TY - JOUR
T1 - RNA interference screen reveals an essential role of Nedd4-2 in dopamine transporter ubiquitination and endocytosis
AU - Sorkina, Tatiana
AU - Miranda, Manuel
AU - Dionne, Kalen R.
AU - Hoover, Brian R.
AU - Zahniser, Nancy R.
AU - Sorkin, Alexander
PY - 2006/8/2
Y1 - 2006/8/2
N2 - 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.
AB - 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.
KW - Dopamine transporter
KW - Endocytosis
KW - HA epitope tag
KW - Nedd4-2
KW - RNA interference
KW - Ubiquitination
UR - http://www.scopus.com/inward/record.url?scp=33748146552&partnerID=8YFLogxK
U2 - 10.1523/JNEUROSCI.1301-06.2006
DO - 10.1523/JNEUROSCI.1301-06.2006
M3 - Article
C2 - 16885233
AN - SCOPUS:33748146552
SN - 0270-6474
VL - 26
SP - 8195
EP - 8205
JO - Journal of Neuroscience
JF - Journal of Neuroscience
IS - 31
ER -