Document Detail


Flotillins regulate membrane mobility of the dopamine transporter but are not required for its protein kinase C dependent endocytosis.
MedLine Citation:
PMID:  23418867     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Flotillins were proposed to mediate clathrin-independent endocytosis, and recently, flotillin-1 was implicated in the protein kinase C (PKC)-triggered endocytosis of the dopamine transporter (DAT). Since endocytosis of DAT was previously shown to be clathrin-mediated, we re-examined the role of clathrin coat proteins and flotillin in DAT endocytosis using DAT tagged with the hemagglutinin epitope (HA) in the extracellular loop and a quantitative HA antibody uptake assay. Depletion of flotillin-1, flotillin-2 or both flotillins together by small interfering RNAs (siRNAs) did not inhibit PKC-dependent internalization and degradation of HA-DAT. In contrast, siRNAs to clathrin heavy chain and μ2 subunit of clathrin adaptor complex AP-2 as well as a dynamin inhibitor Dyngo-4A significantly decreased PKC-dependent endocytosis of HA-DAT. Similarly, endocytosis and degradation of DAT that is not epitope-tagged were highly sensitive to the clathrin siRNAs and dynamin inhibition but were not affected by flotillin knockdown. Very little co-localization of DAT with flotillins was observed in cells ectopically expressing DAT and in cultured mouse dopaminergic neurons. Depletion of flotillins increased diffusion rates of HA-DAT in the plasma membrane, suggesting that flotillin-organized microdomains may regulate the lateral mobility of DAT. We propose that clathrin-mediated endocytosis is the major pathway of PKC-dependent internalization of DAT, and that flotillins may modulate functional association of DAT with plasma membrane rafts rather than mediate DAT endocytosis.
Authors:
Tatiana Sorkina; John Caltagarone; Alexander Sorkin
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural     Date:  2013-03-11
Journal Detail:
Title:  Traffic (Copenhagen, Denmark)     Volume:  14     ISSN:  1600-0854     ISO Abbreviation:  Traffic     Publication Date:  2013 Jun 
Date Detail:
Created Date:  2013-05-20     Completed Date:  2013-11-19     Revised Date:  2014-06-03    
Medline Journal Info:
Nlm Unique ID:  100939340     Medline TA:  Traffic     Country:  England    
Other Details:
Languages:  eng     Pagination:  709-24     Citation Subset:  IM    
Copyright Information:
© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
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MeSH Terms
Descriptor/Qualifier:
Animals
Cell Membrane / metabolism
Cells, Cultured
Clathrin / genetics,  metabolism
Dopamine Plasma Membrane Transport Proteins / metabolism*
Dopaminergic Neurons / metabolism
Dynamins / antagonists & inhibitors
Endocytosis*
HEK293 Cells
Humans
Hydrazones / pharmacology
Membrane Proteins / genetics,  metabolism*
Mice
Naphthols / pharmacology
Protein Kinase C / genetics,  metabolism*
Protein Transport
Proteolysis
RNA, Small Interfering / genetics
Grant Support
ID/Acronym/Agency:
DA014204/DA/NIDA NIH HHS; R01 DA014204/DA/NIDA NIH HHS; T32DA031111/DA/NIDA NIH HHS
Chemical
Reg. No./Substance:
0/Clathrin; 0/Dopamine Plasma Membrane Transport Proteins; 0/Hydrazones; 0/Membrane Proteins; 0/Naphthols; 0/RNA, Small Interfering; 0/dyngo-4a; 0/flotillins; EC 2.7.11.13/Protein Kinase C; EC 3.6.5.5/Dynamins
Comments/Corrections

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