Trace Amine‐Associated Receptor Intracellular Localization and Trafficking

Abstract

Trace amine-associated receptor 1 (TAAR1) is a G-protein coupled receptor (GPCR) found in monoaminergic neurons of the central nervous system. Amphetamine-like psychostimulants activate TAAR1, which couples to Gαs and Gα13 downstream signaling cascades, and the latter regulates the RhoA-mediated internalization of neurotransmitter transporters. TAAR1 stimulation is crucial to methamphetamine (MA) mechanism of action, and a common variant of TAAR1 is associated with increased MA craving in MA users. Although the impact of TAAR1 on behavior has been characterized, the molecular mechanisms underlying TAAR1 pharmacology are incompletely understood. TAAR1 is a unique GPCR as it resides inside the cell rather than at the cell surface, though the precise location of TAAR1 within the cell is unknown. Understanding the intracellular localization, orientation, and trafficking of TAAR1 will further inform the molecular mechanisms of TAAR1 signaling. However, available antibodies and fluorescent fusion proteins for imaging TAAR1 are inadequate for such studies. Therefore, we have developed a novel method for imaging human TAAR1 (hTAAR1) through both light and electron microscopy using the miniVIPER peptide labeling system for multiscale microscopy. The hTAAR1-miniVIPER system has been applied to in vitro systems and has been used in conjunction with light microscopy to explore subcellular localization of the recombinant receptor in Chinese hamster ovary cells. Further investigation with electron microscopy will be carried out to determine the orientation of hTAAR1 on internal membranes and will indicate how TAAR1 mobilizes spatially distinct signaling cascades within the cell. In order to study the receptor in native tissue, AAV constructs will be employed to express the hTAAR1-miniVIPER construct in the dopamine neurons of mice. Tissue from these animals will be imaged through correlative light and electron microscopy to determine the subcellular localization and orientation of TAAR1. Animals expressing hTAAR1-miniR will be subjected to acute and chronic drug treatments to determine how TAAR1 agonism affects receptor localization and trafficking. Together these studies will expand our current understanding of molecular mechanisms of TAAR1 signaling and will inform future work concerning the role TAAR1 plays in the modulation of neural circuity relevant to addiction and methamphetamine use disorder.