Background: Receptor trafficking and cross-talk regulate 7TM/GPCR signaling capacity. Results: Inhibition of GLP-1R internalization in the presence of GIPR reduces GLP-1R signaling. Conclusion: GLP-1R internalization is essential for full receptor functionality and is abrogated upon cross-talk with GIPR. Significance: GLP-1R cross-talk with GIPR has functional consequences for GLP-1R signaling.

GPR139 is an orphan G protein-coupled receptor that is expressed primarily in the brain. Not much is known regarding the function of GPR139. Recently we have shown that GPR139 is activated by the amino acids l-tryptophan and l-phenylalanine (EC50 values of 220 μM and 320 μM, respectively), as well as di-peptides comprised of aromatic amino acids. This led us to hypothesize that GPR139 may be activated by peptides. Sequence alignment of the binding cavities of all class A GPCRs, revealed that the binding pocket of the melanocortin 4 receptor is similar to that of GPR139. Based on the chemogenomics principle “similar targets bind similar ligands”, we tested three known endogenous melanocortin 4 receptor agonists; adrenocorticotropic hormone (ACTH) and α- and β-melanocyte stimulating hormone (α-MSH and β-MSH) on CHO-k1 cells stably expressing the human GPR139 in a Fluo-4 Ca2+-assay. All three peptides, as well as their conserved core motif HFRW, were found to activate GPR139 in the low micromolar range. Moreover, we found that peptides consisting of nine or ten N-terminal residues of α-MSH activate GPR139 in the submicromolar range. α-MSH1-9 was found to correspond to the product of a predicted cleavage site in the pre-pro-protein pro-opiomelanocortin (POMC). Our results demonstrate that GPR139 is a peptide receptor, activated by ACTH, α-MSH, β-MSH, the conserved core motif HFRW as well as a potential endogenous peptide α-MSH1-9. Further studies are needed to determine the functional relevance of GPR139 mediated signaling by these peptides.

GPR139 is an orphan G protein-coupled receptor expressed in the brain, in particular in the habenula, hypothalamus and striatum. It has therefore been suggested that GPR139 is a possible target for metabolic disorders and Parkinson’s disease. Several surrogate agonist series have been published for GPR139. Two series published by Shi et al. and Dvorak et al. included agonists 1a and 7c respectively, with potencies in the ten-nanomolar range. Furthermore, Isberg et al. and Liu et al. have previously shown that tryptophan (Trp) and phenylalanine (Phe) can activate GPR139 in the hundred-micromolar range. In this study, we produced a mutagenesis-guided model of the GPR139 binding site to form a foundation for future structure-based ligand optimization. Receptor mutants studied in a Ca2+ assay demonstrated that residues F1093×33, H1875×43, W2416×48 and N2717×38, but not E1083×32, are highly important for the activation of GPR139 as predicted by the receptor model. The initial ligand-receptor complex was optimized through free energy perturbation simulations, generating a refined GPR139 model in agreement with experimental data. In summary, the GPR139 reference surrogate agonists 1a and 7c, and the endogenous amino acids l-Trp and l-Phe share a common binding site, as demonstrated by mutagenesis, ligand docking and free energy calculations.

GPR139 is an orphan class A G protein-coupled receptor found mainly in the central nervous system. It has its highest expression levels in the hypothalamus and striatum, regions regulating metabolism and locomotion, respectively, and has therefore been suggested as a potential target for obesity and Parkinson’s disease. The two aromatic amino acids L-Trp and L-Phe have been proposed as putative endogenous agonists, and three structurally related benzohydrazide, glycine benzamide, and benzotriazine surrogate agonist series have been published. Herein, we assayed 158 new analogues selected from a pharmacophore model, and identified 12 new GPR139 agonists, containing previously untested bioisosteres. Furthermore, we present the first combined structure-activity relationships, and a refined pharmacophore model to serve as a rationale for future ligand identification and optimization.

RECEPTORSG protein-coupled receptors (GPCRs) mediate many of the physiological responses to endogenous ligands such as neurotransmitters, hormones, metabolites, ions and sensory signals. 1 Although their ligands are structurally very diverse, GPCRs share a common molecular structure of seven transmembrane-spanning α-helices connected by three intracellular and three extracellular loops, with an extracellular N terminus and intracellular C terminus. 2 Amongst all of the drugs approved by the Food and Drug Administration (FDA), 34% target GPCRs 3 making this the largest class of drug targets. However, despite this, only 27% of all non-olfactory GPCRs are presently drug targets. Based on previous success with targeting this protein family and strongThis is an open access article under the terms of the Creat ive Commo ns Attri butio n NonCo mmercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. AbstractG protein-coupled receptors (GPCRs) constitute the largest family of receptors and membrane proteins in the human genome with ~800 members of which half are olfactory.GPCRs are activated by a very broad range of endogenous signalling molecules and are involved in a plethora of physiological functions. All GPCRs contain a transmembrane domain, consisting of a bundle of seven α-helices spanning the cell membrane, and forming the majority of the known ortho-or allosteric ligand binding sites. Due to their many physiological functions and the accessible and druggable transmembrane pocket, GPCRs constitute the largest family of drug targets mediating the actions of 34% of currently