Date

March 2008

Document Type

Dissertation

Degree Name

Ph.D.

Institution

Oregon Health & Science University

Abstract

Trace amine-associated receptor 1 (TAAR1) is a member of a distinct family of G-protein coupled receptors. TAAR1 is activated by endogenous trace amines (TA) such as β-phenylethylamine (PEA) and para-tyramine (p-TYR). Abnormal levels of TAs have been associated with mood disorders, but the physiological role of TAAR1 remains unclear. My thesis is that TAAR1 represents a novel target of amphetamine (AMPH) and methamphetamine (METH). The four studies described here were designed to test hypotheses derived from this thesis. In the first study I tested the hypothesis that AMPH and METH are agonists at several mammalian species of TAAR1. A pharmacological characterization of the activation of TAAR1 by trace amines, AMPH, and METH was carried out using an in vitro cAMP assay. The results show that PEA and p-TYR are potent agonists at TAAR1, that there is a significant difference in stereoselectivity with respect to isomers of AMPH and METH, and the stereoselectivity is species-dependent. In the second study I explored the determinants of species-specific TAAR1 stereoselectivity. I hypothesized amino acids that differ between species in key locations of the putative ligand-binding domain for TAAR1 are responsible for the observed species-dependent stereoselectivity. Using site-directed mutagenesis, a mutation to transmembrane 6, M6.55T for the rat and T6.55M in the case of the mouse, caused a decrease of potency in the rat while increasing potency in the mouse for both the S(+) and R(-) enantiomers of AMPH and METH. A mutation in transmembrane 7, N7.39Y for the rat and Y7.39N for the mouse, reversed the species dependent stereoselectivity of the rat and mouse TAAR1 to enantiomers of AMPH and METH. My third study tested the hypothesis that METH and AMPH activate the wildtype human TAAR1 (hTAAR1). Using a cAMP-dependent reporter assay I demonstrated that PEA as well as both isomers of METH and AMPH activated the wild-type hTAAR1 in a dose-dependent manner at concentrations that are exceeded by users of these drugs. The fourth study investigated the hypothesis that the human dopamine transporter (hDAT) and hTAAR1 co-localize in cells. HEK-293 cells, stably expressing hDAT fused to green fluorescent protein (hDAT-GFP), were treated with METH resulting in redistribution of hDAT-GFP from the cell surface to the cytoplasm. There was also some preliminary evidence of hTAAR1 and hDAT co-localization. In conclusion, TAAR1 is potently activated by METH in vitro at concentrations less than those commonly observed in drug abusers. Preliminary evidence of DAT co-localization with hTAAR1 and METH induced internalization of DAT suggests that hTAAR1 could play a role in mediating some of METH’s mental and physical effects. Therefore, I conclude that TAAR1 represents a novel target for potential anti-METH medication and for improved psychotherapeutics in some mood disorders.

Identifier

doi:10.6083/M4ZK5DNR

Division

Neuroscience Graduate Program

School

School of Medicine

Share

COinS
 
 

To view the content in your browser, please download Adobe Reader or, alternately,
you may Download the file to your hard drive.

NOTE: The latest versions of Adobe Reader do not support viewing PDF files within Firefox on Mac OS and if you are using a modern (Intel) Mac, there is no official plugin for viewing PDF files within the browser window.