Oligomerization of the Dopamine Transporter: cocaine-analog- induced conformational changes at a homodimer interface Jonathan A. Javitch, MD,PhD Columbia University

The Dopamine Transporter (DAT) is responsible for re-uptake of dopamine from the synaptic cleft Amph

Although the inhibition of DAT by cocaine and amphetamine is widely documented, the structural basis of DA transport and its inhibition by cocaine and other psychostimulants such as amphetamine is poorly understood. Accessibility of endogenous cysteines and DAT topology Conformational changes associated with substrate transport and inhibitor binding to DAT Oligomerization of DAT Trafficking of DAT induced by amphetamine, cocaine, and manipulation of signal transduction pathways Mechanism of amphetamine-mediated DA efflux Bacterial and archaeal sodium-dependent transporters as model systems for structural studies

Oligomerization of neurotransmitter transporters?? (The horror! The horror!) Serotonin transporter: Co-IP effects of cysteine modification FRET GABA transporter FRET Glycine transporter: dimer intracellular and not on surface DAT: Radiation inactivation consistent with dimer and/or tetramer FRET

Flag-HA-synDAT

Cross-linking of Flag-HA-DAT

DAT runs as a broad band ~85k Da DAT is cross-linked by copper phenanthroline to a broad band ~195k Da DAT is cross-linked by bis-MTSEA to a broad band ~195k Da Cross-linking is reversed by reduction with DTT Are we cross-linking a DAT homodimer or a heterodimer between DAT and another protein?

Myc-His-DAT

Coimmunoprecipitation of Flag-HA-DAT and Myc-His-DAT

DAT is cross-linked in mouse striatal membranes

Which cysteine is responsible? MTSET blocks cross-linking so…

Cys306 is necessary for DAT cross-linking

Flag-HA-CD-DAT

Cys306 is sufficient for DAT cross-linking

The G(V/I)XXG(V/I)XX(A/T) motif is conserved in TM6 of neurotransmitter transporters

GVXXGVXXA/T Dimerization motif Deviation from helical periodicity TM6

Mutation of Gly323 and Gly327 abolished uptake

Summary of Dimerization DAT in the plasma membrane can be cross-linked into a dimer by bis-EA or CuP. Cys-306 at the extracellular end of TM6 is necessary and sufficient for cross-linking. DAT is a symmetrical dimer (at least). Cross-linking does not alter uptake or binding. The GVXXGCXXA motif is presumably involved in dimerization of TM6. “Only” 24 TMs to sort out…. But wait!

DAT is a tetramer in the membrane: a second symmetrical interface in TM4 is crosslinked by Cu ++ or HgCl 2

Cocaine analogs block crosslinking of the TM4 interface but not the TM6 interface – conformational change

Targeting and trafficking of the dopamine transporter Activation of PKC leads to acute ’downregulation’ of DAT

DAT internalization upon direct PKC activation and upon activation of a co-expressed SP receptor Control200 nM SP 1  M PMA HEK-293 cells co-expressing EGFP-hDAT and the substance P receptor (NK-1)

The hDAT contains multiple putative Ser/Thr phosphorylation sites Abbr.Full name of mutant transporterV 0 (%) WT FLAG-hDAT 100 ± 10  1-22  1-22FLAG-HA-hDAT 71 ± 2 N’N’  1-22FLAG-HA-hDAT T43A/S44A/S45A/T46A/T48A/- S53A/T62A 36 ± 7 C  1-22FLAG-HA-hDAT Y593A/S582A/S586A/T613A 43 ± 8 N+C  1-22FLAG-HA-hDAT S44A/ S45A/S53A/T62A/S582A/-S586A/T613A 18 ± 6 N’+C  1-22FLAG-HA-hDAT T43A/S44A/S45A/T46A/T48A/- S53A/T62A/Y593A/S582A/S586A/T613A 15 ± 4 ICL  1-22FLAG-HA-hDAT S261A/S262A/T339A/S517A 16 ± 10 N+ICL  1-22FLAG-HA-hDAT S44A/ S45A/S53A/T62A/S262A/-T339A/S517A 18 ± 8 C+ICL  1-22FLAG-HA-hDAT S262A/T339A/S517A/S582A/- S586A/T613A 10 ± 5 XPK8  1-22FLAG-HA-hDAT S53A/T62A/S262A/T339A/- S517A/S582A/S586A/T613A 1.5 ± 0.4 PKC consensus sites Other putative internalization motifs Non-consensus sites

Surface biotinylation Uptake No effect of mutating multiple serines and threonines in different combinations

ControlFLAG-hDAT 0SPPMA 0SPPMA 105 kD 0SPPMA FLAG-HA-hDAT-  1-22 Direct protein kinase C activation and activation of a co- expressed SP receptor increase phosphorylation of DAT Western blot Phosphorylation

Phosphorylation of a non-DAT substrate mediates internalization Role of phosphorylation?

Properties of N-terminal truncation mutant Internalization is normal in response to PMA Internalization is normal in response to SP Surface expression is normal/enhanced Km of dopamine and tyramine are normal Vmax/surface DAT is normal Oligomerization is normal

Acknowledgements Charlotta Grånäs Claus Juul Loland Ulrik Gether Habibeh Khoshbouei Aurelio Galli Bipasha Guptaroy L'Aurelle Johnson David Lund, Margaret E. Gnegy Mu Fa Zhou Amy Newman Shonit Das – bacterial transporters Yvette Dehnes – DAT IL3 Jasmine Ferrer – DAT - the beginning Naomi Goldberg - bacterial transporters Wen Guo – receptor Hanne Hastrup – DAT X-linking George Liapakis – receptor Matthias Quick - bacterial transporters Namita Sen – DAT regulation Lei Shi - receptor Merrill Simpson - receptor Mark Sonders - DAT Arthur KarlinNIDA