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.
Highlights
The ECL2 of family B G protein-coupled receptor (GPCR) has been suggested to contribute to biological activity
As exendin(9 –39) affinity was unaltered at all mutant receptors, reductions in 125I-exendin(9 –39) binding but not antibody labeling may suggest a subpopulation of receptors for which the ligand binding domain of the receptor is misfolded, leading to loss of functional receptors at the cell surface
It is widely accepted that the extracellular loop (ECL) of family A GPCRs, ECL2, are an important component for receptor function, with roles that include facilitating ligand binding, receptor trafficking, communication of ligand signal, and/or stabilization of an active ligand-receptor complex
Summary
The ECL2 of family B GPCRs has been suggested to contribute to biological activity. Results: Mutation of most ECL2 residues to alanine results in changes in binding and/or efficacy of GLP-1 peptide agonists. The data available highlight the significance of the receptor core region in both peptide binding and receptor activation [34, 35], including residues within the putative ECL2 of the GLP-1R [36, 37], secretin receptor [18], corticotrophin-releasing factor receptors [38, 39], and parathyroid hormone receptors [16], suggesting this potentially forms a significant site of interaction for the N-terminal amino acids of the peptide ligands and/or plays an important role in stabilizing active state conformations in the presence of ligand. We have performed systematic substitution of each residue of ECL2 of the human GLP-1R by alanine and assessed the effects across a series of pharmacological outputs, which demonstrated critical residues for receptor activation that vary in an agonist peptide- or pathway-specific manner
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