title: Amphetamine activates Rho GTPase signaling to mediate dopamine transporter internalization and acute behavioral effects of amphetamine.
abstract: Acute amphetamine (AMPH) exposure elevates extracellular dopamine through a variety of mechanisms that include inhibition of dopamine reuptake, depletion of vesicular stores, and facilitation of dopamine efflux across the plasma membrane. Recent work has shown that the DAT substrate AMPH, unlike cocaine and other nontransported blockers, can also stimulate endocytosis of the plasma membrane dopamine transporter (DAT). Here, we show that when AMPH enters the cytoplasm it rapidly stimulates DAT internalization through a dynamin-dependent, clathrin-independent process. This effect, which can be observed in transfected cells, cultured dopamine neurons, and midbrain slices, is mediated by activation of the small GTPase RhoA. Inhibition of RhoA activity with C3 exotoxin or a dominant-negative RhoA blocks AMPH-induced DAT internalization. These actions depend on AMPH entry into the cell and are blocked by the DAT inhibitor cocaine. AMPH also stimulates cAMP accumulation and PKA-dependent inactivation of RhoA, thus providing a mechanism whereby PKA- and 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 couple to PKA in dopamine neurons antagonizes RhoA activation, DAT internalization, and hyperlocomotion observed in mice after AMPH treatment. These observations support the existence of an unanticipated intracellular target that mediates the effects of AMPH on RhoA and cAMP signaling and suggest new pathways to target to disrupt AMPH action.
authors.affiliation: Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, PA 15213;
authors.forename: David S
authors.initials: DS
authors.lastname: Wheeler
authors.affiliation: Laboratory of Molecular and Cellular Neurobiology, National Institute of Mental Health, National Institutes of Health, Bethesda, MD 20892; susan.amara@nih.gov smunderhill@yahoo.com.
authors.forename: Suzanne M
authors.initials: SM
authors.lastname: Underhill
authors.affiliation: Center for Biologic Imaging, University of Pittsburgh, Pittsburgh, PA 15261;
authors.forename: Donna B
authors.initials: DB
authors.lastname: Stolz
authors.affiliation: Department of Pathology, Division of Neuropathology, University of Pittsburgh, Pittsburgh, PA 15213;
authors.forename: Geoffrey H
authors.initials: GH
authors.lastname: Murdoch
authors.affiliation: Department of Neurobiology, Center for the Neural Basis of Cognition, Center for Neuroscience, University of Pittsburgh, Pittsburgh, PA 15260.
authors.forename: Edda
authors.initials: E
authors.lastname: Thiels
authors.affiliation: Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, PA 15213;
authors.forename: Guillermo
authors.initials: G
authors.lastname: Romero
authors.affiliation: Laboratory of Molecular and Cellular Neurobiology, National Institute of Mental Health, National Institutes of Health, Bethesda, MD 20892; susan.amara@nih.gov smunderhill@yahoo.com.
authors.forename: Susan G
authors.initials: SG
authors.lastname: Amara
mesh_terms: D015647:2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine / Q000494:pharmacology; D000661:Amphetamine / Q000494:pharmacology*; D000818:Animals; D001522:Behavior, Animal / Q000187:drug effects / Q000502:physiology; D001693:Biological Transport, Active / Q000187:drug effects; D000697:Central Nervous System Stimulants / Q000494:pharmacology; D002966:Clathrin / Q000378:metabolism; D003042:Cocaine / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D018491:Dopamine Agonists / Q000494:pharmacology; D050483:Dopamine Plasma Membrane Transport Proteins / Q000378:metabolism*; D059290:Dopaminergic Neurons / Q000187:drug effects / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects; D008124:Locomotion / Q000187:drug effects / Q000502:physiology; D008297:Male; D008636:Mesencephalon / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D015398:Signal Transduction / Q000187:drug effects; D020744:rac GTP-Binding Proteins / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000378:metabolism*
chemical_list: D000697:Central Nervous System Stimulants; D002966:Clathrin; D018491:Dopamine Agonists; D050483:Dopamine Plasma Membrane Transport Proteins; D015647:2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine; D000661:Amphetamine; D017868:Cyclic AMP-Dependent Protein Kinases; D020744:rac GTP-Binding Proteins; D020741:rho GTP-Binding Proteins; D003042:Cocaine
keywords: Rho GTPase; amphetamine; dopamine transporter; endocytosis; protein kinase A