Amphetamine activates Rho GTPase signaling to mediate dopamine transporter internalization and acute behavioral effects of amphetamine David S. Wheelera,1,2, Suzanne M. Underhillb,2,3, Donna B. Stolzc, Geoffrey H. Murdochd, Edda Thielse, Guillermo Romeroa, and Susan G. Amarab,3 aDepartment of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, PA 15213; bLaboratory of Molecular and Cellular Neurobiology, National Institute of Mental Health, National Institutes of Health, Bethesda, MD 20892; cCenter for Biologic Imaging, University of Pittsburgh, Pittsburgh, PA 15261; dDepartment of Pathology, Division of Neuropathology, University of Pittsburgh, Pittsburgh, PA 15213; and eDepartment of Neurobiology, Center for the Neural Basis of Cognition, Center for Neuroscience, University of Pittsburgh, Pittsburgh, PA 15260 Contributed by Susan G. Amara, October 2, 2015 (sent for review September 3, 2015; reviewed by Aurelio Galli, Harald H. Sitte, and Roxanne A. Vaughan) Acute amphetamine (AMPH) exposure elevates extracellular do- the same machinery would be involved in AMPH-mediated in- pamine through a variety of mechanisms that include inhibition of ternalization. A secondary pathway of dynamin-mediated inter- dopamine reuptake, depletion of vesicular stores, and facilitation nalization of membrane-localized proteins involves small GTPases of dopamine efflux across the plasma membrane. Recent work has of the Rho family. Rho proteins are composed of three subfam- shown that the DAT substrate AMPH, unlike cocaine and other ilies (Rho, Rac, and CDC42), each of which play a major role in nontransported blockers, can also stimulate endocytosis of the actin cytoskeletal reorganization and vesicular trafficking (10). To plasma membrane dopamine transporter (DAT). Here, we show further characterize the dynamin-dependent mechanism of DAT that when AMPH enters the cytoplasm it rapidly stimulates DAT internalization in response to AMPH, we examined if it was me- internalization through a dynamin-dependent, clathrin-indepen- diated by clathrin or whether it was a Rho-dependent process. dent process. This effect, which can be observed in transfected We also sought to resolve which signaling pathways activated cells, cultured dopamine neurons, and midbrain slices, is mediated by AMPH trigger internalization, whether these effects reflect a by activation of the small GTPase RhoA. Inhibition of RhoA activity direct intracellular action of AMPH, and whether they depend with C3 exotoxin or a dominant-negative RhoA blocks AMPH- on the elevation of extracellular dopamine. induced DAT internalization. These actions depend on AMPH entry Here, we demonstrate that AMPH [(+)-α-methyl phenylethyl- into the cell and are blocked by the DAT inhibitor cocaine. AMPH amine] acts within the cytoplasm to stimulate Rho family GTPases also stimulates cAMP accumulation and PKA-dependent inactiva- and trigger DAT internalization through a Rho- and dynamin- tion of RhoA, thus providing a mechanism whereby PKA- and dependent pathway that does not involve clathrin. We also show RhoA-dependent signaling pathways can interact to regulate the timing and robustness of AMPH’s effects on DAT internalization. Consistent with this model, the activation of D1/D5 receptors that Significance couple to PKA in dopamine neurons antagonizes RhoA activation, DAT internalization, and hyperlocomotion observed in mice after The dopamine transporter (DAT), a major target for psychosti- AMPH treatment. These observations support the existence of an mulant drugs, including cocaine and amphetamines, clears ex- unanticipated intracellular target that mediates the effects of AMPH tracellular dopamine and restricts the temporal and spatial on RhoA and cAMP signaling and suggest new pathways to target extent of neurotransmitter signaling. This study examines the to disrupt AMPH action. mechanism through which amphetamines trigger internalization of DAT and demonstrates that amphetamine activates the small dopamine transporter | amphetamine | Rho GTPase | protein kinase A | GTPases, Rho and Rac. Rho activation triggers endocytosis of endocytosis DAT by a dynamin-dependent, clathrin-independent pathway. Intriguingly, amphetamine must enter the cell to have these effects, and it also increases cAMP, which in turn inactivates Rho oth cocaine and amphetamine (AMPH) are psychostimulants and limits carrier internalization. Consistent with these obser- that induce euphoria and hyperactivity by increasing extra- B vations, the activation of receptors that couple to protein kinase A cellular dopamine. Despite the apparent commonalities in the in dopamine neurons also antagonizes the behavioral effects effects and mechanism of action of the two drugs, amphetamine of amphetamine in mice, suggesting new pathways to target exposure has behavioral, neuroadaptive, and neurotoxic conse- to disrupt amphetamine action. quences not associated with cocaine use (1–5). These dissimi- larities likely stem from several differences in the mechanism of Author contributions: D.S.W., S.M.U., G.H.M., E.T., G.R., and S.G.A. designed research; D.S.W., action of the two drugs. Amphetamines, unlike cocaine and S.M.U., D.B.S., G.H.M., and E.T. performed research; G.R. and S.G.A. contributed new other nontransported inhibitors of the dopamine transporter reagents/analytic tools; D.S.W., S.M.U., G.H.M., E.T., G.R., and S.G.A. analyzed data; and (DAT), are transporter substrates that compete with dopamine D.S.W., S.M.U., G.H.M., G.R., and S.G.A. wrote the paper. for transport, enter dopamine neurons, enhance efflux of dopa- Reviewers: A.G., Vanderbilt University; H.H.S., Medical University Vienna, Center for Physiology and Pharmacology; and R.A.V., University of North Dakota School of Medicine & mine, and also stimulate internalization of the DAT from the cell Health Sciences. surface (6, 7). To better understand how amphetamines mediate The authors declare no conflict of interest. their effects within the cell, we undertook a series of studies to See Commentary on page 15538. establish the cellular pathways and signaling mechanisms that 1Present address: Department of Medicine, Einstein Medical Center Philadelphia, Philadelphia, underlie the endocytosis of the DAT triggered by amphetamines. PA 19141. AMPH-mediated DAT internalization has been shown to be 2D.S.W. and S.M.U. contributed equally to this work. dynamin-dependent (6). Dynamin-dependence is a key feature 3 To whom correspondence may be addressed. Email: [email protected] or smunderhill@ of clathrin-mediated endocytosis, and, indeed, previous reports yahoo.com. of clathrin-dependent constitutive cycling (8) and PKC-mediated This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. internalization (9) of DAT make it reasonable to consider that 1073/pnas.1511670112/-/DCSupplemental. E7138–E7147 | PNAS | Published online November 9, 2015 www.pnas.org/cgi/doi/10.1073/pnas.1511670112 Downloaded by guest on October 1, 2021 that cytoplasmic AMPH stimulates a secondary pathway of cAMP total internal reflection fluorescence (TIRF) microscopy dem- PNAS PLUS production, which leads to Rho inactivation by PKA-dependent onstrate that less than 0.5% of internalizing DAT colocalizes phosphorylation. This transient period of Rho activation corre- with clathrin (Movie S1). In contrast, parallel experiments lates with ongoing DAT internalization, and this temporal window monitoring transferrin accumulation confirm previous studies of activation can be shortened by stimulating cell-surface Gs- that over 80% of internalizing transferrin colocalizes with clathrin coupled receptors to raise cAMP and increase the rate of Rho (Fig. 1B). Thus, AMPH-stimulated DAT internalization in inactivation. We also show in vivo that preemptive D1/D5 re- SK-N-SH cells is dynamin-dependent, but clathrin-independent. ceptor stimulation to activate PKA can reduce AMPH-evoked Dynamin-dependent, clathrin-independent internalization is gen- hyperlocomotion without altering the increase in locomotion in- erally divided into two categories depending on whether Rho ac- duced by cocaine. Because these signaling events occur in trans- tivation is required (11). Coexpression of the DAT with a plasmid fected cell lines as well as in neurons, the results indicate that, once encoding GFP-tagged C3 exotoxin, a clostridial enzyme that ADP SEE COMMENTARY AMPH enters the cell, it activates Rho, Rac, and PKA signaling ribosylates and inactivates Rho family GTPases, prevents AMPH- independently of any actions to elevate intracellular dopamine. The induced clustering and internalization of DAT in SK-N-SH cells direct activation of cytoplasmic signaling cascades by AMPH within (Fig. 1 C–E and Movie S2). Using TIRF microscopy we observed the cell can contribute to the behavioral effects of acute amphet- that expression of C3 for 18–24 h in SK-N-SH cells reduces the amine exposure and may explain some of the unique neurobiolog- number of focal adhesions, consistent with Rho inhibition. The ical consequences associated with amphetamine use and abuse. continuing spontaneous movement of the C3(+) cells and their ability to transport dopamine (Fig. 1F) indicate that the cells are Results still viable. The presence of the Rho inhibitor prevented the AMPH-Induced DAT Internalization Is Clathrin-Independent. Previous increase in DAT endocytosis observed with AMPH, supporting a studies have indicated that the endocytosis of DAT activated by role for Rho-GTPases in DAT internalization. AMPH