NIDA Mini Convention: Structure, Function and Regulation of the Dopamine Transporter

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NIDA Mini Convention Structure, Function and
Regulation of the Dopamine Transporter Protein-P
rotein Interactions Defining New Pathways
Involved in the Regulation of the Dopamine
Transporter Gonzalo E. Torres, Ph.D. Department
of Cell Biology Duke University November 7, 2003
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Dopamine Projections
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The Dopamine Terminal
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• DAT Belongs to a Family of Na/Cl--dependent
  Neurotransmitter Transporters
• Plasma Membrane
• DAT, NET, SERT
• GABA Transporter
• Glycine Transporters
• Orphan Transporters

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Cell Biology of DAT
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Cell Biology of DAT
1. Assembly and Transport to PM
Oligomerization
2. Proper Subcellular Targeting
Interacting protein PICK1
3. Modulation of Function and Signaling
Interacting protein Hic5
.
4. Plasma Membrane Trafficking
Internalization?
.
.
.
.
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Cell Biology of DAT
1. Assembly and Transport to PM
Oligomerization
2. Proper Subcellular Targeting
Interacting protein-PICK1
3. Modulation of Function and Signaling
Interacting protein Hic5
.
4. Plasma Membrane Recycling Clathrin
coated vesicles
.
.
.
.
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Does DAT exist as an oligomeric complex?

• Evidence supporting
  oligomerization of Monoamine
• Plasma Membrane
  Transporters
• Radiation inactivation
  (DAT)
• Cross-linking experiments
  (SERT)
• Functional expression of
  concatamers (SERT)

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Does 6His-tagged DAT interact with HA-tagged DAT
HA
6His
?
HA
6His
Ni
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Interaction of epitope-tagged DAT proteins
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Topology of the human DAT
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DATY335A is not functional but expressed at the
cell surface
DATY335A acts as dominant-negative mutant on
wt-DAT function
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Topology of the human DAT
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Amino terminal truncations of DAT function as
dominant-negative mutants
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Topology of the human DAT
Leucine repeat TM2
Leucine repeat TM9
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Disruption of the Leucine-repeat from TM2 results
in a non-functional transporter devoid of
dominant-negative activity
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TM2 is required for DAT assembly
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Oligomerization of DAT
.
Biochemical and functional evidence demonstrate
that DAT exists as an oligomeric complex in
cells The second transmembrane domains is
involved in DAT assembly and trafficking Oligome
rization of DAT proteins seems to be required
for the proper trafficking of the transporter to
the plasma membrane
.
.
Torres et al., Submitted
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Cell Biology of DAT
1. Assembly and Transport to PM
Oligomerization
2. Proper Subcellular Targeting
Interacting protein-PICK1
3. Modulation of Function and Signaling
Interacting protein Hic5
.
4. Plasma Membrane Recycling Clathrin
coated vesicles
.
.
.
.
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Identification of DAT interacting proteins using
the Yeast 2-H system

brain cDNA library (20 million clones)
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Putative DAT Interacting Proteins PICK1 Shan
k PDZ domain-containing
proteins Syntenin Hic-5 ? subunit AP2
Adaptor proteins Calmodulin SNAP-25
Transmitter release protein TorsinA
Unknown
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Identification of PICK1 as a DAT interacting
protein
PDZ domain coiled coil
N
C
KD
20 110 139
166
415
.
PICK1 is a PDZ domain-containing protein
identified as a PKC interacting
protein Interacts with AMPAR, mGLUR7R, ASIC, and
EphR Involved in clustering, trafficking, and
targeting
.
.
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The Interaction between DAT and PICK1 requires
the PDZ domain of PICK1 and the PDZ binding site
of DAT
BAIT
PREY
INTERACTION pAS2.1-DATC pGAD10-PICK1
pAS2.1-DATC pGAD10-PICK1(K27A/
D28A) - -------RHWLKV pGAD10-PICK1
-------RHW pGAD10-PICK1
- -------RHWLK pGAD10-PICK1
- -------RHWLKVY pGAD10-PICK1 -
PDZ domain
Class II PDZ binding site


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Interaction between full-length DAT and PICK1
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DAT and PICK1 form clusters in HEK 293 cells
PICK1 DAT PICK1
DAT PICK1 DAT
PICK1
?-DAT
?- PICK1
overlay
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Functional Interaction between DAT and PICK1 in
HEK293 cells
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DAT and PICK1 co-localize in dopamine neurons
?-DAT
?-DAT surface
?-DAT
?-DAT
?-PICK1
?-PICK1
?-VMAT2
?-synapsin
overlay
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The PICK1 binding site in DAT is critical for the
targeting of the transporter to neuronal
processes
?-HA ?-PICK1
overlay
HA-DAT
HA-DATW617?
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DAT and PICK1 form a protein complex in brain
DAT / DAT -/-
DAT/ DAT -/-
DAT

PICK1
IP ? -PICK1 IP ? -DAT IB ? -DAT IB
? -PICK1
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DAT and PICK1 Interaction

• PICK1 may contribute to the targeting of DAT to
  their
• appropriate location at nerve terminals through
  an
• interaction involving the PDZ domain of PICK1
  and a
• PDZ binding site located at the end of the
  transporter
• Monoamine transporters represent a novel class
• of membrane-bound proteins that are regulated by
  
• PDZ domain-containing proteins
• 
  

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Putative DAT Interacting Proteins PICK1 Shan
k PDZ domain-containing
proteins Syntenin Hic-5 ? subunit AP2
Adaptor proteins Calmodulin SNAP-25
Transmitter release protein TorsinA
Unknown
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Regulation of DAT Function by Calmodulin
Cell Surface
Total
IB DAT
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Function of monoamine transporters using the
protein-protein interaction approach
signaling, multi-protein complex?
Hic-5
targeting, clustering
PICK1
AP2
calmodulin
snap25
PDZ domain-containing proteins Adaptor
proteins Neurotransmitter release
proteins Uncharacterized proteins
torsinA
syntenin
shank
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Proteomic Approach to Identify DAT interacting
proteins
A proteomic approach to identify the entire
network of proteins associated with DAT

• - synaptosomal
• preparation
• Immuno
• precipitation

DAT / (DAT -/-)
Mass Spectrometry
2D-gel separation
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Future Studies Identification of the protein
network associated with monoamine
transporters Relevance of these mechanisms to
drug addiction
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thanks to.

• Marc G. Caron Jeff Staudinger
• Ava Sweeney
• Ana Marin Susan Amara
• Veronica Sandoval
• Wei-Dong Yao Susan Ingram
• Sheila Thomas

Kansas University
University of Pittsburgh
Washington State University
Duke University Medical Center
Harvard Medical School
NIDA/NIH
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The Neurotransmitter Cycle
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The leucine-repeat in TM2 is important for DAT
assembly and trafficking
HA-
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N-linked Glycosylation is not essential for
oligomerization
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Importance of Transporter Oligomerization
Hastrup et al., 2001.Symmetrical dimer of the
human dopamine transporter revealed by
cross-linking Cys-306 at the extracellular end of
the sixth transmembrane segment. Sorkina et al.,
2003 Oligomerization of dopamine transporters
visualized in living cells by fluorescence
resonance energy transfer microscopy. Kilic and
Rudnick, 2000 Oligomerization of serotonin
transporter and its functional consequences. Shol
ze et al., 2002. Mutations within an
intramembrane leucine heptad repeat disrupt
oligomer formation of the rat GABA transporter
1. Hahn et al., 2003 A mutation in the human
norepinephrine transporter gene (SLC6A2)
associated with orthostatic intolerance disrupts
surface expression of mutant and wild-type
transporters.
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