Oligomerization of Dopamine Transporters Visualized in Living Cells by Fluorescence Resonance Energy Transfer Microscopy

摘要: To examine the oligomeric state and trafficking of dopamine transporter (DAT) in different compartments living cells, human DAT was fused to yellow (YFP) or cyan fluorescent protein (CFP). YFP-DAT CFP-DAT were transiently stably expressed porcine aortic endothelial (PAE) embryonic kidney (HEK) 293 an immortalized dopaminergic cell line 1RB3AN27. Fluorescence microscopic imaging cells co-expressing revealed fluorescence resonance energy transfer (FRET) between CFP YFP, which is consistent with intermolecular interaction fusion proteins. FRET signals detected CFP- located at plasma membrane intracellular compartments. Phorbol esters amphetamine induced endocytosis YFP/CFP-DAT early recycling endosomes, identified by Rab5, Rab11, Hrs EEA.1 Interestingly, however, mainly excluded from Rab5- Hrs-containing microdomains within endosomes. The strongest measured indicative efficient oligomerization internalized DAT. interactions confirmed co-immunoprecipitation. A mutant that retained endoplasmic reticulum (ER) after biosynthesis used show ER. Moreover, co-expression ER-retained wild-type resulted retention These data suggest oligomers are formed ER then constitutively maintained both surface during