Spatial Approximations between Residues 6 and 12 in the Amino-terminal Region of Glucagon-like Peptide 1 and Its Receptor

Abstract

Understanding the molecular basis of natural ligand binding and activation of the glucagon-like peptide 1 (GLP1) receptor may facilitate the development of agonist drugs useful for the management of type 2 diabetes mellitus. We previously reported molecular approximations between carboxyl-terminal residues 24 and 35 within GLP1 and its receptor. In this work, we have focused on the amino-terminal region of GLP1, known to be critical for receptor activation. We developed two high-affinity, full agonist photolabile GLP1 probes having sites of covalent attachment in positions 6 and 12 of the 30-residue peptide (GLP1(7-36)). Both probes bound to the receptor specifically and covalently labeled single distinct sites. Glucagon-like peptide 1 (GLP1), 2 secreted by the intestinal L cells in response to food ingestion, is a glucoincretin hormone that possesses multiple physiological functions, including stimulation of glucose-dependent insulin secretion, inhibition of glucagon secretion and gastric emptying, and reduction in food intake, augmenting insulin biosynthesis, restoring ␤-cell sensitivity to glucose, increasing ␤-cell proliferation, and reducing apoptosis (1, 2). Because of these multiple antidiabetic actions and its ability to lower body weight, GLP1 receptor agonists have attracted much interest as a treatment for type 2 diabetes (3).The GLP1 receptor is a member of the family B G proteincoupled receptors (GPCRs) that includes many important drug targets such as receptors for glucagon, glucagon-like peptide 2, gastric inhibitory polypeptide, secretin, vasoactive intestinal polypeptide, corticotropin-releasing factor, parathyroid hormone and calcitonin (4). A signature structural feature of this family is a long and structurally complex extracellular aminoterminal domain containing six conserved cysteine residues that form disulfide bonds that contribute to the development of a highly folded structure (5-9). This domain has been suggested to be the predominant domain for natural ligand binding and this is a consistent theme throughout the family (10 -16). Natural ligands for family B receptors are all moderately long peptides in excess of 25 residues that have diffuse pharmacophoric domains, contributing to the complexity of their flexible interactions with the receptor amino-terminal domain. Like natural ligands for other members in the family B GPCRs, the aminoterminal portion of GLP1 is critical for the receptor selectivity and activation, whereas the carboxyl-terminal portion is critical for high affinity ligand binding (17). A tethering mechanism involving two domains of binding, with the carboxyl-terminal region of the ligand binding to the receptor amino terminus, and the amino-terminal region of the ligand possibly interacting with the receptor body, has been proposed for activation of this family of receptors (18 -20).Recently, our understanding of the molecular basis of ligand binding of the family B GPCRs has been substantially advanced with the solution of the NMR and crystal structures of the amin...

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

“…Experiments with photolabile probes for GLP-1 provide only partial information regarding the spa-tial approximation of the residues in the peptide and receptor that interact with each other. Phe 6 of GLP-1 is in close proximity to Tyr 145 of GLP1R according to that study (46). Recent results from another study that used the same method showed discrepancies compared with crystallography experiments.…”

“…Thus, many approaches, such as alanine scanning, photoaffinity labeling, and molecular docking, have been attempted to identify specific amino acids that are required for this interaction (28,(43)(44)(45)(46)(47) (24,28,43,44,48). However, these studies do not explain how individual residues in the peptide interact with residues in the receptor.…”

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

“…Similar to the naturally occurring polymorphism M149, these residues are considerably more hydrophobic and bulky than the wild-type Thr; consistent with M149, they have large detrimental effects, which is perhaps not surprising given the recent structural information emerging for the GLP-1R. Recently, Miller and colleagues used photoaffinity labeling that detected close proximity between residues 16 and 12 of the GLP-1 peptide with residues 141 and 145 of the GLP-1R, respectively (Chen et al, 2010;Miller et al, 2011). Given that these residues precede, and would neighbor residue 149 in an a-helical tertiary structure, it indicates that 149 is near the endogenous peptide binding pocket.…”

“…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

“…These templates were linked by a global alignment, which was used by Modeler to generate a full GLP-1R model with GLP-1(7-36)NH 2 bound. Modeler used 4 p-benzoylphenylalanine (Bpa) photoaffinity crosslinking-derived distance constraints between GLP-1 and GLP-1R (Chen et al, 2009(Chen et al, , 2010Miller et al, 2011) and two sets of constraints derived from reciprocal mutagenesis experiments that resulted in gain of function Vertongen et al, 2001;Moon et al, 2015), as shown in Table 1; the effect of these constraints was to provide additional information on the peptide-receptor interaction in the region between the TM and ECD templates. These reciprocal mutagenesis results were essentially the only mutagenesis results used in the generation of the model.…”