Methamphetamine Activates Small GTPase Rac1 Independently from RhoA and Trace Amine-Associated Receptor 1 (TAAR1)

Abstract ID 129663 Poster Board 153 Amphetamines are a broad class of psychostimulants that have become important treatments for attention deficit hyperactivity disorder. Despite their therapeutic qualities, however, amphetamines have strong abuse potential that can lead to detrimental behavioral and psychological changes. Our lab has previously established that amphetamines induce internalization of the dopamine transporter (DAT) in dopamine neurons by activating trace amine-associated receptor 1 (TAAR1) inside the cell. In particular, we demonstrated that amphetamines activate small guanosine triphosphatase (GTPase) RhoA in a TAAR1-dependent manner to initiate DAT internalization. Interestingly, we also observed that amphetamines activate another Rho-family GTPase, Rac1, but Rac1 is not required for DAT internalization. Here, we explore three potential pathways of Rac1 activation after methamphetamine (METH) exposure. Given that guanine nucleotide exchange factors (GEFs) are responsible for inducing the GTP-bound state of small GTPases, we first inhibited three different Rac1 GEFs: Tiam1, TrioN, and P-Rex1 using Rac1 inhibitors NSC 23766 (blocks Tiam1 and TrioN) and 1A-116 (blocks P-Rex1). We also tested a third inhibitor, EHT 1864, which alternatively inhibits activity by binding directly to Rac1. Data from fluorescence resonance energy transfer (FRET) imaging show that EHT 1864 is the best inhibitor of METH induced Rac1 activity, 1A-116 moderately inhibits Rac1, and NSC 23766 does not inhibit Rac1 activity. This indicates that P-Rex1 may be important for METH induced Rac1 activation, but TrioN and Tiam1 are not. Second, we tested whether RhoA activation is necessary for Rac1 activation by measuring Rac1 activity after pre-treatment with a RhoA inhibitor. Using Pak1-GST beads, we show that Rac1 activity was not affected by the RhoA inhibitor, suggesting that RhoA and Rac1 act independently after METH exposure. Third, we analyzed whether TAAR1 is necessary for Rac1 activation by comparing METH induced Rac1 activity in TAAR1 knockout cells with wildtype cells. FRET imaging data show that Rac1 activity does not differ between TAAR1 knockout cells and wildtype cells after METH exposure, demonstrating that unlike RhoA, Rac1 activation is not TAAR1-dependent. Taken together, our data support the notion that amphetamines activate Rac1 via an alternative pathway independent of TAAR1 and RhoA. However, the regulators of Rac1 activation and its downstream effects remain unknown. Further exploration into the mechanisms of action behind amphetamines is therefore crucial to our understanding of these how these drugs alter dopamine concentrations in the brain and how amphetamines contribute to beneficial and harmful therapeutic outcomes. This work was supported by Intramural Research at the NIH, NIMH