Glucagon-like peptide-1 receptor dimerization differentially regulates agonist signaling but does not affect small molecule allostery

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 109; no. 45; pp. 18607 - 18612
• Main Authors: Harikumar, Kaleeckal G, Wootten, Denise, Pinon, Delia I, Koole, Cassandra, Ball, Alicja M, Furness, Sebastian G. B, Graham, Bim, Dong, Maoqing, Christopoulos, Arthur, Miller, Laurence J, Sexton, Patrick M
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 06-11-2012; National Acad Sciences
• Subjects: Agonists; Allosteric Regulation - drug effects; Animals; Biological Sciences; Calcium; Cercopithecus aethiops; CHO Cells; COS Cells; Cricetinae; cyclic AMP; Cyclic AMP - metabolism; Diabetes; Dimerization; Dimers; drugs; energy transfer; G-protein coupled receptors; Genes; glucagon-like peptide 1; Glucagon-Like Peptide-1 Receptor; Humans; insulin secretion; Ligands; mitogen-activated protein kinase; Modulated signal processing; Molecules; Mutation; noninsulin-dependent diabetes mellitus; peptide receptors; Peptides - pharmacology; Phosphorylation; Protein Multimerization - drug effects; Protein subunits; Protein Subunits - metabolism; Proteins; Receptors; Receptors, Glucagon - agonists; Receptors, Glucagon - metabolism; Signal Transduction - drug effects; Small Molecule Libraries - pharmacology; therapeutics
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1205227109

The glucagon-like peptide-1 receptor (GLP-1R) is a family B G protein-coupled receptor and an important drug target for the treatment of type II diabetes, with activation of pancreatic GLP-1Rs eliciting glucose-dependent insulin secretion. Currently, approved therapeutics acting at this receptor are peptide based, and there is substantial interest in small molecule modulators for the GLP-1R. Using a variety of resonance energy transfer techniques, we demonstrate that the GLP-1R forms homodimers and that transmembrane helix 4 (TM4) provides the primary dimerization interface. We show that disruption of dimerization using a TM4 peptide, a minigene construct encoding TM4, or by mutation of TM4, eliminates G protein-dependent high-affinity binding to GLP-1(7-36)NH ₂ but has selective effects on receptor signaling. There was <10-fold decrease in potency in cAMP accumulation or ERK1/2 phosphorylation assays but marked loss of intracellular calcium mobilization by peptide agonists. In contrast, there was near-complete abrogation of the cAMP response to an allosteric agonist, compound 2, but preservation of ERK phosphorylation. Collectively, this indicates that GLP-1R dimerization is important for control of signal bias. Furthermore, we reveal that two small molecule ligands are unaltered in their ability to allosterically modulate signaling from peptide ligands, demonstrating that these modulators act in cis within a single receptor protomer, and this has important implications for small molecule drug design.