Abstract

The glucagon-like peptide 1 (GLP-1) receptor is a class B G protein-coupled receptor (GPCR) that is a key target for treatments for type II diabetes and obesity. This receptor, like other class B GPCRs, displays biased agonism, though the physiological significance of this is yet to be elucidated. Previous work has implicated R2.60¹⁹⁰, N3.43²⁴⁰, Q7.49³⁹⁴ and H6.52³⁶³ as key residues involved in peptide-mediated biased agonism (Wootten et al., 2013a), with R2.60¹⁹⁰, N3.43²⁴⁰ and Q7.49³⁹⁴ predicted to form a polar interaction network. In this study, we used novel insight gained from recent crystal structures of the transmembrane domains of the glucagon and corticotropin releasing factor 1 (CRF1) receptors to develop improved models of the GLP-1 receptor that predict additional key molecular interactions with these amino acids. We have introduced E6.53³⁶⁴A, N3.43²⁴⁰Q, Q7.49³⁹⁴N and N3.43²⁴⁰Q/ Q7.49³⁹⁴N mutations to probe the role of predicted H-bonding and charge-charge interactions in driving cAMP, calcium or ERK signaling. A polar interaction between E6.53³⁶⁴ and R2.60¹⁹⁰ was predicted to be important for GLP-1- and exendin-4-, but not oxyntomodulin-mediated cAMP formation and also ERK1/2 phosphorylation. In contrast, Q7.49³⁹⁴, but not R2.60¹⁹⁰/ E6.53³⁶⁴ was critical for calcium mobilisation for all three peptides. Mutation of N3.43²⁴⁰ and Q7.49³⁹⁴ had differential effects on individual peptides providing evidence for molecular differences in activation transition. Collectively, this work expands our understanding of peptide-mediated signaling from the GLP-1 receptor and the key role that the central polar network plays in these events.