Second extracellular loop of human glucagon-like peptide-1 receptor (GLP-1R) has a critical role in GLP-1 peptide binding and receptor activation

Abstract

The glucagon-like peptide-1 receptor (GLP-1R) is a therapeutically important family B G protein-coupled receptor (GPCR) that is pleiotropically coupled to multiple signaling effectors and, with actions including regulation of insulin biosynthesis and secretion, is one of the key targets in the management of type II diabetes mellitus. However, there is limited understanding of the role of the receptor core in orthosteric ligand binding and biological activity. To assess involvement of the extracellular loop (ECL) 2 in ligand-receptor interactions and receptor activation, we performed alanine scanning mutagenesis of loop residues and assessed the impact on receptor expression and GLP-1(1-36)-NH(2) or GLP-1(7-36)-NH(2) binding and activation of three physiologically relevant signaling pathways as follows: cAMP formation, intracellular Ca(2+) (Ca(2+)(i)) mobilization, and phosphorylation of extracellular signal-regulated kinases 1 and 2 (pERK1/2). Although antagonist peptide binding was unaltered, almost all mutations affected GLP-1 peptide agonist binding and/or coupling efficacy, indicating an important role in receptor activation. However, mutation of several residues displayed distinct pathway responses with respect to wild type receptor, including Arg-299 and Tyr-305, where mutation significantly enhanced both GLP-1(1-36)-NH(2)- and GLP-1(7-36)-NH(2)-mediated signaling bias for pERK1/2. In addition, mutation of Cys-296, Trp-297, Asn-300, Asn-302, and Leu-307 significantly increased GLP-1(7-36)-NH(2)-mediated signaling bias toward pERK1/2. Of all mutants studied, only mutation of Trp-306 to alanine abolished all biological activity. These data suggest a critical role of ECL2 of the GLP-1R in the activation transition(s) of the receptor and the importance of this region in the determination of both GLP-1 peptide- and pathway-specific effects.

FIGURE 1.

Amino acid sequence alignments. A, alignments of putative ECL2 of human family B GPCRs, with absolute conservation of residues with respect to human GLP-1R, are highlighted in boldface, and putative TM/ECL2 boundaries are indicated; B, human family B peptides, with absolute conservation of residues with respect to human GLP-1 peptide, are highlighted in boldface.

FIGURE 2.

Cell surface expression profiles of human GLP-1R ECL2 alanine mutants. Cell surface expression profiles of each of the human GLP-1R ECL2 alanine mutants are compared with wild type stably transfected into FlpInCHO cells as determined through antibody detection of the N-terminal c-Myc epitope label (A) and by specific ¹²⁵I-exendin(9–39) binding (B). Statistical significance of changes in total cell surface expression in comparison with wild type human GLP-1R expression (100%) was determined by one-way analysis of variance and Dunnett's post-test and are indicated with an asterisk (*, p < 0.05). All data are means ± S.E. of seven to nine or three to four independent experiments conducted in duplicate for antibody detection and specific ¹²⁵I-exendin(9–39) binding, respectively.

FIGURE 3.

Agonist binding profiles of human GLP-1R ECL2 alanine mutants. Characterization of the binding of GLP-1(1–36)-NH₂ (A and B) and GLP-1(7–36)-NH₂ (C and D) in competition with the radiolabeled antagonist, ¹²⁵I-exendin(9–39), in whole FlpInCHO cells stably expressing the wild type human GLP-1R or each of the human GLP-1R ECL2 alanine mutants. Data are normalized to maximum ¹²⁵I-exendin(9–39) binding, with nonspecific binding measured in the presence of 1 μm exendin(9–39) and analyzed with a three-parameter logistic equation as defined in Equation 1. All values are means ± S.E. of three to four independent experiments, conducted in duplicate.

FIGURE 4.

cAMP accumulation profiles of human GLP-1R ECL2 alanine mutants. Characterization of cAMP accumulation in the presence of GLP-1(1–36)-NH₂ (A and C) and GLP-1(7–36)-NH₂ (B and D) in FlpInCHO cells stably expressing the wild type human GLP-1R or each of the human GLP-1R ECL2 alanine mutants that effect peptide binding affinity (A and B) or has no significant effect on peptide binding affinity (C and D) is shown. Data are normalized to the response elicited by 100 μm forskolin and analyzed with an operational model of agonism as defined in Equation 2. All values are means ± S.E. of four to seven independent experiments, conducted in duplicate. Visual representation of cAMP pathway coupling efficacy (logτ_c) in the presence of GLP-1(1–36)-NH₂ (E) GLP-1(7–36)-NH₂ and (F) is shown. Statistical significance of changes in coupling efficacy in comparison with wild type human GLP-1R was determined by one-way analysis of variance and Dunnett's post-test and is indicated with an asterisk (*, p < 0.05). All values are logτ_c ± S.E of four to seven independent experiments, conducted in duplicate.

FIGURE 5.

pERK1/2 profiles of human GLP-1R ECL2 alanine mutants. Characterization of pERK1/2 in the presence of GLP-1(1–36)-NH₂ (A and C) and GLP-1(7–36)-NH₂ (B and D) in FlpInCHO cells stably expressing the wild type human GLP-1R or each of the human GLP-1R ECL2 alanine mutants that effect peptide binding affinity (A and B) or has no significant effect on peptide binding affinity (C and D) is shown. Data are normalized to the maximal response elicited by 10% FBS and analyzed with an operational model of agonism as defined in Equation 2. All values are means ± S.E. of five to seven independent experiments, conducted in duplicate. Visual representation of ERK1/2 coupling efficacy (logτ_c) in the presence of GLP-1(1–36)-NH₂ (E) and GLP-1(7–36)-NH₂ (F). Statistical significance of changes in coupling efficacy in comparison with wild type human GLP-1R was determined by one-way analysis of variance and Dunnett's post-test and is indicated with an asterisk (*, p < 0.05). All values are logτ_c ± S.E. five to seven independent experiments, conducted in duplicate.

FIGURE 6.

Ca²⁺_i mobilization profiles of human GLP-1R ECL2 alanine mutants. Characterization of Ca²⁺_i mobilization in the presence of GLP-1(7–36)-NH₂ in FlpInCHO cells stably expressing the wild type human GLP-1R or each of the human GLP-1R ECL2 alanine mutants that effect peptide binding affinity (A) or has no significant effect on peptide binding affinity (B) is shown. Data are normalized to the maximal response elicited by 100 μm ATP and analyzed with an operational model of agonism as defined in Equation 2. All values are means ± S.E. of three to five independent experiments, conducted in duplicate. Visual representation of Ca²⁺_i coupling efficacy (logτ_c) in the presence of GLP-1(7–36)-NH₂ (C) is shown. Statistical significance of changes in coupling efficacy in comparison with wild type human GLP-1R was determined by one-way analysis of variance and Dunnett's post-test and is indicated with an asterisk (*, p < 0.05). All values are logτ_c ± S.E. of three to five independent experiments, conducted in duplicate.

FIGURE 7.

Correlation plots of pathway efficacy (logτ_c) of human GLP-1R ECL2 alanine mutants. Correlation plots of changes in pathway coupling efficacy (logτ_c) of human GLP-1R ECL2 alanine mutants with respect to wild type receptor are shown. A, cAMP versus pERK1/2 for GLP-1(1–36)-NH₂; B, cAMP versus pERK1/2 for GLP-1(7–36)-NH₂; C, Ca²⁺_i versus cAMP for GLP-1(7–36)-NH₂; D, Ca²⁺_i versus cAMP for GLP-1(7–36)-NH₂ after exclusion of D293A and Y305A; and E, pERK1/2 versus Ca²⁺_i for GLP-1(7–36)-NH₂. Data were fit by linear regression. The line of regression and 99% confidence intervals are displayed.