Abstract
SummaryLigand-directed signal bias offers opportunities for sculpting molecular events, with the promise of better, safer therapeutics. Critical to the exploitation of signal bias is an understanding of the molecular events coupling ligand binding to intracellular signaling. Activation of class B G protein-coupled receptors is driven by interaction of the peptide N terminus with the receptor core. To understand how this drives signaling, we have used advanced analytical methods that enable separation of effects on pathway-specific signaling from those that modify agonist affinity and mapped the functional consequence of receptor modification onto three-dimensional models of a receptor-ligand complex. This yields molecular insights into the initiation of receptor activation and the mechanistic basis for biased agonism. Our data reveal that peptide agonists can engage different elements of the receptor extracellular face to achieve effector coupling and biased signaling providing a foundation for rational design of biased agonists.
Highlights
G protein-coupled receptors (GPCRs) are critical for the transmission of extracellular signals across the cell membrane to initiate intracellular responses (Fredriksson et al, 2003) and are the leading targets of currently marketed therapeutics (Overington et al, 2006)
To understand the functional interface at the GLP-1R extracellular surface, we completed alanine-scanning mutagenesis of the ECLs and adjacent TM residues, coupled with analysis of ligand affinity and signaling for three key pathways that are involved in GLP-1R function and rely on different effector engagement (Figure S1)
We assessed three peptides (GLP1, oxyntomodulin, and exendin-4) with highly conserved N-terminal sequences that display biased agonism (Figure S2). This biased agonism can be observed in both the recombinant cells used in this mutagenesis study and natively expressing insulinoma cells that display key features of b islets, where both exendin-4 and oxyntomodulin were biased away from GLP-1 in promotion of cellular proliferation and reducing apoptosis, compared to cAMP signaling (Figure S2)
Summary
G protein-coupled receptors (GPCRs) are critical for the transmission of extracellular signals across the cell membrane to initiate intracellular responses (Fredriksson et al, 2003) and are the leading targets of currently marketed therapeutics (Overington et al, 2006). Class B peptide hormone receptors are a subfamily of GPCRs that are major targets for the treatment of chronic disease, including type 2 diabetes, obesity, and dis-regulated bone metabolism (Couvineau and Laburthe, 2012). They include receptors that bind calcitonin, calcitonin gene-related peptide, vasoactive intestinal polypeptide, pituitary adenylate cyclaseactivating polypeptide, corticotropin releasing factor (CRF), gastric inhibitory polypeptide, parathyroid hormone, glucagon, and glucagon-like peptides (GLP-1 and GLP-2). Despite sequence divergence in this region between different receptors, this extracellular domain (ECD) contains key conserved residues, including three disulphide bonds that aid in stability and confer structural similarities between receptors
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