This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.The goal of the current proposal is to develop molecular models of possible binding sites for agonists and antagonists within the transmembrane (TM) domain of the human sweet taste receptor, the heterodimer of human T1R2 (hT1R2) and hT1R3, which belongs to the G protein coupled receptor family. Recent crystal structures of bovine rhodopsin provide an opportunity to construct models of GPCRs that have been shown to be reliable predictors of selectivity and of activity in these receptors. We hypothesize that: 1. Agonists and antagonists bind in the same TM pocket; and 2. Through binding, agonists induce specific conformational changes that lead to activation and are blocked by the binding of antagonists. We will identify the binding pockets in the receptor for artificial sweeteners, (e.g., cyclamate) and inhibitors, (e.g., lactisole) by computational molecular docking and test their validity via mutagenesis studies. The experimentally confirmed models will be used to conduct molecular dynamics simulations on occupied and unoccupied receptors to develop an understanding of the molecular basis for activation and inhibition mechanisms of the sweet taste response. The mechanisms inferred from simulations will be tested by mutations designed to produce a constitutively active receptor. These studies should lead to a better understanding of receptor function and will provide the basis for design of novel sweet taste modulators.
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