Structural basis for constitutive activity and agonist‐induced activation of the enteroendocrine fat sensor GPR119

Abstract

BACKGROUND AND PURPOSEGPR119 is a Gαs-coupled 7TM receptor activated by endogenous lipids such as oleoylethanolamide (OEA) and by the dietary triglyceride metabolite 2-monoacylglycerol. GPR119 stimulates enteroendocrine hormone and insulin secretion. But despite massive drug discovery efforts in the field, very little is known about the basic molecular pharmacology of GPR119. EXPERIMENTAL APPROACHGPR119 receptor signalling was studied in transfected cells. Mutational mapping (30 mutations in 23 positions) was performed on residues required for ligand-independent and agonist-induced GPR119 activation (AR231453 and OEA). Novel Rosetta-based receptor modelling was applied, using a composite template approach with segments from different X-ray structures and fully flexible ligand docking. KEY RESULTSThe increased signalling induced by increasing the cell surface expression of GPR119 in the absence of agonist and the inhibitory effect of two synthetic inverse agonists demonstrated that GRP119 signals with a high degree of constitutive activity through the Gαs pathway. The mutational maps for AR231453 and OEA were very similar and, surprisingly, also similar to the mutational map for residues affecting the constitutive signalling -albeit with key differences. Surprisingly, almost all residues in extracellular loop-2b were important for the constitutive activity. The molecular modelling and docking demonstrated that AR231453 binds in a 'vertical' pocket in between mutational hits reaching from the centre of the receptor out to extracellular loop-2b. CONCLUSIONS AND IMPLICATIONSThe high constitutive activity of GPR119 should be taken into account in future drug discovery efforts, which can now be guided by the detailed knowledge of the physiochemical properties of the extended ligand-binding pocket. Abbreviations7TM, seven transmembrane; ECL, extracellular loop; OEA, oleoylethanolamide; REU, Rosetta energy units; RMSD, root mean square deviation.

Regulation of gut hormone secretion. Studies using isolated perfused intestines

“…All of these receptorshave been found to be expressed in enteroendocrine cells, including GLP-1 producing cells 16,17,41,46,66 .…”

“…GPR119 signals through Gs-coupling and cAMP leading to hormone secretion16 . So far most evidence indicating a role for GPR119 in GLP-1 secretion has been gathered from single cell studies.…”

Mutation-Guided Unbiased Modeling of the Fat Sensor GPR119 for High-Yield Agonist Screening

“…Two assays were used: Alumnia (A) and Hithunter (H). We described data derived from the Alumnia assay in a previous study (Engelstoft et al, 2014).…”

“…AR231453) reported to promote pancreatic postprandial insulin and incretin secretion in a glucose-dependent fashion (Semple et al, 2011). In a previous study we applied classical docking methods to elucidate the binding mode of AR231453 to GPR119, but were unable to discriminate between multiple possible binding conformations (Engelstoft et al, 2014).…”

Dissecting the Physiology and Pathophysiology of Glucagon-Like Peptide-1

“…This receptor is also expressed, and hence can be studied, in vitro , by the human enteroendocrine cell model NCI-H716 or by the murine GLUTag cell line ( 320 ). Heterologous expression in vitro unveiled its constitutive activity capable to raise intracellular cAMP levels through Gαs ( 321 ) and lead to the secretion of GLP-1 and PYY ( 89 ). Rodents, contrarily to humans, express GPR119 also in some regions of the brain ( 316 ).…”