Mutations in the lipid-binding domain of alpha-synuclein confer overlapping, yet distinct, functional properties in the regulation of dopamine transporter activity.
Alpha-synuclein and its missense mutants (A30P, A53T) have been linked to the genesis of idiopathic and rare familial forms of Parkinson's disease, respectively. Here we show that, similar to the wild-type alpha-synuclein, the A30P mutant forms a strong complex with the human dopamine transporter (hDAT), through direct protein:protein interactions between the nonamyloid beta component (NAC) domain of the A30P mutant and the last 22 aminoacyl residues of the carboxy-terminal tail of hDAT. The A30P mutant negatively modulates hDAT functional activity and to a greater extent than wild-type alpha-synuclein, with reduced uptake of extracellular dopamine and dopamine-mediated, hDAT-dependent cytotoxicity. By contrast, the A53T mutant neither forms a strong protein:protein complex with hDAT nor modulates dopamine uptake by hDAT, and dopamine-mediated, hDAT-dependent cytotoxicity is higher than with either wild-type or the A30P variant of alpha-synuclein, but not significantly different from that of cells expressing hDAT alone. Confocal microscopy shows substantial overlap in colocalization of all three alpha-synuclein variants with hDAT, with only minor differences. Although the complex formation with hDAT occurs through the NAC domain of the alpha-synuclein variants, it is the familial Parkinson's disease-linked missense mutations present in the amino-terminal lipid binding domain of the alpha-synuclein variants that dictate the extent of the regulation of hDAT function. These studies highlight previously unknown properties of the A30P and the A53T mutants of alpha-synuclein with respect to the modulation of hDAT activity and/or regulation, and its subsequent functional outcome, which are uniquely distinct.
Wersinger
Christophe
C
Department of Pediatrics, Georgetown University Medical Center, 3870 Reservoir Road NW, Washington, DC 20007, USA.
Prou
Delphine
D
Vernier
Philippe
P
Niznik
Hyman B
HB
Sidhu
Anita
A
D000818:Animals; D016923:Cell Death / Q000235:genetics*; D002460:Cell Line; D004298:Dopamine / Q000378:metabolism*; D050483:Dopamine Plasma Membrane Transport Proteins; D006801:Humans; D050356:Lipid Metabolism; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins / Q000378:metabolism*; D051379:Mice; D020125:Mutation, Missense / Q000235:genetics*; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics*; D019636:Neurodegenerative Diseases / Q000235:genetics / Q000378:metabolism; D018384:Oxidative Stress / Q000187:drug effects / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D051843:Synucleins; D051844:alpha-Synuclein
D050483:Dopamine Plasma Membrane Transport Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; C497604:SNCA protein, human; C497605:Snca protein, mouse; D051843:Synucleins; D051844:alpha-Synuclein; D004298:Dopamine