Proteomic Tools for the Comprehensive Analysis of Dopamine Transporter Topology

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DESCRIPTION (provided by applicant): This R21 application (10 pages max) is being submitted in response to PAR-03-017 "Cutting-Edge Basic Research Awards (CEBRA)" which provides support for highly innovative or conceptually creative research to advance the understanding of drug abuse and addiction. Accurate assessments of the lipid embedded residues of membrane proteins are crucial for the prediction of membrane protein function and the generation of structural models. Currently, membrane protein topology prediction algorithms are accurate to +/- 5 to 10 amino acids at best. We propose to develop a shotgun proteomic approach for the global analysis of lipid embedded residues of integral membrane proteins within a complex biological sample. We will apply these methods to: 1) a directed approach to characterize the topology of the human dopamine transporter (hDAT), trafficking mutants of hDAT, and native DAT in rat brain tissue and 2) an undirected global approach to characterize the topology of membrane proteins enriched from cell and tissue homogenates.