster ovary
- compound 2
- corticotropin-releasing factor
- diabetes mellitus
- fetal bovine serum
- glucagon-like peptide-1 receptor
- G protein–coupled receptor
- intracellular loop
- phosphorylated extracellular signal regulated kinase 1 and 2
- single nucleotide polymorphism
- Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics