The catecholamine dopamine plays a major role in the regulation of cognitive, emotional and behavioral functions. Abnormalities in its regulation have been implicated in a number of psychiatric and neurological disorders. Dopamine exerts its actions at a number of G-protein-coupled receptors, including the D24-like (D2, D3, D4) and D1-like (D1, D5) receptors. Antipsychotic medications potently inhibit D2-like receptors, and it is unclear which types or combinations of types are relevant to the etiology and treatment of psychosis. Furthermore, the structural bases of the differences in pharmacological specificity among these receptors is unknown. After dopamine's release, its concentration in and around the synapse is rapidly reduced by the dopamine transporter (DAT). This protein is the major molecular target of several psychoactive drugs, including cocaine. The availability of a cocaine antagonist which was itself not an uptake blocker, and therefore not rewarding, would be a valuable addition to the therapeutic armamentarium against cocaine abuse and its attendant complications. The rational design of such agents would be greatly facilitated by an appreciation of the structural bases of substrate recognition and inhibition by cocaine and other psychostimulants. The candidate has used a new approach to obtain information about the structure of binding sites by systematically identifying the residues which line the site. This approach combines site-directed mutagenesis to substitute cysteine for putative membrane-spanning segment residues, expression of the mutant, and probing the aqueous surface accessibility of the cysteine residue by its ability to react with small, polar, charged, sulfhydryl-specific reagents. The long-term goals of this project are (a) to understand the structural bases of agonist and antagonist binding and specificity in the dopamine D2-like receptors and related biogenic amine receptors, (b) to determine how agonist binding is transduced into G-protein activation, and (c) to determine the structural bases of the transport of substrate by the dopamine transporter and its inhibition by drugs such as cocaine. Thus, I propose the following specific aims: (1) To identify the amino acid residues in membrane-spanning segments forming the surface of the binding-site crevice of the dopamine D2 receptor. (2) To identify conformational changes of the membrane-spanning segments concomitant with changes in the functional state of the receptor. (3) To identify amino acid residues forming the surface of the binding site and transport pathway of the DAT, focusing on residues which have a greater effect on cocaine binding than on transport. (4) To use computational molecular modeling to interpret our experimental results in a structural context.

Agency
National Institute of Health (NIH)
Institute
National Institute of Mental Health (NIMH)
Type
Research Scientist Development Award - Research (K02)
Project #
5K02MH057324-03
Application #
2890980
Study Section
Molecular, Cellular, and Developmental Neurobiology Review Committee (MCDN)
Program Officer
Brady, Linda S
Project Start
1997-09-01
Project End
2002-04-30
Budget Start
1999-05-01
Budget End
2000-04-30
Support Year
3
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Columbia University (N.Y.)
Department
Psychiatry
Type
Schools of Medicine
DUNS #
167204994
City
New York
State
NY
Country
United States
Zip Code
10032