PT  - JOURNAL ARTICLE
AU  - Tahir Yaqub
AU  - Irina G. Tikhonova
AU  - Jens Lättig
AU  - Remi Magnan
AU  - Marie Laval
AU  - Chantal Escrieut
AU  - Cyril Boulègue
AU  - Chandralal Hewage
AU  - Daniel Fourmy
TI  - Identification of Determinants of Glucose-Dependent Insulinotropic Polypeptide Receptor That Interact with N-Terminal Biologically Active Region of the Natural Ligand
AID  - 10.1124/mol.109.060111
DP  - 2010 Apr 01
TA  - Molecular Pharmacology
PG  - 547--558
VI  - 77
IP  - 4
4099  - http://molpharm.aspetjournals.org/content/77/4/547.short
4100  - http://molpharm.aspetjournals.org/content/77/4/547.full
SO  - Mol Pharmacol2010 Apr 01; 77
AB  - Glucose-dependent insulinotropic polypeptide receptor (GIPR), a member of family B of the G-protein coupled receptors, is a potential therapeutic target for which discovery of nonpeptide ligands is highly desirable. Structure-activity relationship studies indicated that the N-terminal part of glucose-dependent insulinotropic polypeptide (GIP) is crucial for biological activity. Here, we aimed at identification of residues in the GIPR involved in functional interaction with N-terminal moiety of GIP. A homology model of the transmembrane core of GIPR was constructed, whereas a three-dimensional model of the complex formed between GIP and the N-terminal extracellular domain of GIPR was taken from the crystal structure. The latter complex was docked to the transmembrane domains of GIPR, allowing in silico identification of putative residues of the agonist binding/activation site. All mutants were expressed at the surface of human embryonic kidney 293 cells as indicated by flow cytometry and confocal microscopy analysis of fluorescent GIP binding. Mutation of residues Arg183, Arg190, Arg300, and Phe357 caused shifts of 76-, 71-, 42-, and 16-fold in the potency to induce cAMP formation, respectively. Further characterization of these mutants, including tests with alanine-substituted GIP analogs, were in agreement with interaction of Glu3 in GIP with Arg183 in GIPR. Furthermore, they strongly supported a binding mode of GIP to GIPR in which the N-terminal moiety of GIP was sited within transmembrane helices (TMH) 2, 3, 5, and 6 with biologically crucial Tyr1 interacting with Gln224 (TMH3), Arg300 (TMH5), and Phe357 (TMH6). These data represent an important step toward understanding activation of GIPR by GIP, which should facilitate the rational design of therapeutic agents.Copyright © 2010 The American Society for Pharmacology and Experimental Therapeutics