title: RNA interference screen reveals an essential role of Nedd4-2 in dopamine transporter ubiquitination and endocytosis.
abstract: The function of the dopamine transporter (DAT) to terminate dopamine neurotransmission is regulated by endocytic trafficking of DAT. To elucidate the mechanisms of DAT endocytosis, we generated a fully functional mutant of the human DAT in which a hemagglutinin epitope (HA) was incorporated into the second extracellular loop. The endocytosis assay, based on the uptake of an HA antibody, was designed to study constitutive- and protein kinase C (PKC)-dependent internalization of HA-DAT expressed in non-neuronal cells and rat dopaminergic neurons. Large-scale RNA interference analysis of PKC-dependent endocytosis of HA-DAT revealed the essential and specific role of an E3 ubiquitin ligase, Nedd4-2 (neural precursor cell expressed, developmentally downregulated 4-2), as well as the involvement of adaptor proteins present in clathrin-coated pits, such as epsin, Eps15 (epidermal growth factor pathway substrate clone 15), and Eps15R (Eps15-related protein). Depletion of Nedd4-2 resulted in a dramatic reduction of PKC-dependent ubiquitination of DAT. Endogenous Nedd4-2, epsin, and Eps15 were coimmunoprecipitated with heterologously expressed human HA-DAT and endogenous DAT isolated from rat striatum. A new mechanistic model of DAT endocytosis is proposed whereby the PKC-induced ubiquitination of DAT mediated by Nedd4-2 leads to interaction of DAT with adaptor proteins in coated pits and acceleration of DAT endocytosis.
authors.affiliation: Department of Pharmacology, University of Colorado at Denver and Health Sciences Center, Aurora, Colorado 80045, USA.
authors.forename: Tatiana
authors.initials: T
authors.lastname: Sorkina
authors.forename: Manuel
authors.initials: M
authors.lastname: Miranda
authors.forename: Kalen R
authors.initials: KR
authors.lastname: Dionne
authors.forename: Brian R
authors.initials: BR
authors.lastname: Hoover
authors.forename: Nancy R
authors.initials: NR
authors.lastname: Zahniser
authors.forename: Alexander
authors.initials: A
authors.lastname: Sorkin
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D045643:DNA Repair Enzymes; D050483:Dopamine Plasma Membrane Transport Proteins / Q000378:metabolism*; D004705:Endocytosis / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D009474:Neurons / Q000378:metabolism*; D034622:RNA Interference / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D025801:Ubiquitin / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D050483:Dopamine Plasma Membrane Transport Proteins; C476254:N4BP2 protein, human; D025801:Ubiquitin; D045643:DNA Repair Enzymes