title: Flotillins regulate membrane mobility of the dopamine transporter but are not required for its protein kinase C dependent endocytosis.
abstract: 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: Department of Cell Biology, University of Pittsburgh, School of Medicine, Pittsburgh, PA, USA.
authors: Tatiana
authors: T
authors: Sorkina
authors: John
authors: J
authors: Caltagarone
authors: Alexander
authors: A
authors: Sorkin
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002966:Clathrin / Q000235:genetics / Q000378:metabolism; D050483:Dopamine Plasma Membrane Transport Proteins / Q000378:metabolism*; D059290:Dopaminergic Neurons / Q000378:metabolism; D034281:Dynamins / Q000037:antagonists & inhibitors; D004705:Endocytosis; D057809:HEK293 Cells; D006801:Humans; D006835:Hydrazones / Q000494:pharmacology; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D009284:Naphthols / Q000494:pharmacology; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport; D059748:Proteolysis; D034741:RNA, Small Interfering / Q000235:genetics
chemical_list: D002966:Clathrin; D050483:Dopamine Plasma Membrane Transport Proteins; D006835:Hydrazones; D008565:Membrane Proteins; D009284:Naphthols; D034741:RNA, Small Interfering; C568360:dyngo-4a; C106354:flotillins; D011493:Protein Kinase C; D034281:Dynamins