Polar transmembrane interactions drive formation of ligand-specific and signal pathway-biased family B G protein-coupled receptor conformations

Abstract

Recently, the concept of ligand-directed signaling-the ability of different ligands of an individual receptor to promote distinct patterns of cellular response-has gained much traction in the field of drug discovery, with the potential to sculpt biological response to favor therapeutically beneficial signaling pathways over those leading to harmful effects. However, there is limited understanding of the mechanistic basis underlying biased signaling. The glucagon-like peptide-1 receptor is a major target for treatment of type-2 diabetes and is subject to ligand-directed signaling. Here, we demonstrate the importance of polar transmembrane residues conserved within family B G protein-coupled receptors, not only for protein folding and expression, but also in controlling activation transition, ligand-biased, and pathway-biased signaling. Distinct clusters of polar residues were important for receptor activation and signal preference, globally changing the profile of receptor response to distinct peptide ligands, including endogenous ligands glucagon-like peptide-1, oxyntomodulin, and the clinically used mimetic exendin-4.

Cryo-EM structure of the activated GLP-1 receptor in complex with a G protein

“…2). In the extracellular half of the receptor, TM7 bends towards TM6 using as pivot point the kink at G395 7.50b (Wooten numbering in superscript 25 ) (Fig. 3a), equivalent to the conserved G 7.42 (Ballesteros-Weinstein numbering for family A GPCRs in superscript 35 ) in family A GPCRs 36 .…”

Structure of the full-length glucagon class B G-protein-coupled receptor

“…2). The tetrazole ring of NNC0640 inserts into a cleft between helices VI and VII forming hydrogen bonds with S350 6.41b and N404 7.61b (numbers in superscript refer to the modified Ballesteros-Weinstein numbering system for class B GPCRs 14,15 ). The benzamide group of the ligand forms an additional polar interaction with S350 6.41b , while the urea carbonyl oxygen hydrogen bonds with T353 6.44b .…”

A Hydrogen-Bonded Polar Network in the Core of the Glucagon-Like Peptide-1 Receptor Is a Fulcrum for Biased Agonism: Lessons from Class B Crystal Structures

“…Using an early model of the GLP-1 receptor, it was predicted that the R2.60 190 coordinated interactions with Asn and Gln and that these interactions were differentially important for signaling via the individual peptides. Nonetheless, double mutation of Asn3.43 240 and Gln7.49 394 did not fully recapitulate the phenotype of the Arg2.60 190 mutation (Wootten et al, 2013b), suggesting that the model was insufficient to fully explain the differential effects on signaling.…”

“…Nonetheless, class B GPCRs have their own unique set of conserved, intramembranous, polar residues that are likely comparable to those in class A. Prototypical of this receptor class is the GLP-1 receptor that displays pleiotropic coupling and both peptide-and nonpeptidic-biased agonism (Jorgensen et al, 2007;Coopman et al, 2010;Koole et al, 2010;Cheong et al, 2012;Willard et al, 2012;Wootten et al, 2013a;Weston et al, 2014). Recently, the role of the conserved intramembranous polar residues in this receptor was probed by alanine scanning mutagenesis, which revealed clusters of amino acids important for a range of functions, including protein expression and the control of activation transition for both general signal pathway bias and ligand-directed biased signaling (Wootten et al, 2013b). A key network for differential effects on peptide-mediated signaling for GLP-1, exendin-4, and oxyntomodulin was identified and shown to involve R2.…”