Probing the Ligand Binding Pocket within the Transmembrane Domain of Human T1R3

This study aims to advance our understanding of the sweet taste receptor function at the molecular level. The sweet taste receptor has been shown to be a heterodimeric receptor consisting of two closely related family C G-protein coupled receptors (GPCRs), T1R2 and T1R3. Our group previously found that lactisole (antagonist) and cyclamate (agonist) both interact with the transmembrane domain (TMD) of human T1R3 and their binding pockets potentially overlap. Computational models of these pockets were generated based on the bovine rhodopsin X-ray template.;In this work, we have examined the antagonist-binding pocket within the TMD of T1R3 using a panel of lactisole analogs. We have found that lactisole's methoxyl moiety, the CH3-O-group that is linked to the para-carbon of the aromatic ring, is important for lactisole's inhibitory function. We have identified and characterized 2-(1-naphthoxy)propionic acid as a sweet taste receptor inhibitor that is more potent than lactisole. Through correlative replacements of ligand and receptor functional groups, we have identified TM6 residues, Leu-7826.55 and Phe-7786.51 , as potential antagonist recognition sites.;Using a panel of cyclamate analogs we have examined the agonist-binding pocket within the TMD of T1R3. We have identified the physiochemical requirements of cyclamate's cyclohexyl moiety as a key functional group. Through correlative replacements of ligand and receptor functional groups, we have identified TM6 residues, Leu-7826.55 and Phe-7786.51, as potential agonist recognition sites and have identified their potential interacting groups in cyclamate and certain related sulfamate sweeteners.;The experimental results from this work could provide better constraints for modeling studies. Through the concerted use of model building and experimental testing, we hope to come to a reasonable approximation of the ligand-binding pockets within the TMD of T1R3. Such new knowledge could suggest novel strategies to help overcome diet-induced diseases such as diabetes and obesity and lead to significant medical impacts worldwide.