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• FRETGABA transporter• FRETGlycine transporter:

dimer intracellular and not on surfaceDAT:• Radiation inactivation consistent with dimer and/or tetramer• FRET

Flag-HA-synDAT

Cross-linking of Flag-HA-DAT

205

132

90

55

Bis-EA

CuPhe

+

++

+ +

+DTT ++ +PNGase + + +

FlagHA-WT

• 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/TDimerization 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 HgCl2

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

Control 200 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 transporter V0 (%)

WT FLAG-hDAT 100 ± 10

1-22 1-22FLAG-HA-hDAT 71 ± 2

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

Control FLAG-hDAT

0 SP PMA 0 SP PMA

105 kD

0 SP PMA

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 JohnsonDavid Lund, Margaret E. Gnegy

Mu Fa ZhouAmy Newman

Shonit Das – bacterial transportersYvette Dehnes – DAT IL3Jasmine Ferrer – DAT - the beginningNaomi Goldberg - bacterial transportersWen Guo – receptorHanne Hastrup – DAT X-linkingGeorge Liapakis – receptorMatthias Quick - bacterial transportersNamita Sen – DAT regulationLei Shi - receptorMerrill Simpson - receptorMark Sonders - DAT

Arthur Karlin

NIDANIDA