Residues within the Transmembrane Domain of the Glucagon-Like Peptide-1 Receptor Involved in Ligand Binding and Receptor Activation: Modelling the Ligand-Bound Receptor

Abstract

The C-terminal regions of glucagon-like peptide-1 (GLP-1) bind to the N terminus of the GLP-1 receptor (GLP-1R), facilitating interaction of the ligand N terminus with the receptor transmembrane domain. In contrast, the agonist exendin-4 relies less on the transmembrane domain, and truncated antagonist analogs (e.g. exendin 9-39) may interact solely with the receptor N terminus. Here we used mutagenesis to explore the role of residues highly conserved in the predicted transmembrane helices of mammalian GLP-1Rs and conserved in family B G protein coupled receptors in ligand binding and GLP-1R activation. By iteration using information from the mutagenesis, along with the available crystal structure of the receptor N terminus and a model of the active opsin transmembrane domain, we developed a structural receptor model with GLP-1 bound and used this to better understand consequences of mutations. Mutation at Y152 [transmembrane helix (TM) 1], R190 (TM2), Y235 (TM3), H363 (TM6), and E364 (TM6) produced similar reductions in affinity for GLP-1 and exendin 9-39. In contrast, other mutations either preferentially [K197 (TM2), Q234 (TM3), and W284 (extracellular loop 2)] or solely [D198 (TM2) and R310 (TM5)] reduced GLP-1 affinity. Reduced agonist affinity was always associated with reduced potency. However, reductions in potency exceeded reductions in agonist affinity for K197A, W284A, and R310A, while H363A was uncoupled from cAMP generation, highlighting critical roles of these residues in translating binding to activation. Data show important roles in ligand binding and receptor activation of conserved residues within the transmembrane domain of the GLP-1R. The receptor structural model provides insight into the roles of these residues.

Ligand Binding Pocket Formed by Evolutionarily Conserved Residues in the Glucagon-like Peptide-1 (GLP-1) Receptor Core Domain

“…For example, although GLP-1 Ala 18 was proposed to be located close to Glu 133 of the ECD of GLP1R by photoaffinity labeling (45), the ligand-bound crystal structure of the GLP1R ECD shows a hydrophobic interaction between GLP-1 Ala 18 and Leu 32 of the ECD (20,21). Molecular docking studies with biochemical analyses have suggested possible ligand binding pockets in the GLP1R core domain (47,50,51). However, these modeling approaches failed to provide a common consensus for the ligand binding pocket.…”

Differential Impact of Amino Acid Substitutions on Critical Residues of the Human Glucagon-Like Peptide-1 Receptor Involved in Peptide Activity and Small-Molecule Allostery

“…However, with the emergence of two class B TM crystal structures (Hollenstein et al, 2013;Siu et al, 2013) and a plethora of mutagenesis (Lopez de Maturana and Donnelly, 2002;Lopez de Maturana et al, 2004;Coopman et al, 2011;Koole et al, 2012a,b;Wootten et al, 2013) and photoaffinity labeling data (Al-Sabah and Donnelly, 2003;Dong et al, 2004Dong et al, , 2007Dong et al, , 2011Chen et al, 2009Chen et al, , 2010Miller et al, 2011;Coin et al, 2013), structurally and functionally important components of the GLP-1R can begin to be predicted and complementary molecular models can be further refined. In this study, we have created a series of mutations at two GLP-1R residues subject to polymorphic variance (amino acids 149 and 333; Fig.…”

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

“…A had limited effect on GLP-1 affinity and cAMP formation (Coopman et al, 2011), suggesting that other interactions predominate, at least for activation of this pathway. In the current study, we have modeled a GLP-1 bound form of the full-length receptor that incorporates known distance constraints from published crosslinking studies (Fig.…”