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

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Nida mini convention structure

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

Protein-Protein 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


Nida mini convention structure

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


Nida mini convention structure

Assembly

Targeting

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Trafficking

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Cell Biology of DAT


Nida mini convention structure

1

2

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3, 4

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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?

.

.

.


Nida mini convention structure

1

2

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3, 4

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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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DA

DA

?

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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S

Extracellular

P

G

D

G

K

S

S

G

E

S

T

H

I

L

L

L

F

M

L

V

F

V

I

L

C

I

L

L

L

K

V

L

L

G

A

F

L

S

I

I

P

Q

D

Y335A (Y-x-x-)

M

S

S

R

T

V

K

K

P

R

E

A

Intracellular

L

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


Nida mini convention structure

S

Extracellular

P

G

D

G

K

S

S

G

E

S

T

H

I

L

L

L

F

M

L

V

F

V

I

L

C

I

L

L

L

K

V

L

L

G

A

F

L

S

I

I

P

Q

D

M

S

S

R

T

M60

V

K

K

P

R

E

A

Intracellular

L

Topology of the human DAT


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Amino terminal truncations of DAT function as

dominant-negative mutants


Nida mini convention structure

S

Extracellular

P

G

D

G

K

S

S

G

E

S

T

H

I

L

L

L

F

L

M

V

F

V

I

L

C

I

L

L

L

K

V

L

L

G

A

F

L

S

I

I

P

Q

D

M

S

S

R

T

V

K

K

P

R

E

A

Intracellular

L

Topology of the human DAT

Leucine repeat

TM2

Leucine repeat

TM9


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120

80

40

0

.

1

DAT TM2 TM9

DAT

DAT/2AR

DAT/DAT4LA

DAT/pcDNA3.1

DAT/DATY335A

DAT/DATS582stop

Disruption of the Leucine-repeat from TM2 results in a non-functional transporter devoid of dominant-negative activity


Nida mini convention structure

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

Oligomerization of DAT proteins seems to be required for

the proper trafficking of the transporter to the plasma

membrane

.

.

Torres et al., Submitted


Nida mini convention structure

1

2

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.

3, 4

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

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

.

.

.


Nida mini convention structure

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

Shank PDZ domain-containing proteins

Syntenin

Hic-5

 subunit AP2 Adaptor proteins

Calmodulin

SNAP-25 Transmitter release protein

TorsinA Unknown


Identification of pick1 as a dat interacting protein

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-DATCpGAD10-PICK1(K27A/D28A) -

-------RHWLKVpGAD10-PICK1 +

-------RHWpGAD10-PICK1 -

-------RHWLKpGAD10-PICK1 -

-------RHWLKVYpGAD10-PICK1 -

PDZ domain

Class II PDZ binding site

*

*


Nida mini convention structure

DAT + + +

PICK1 - + +

DATW617* +

PICK1 +

IP: PICK1 total

IB: DAT DAT

DAT

PICK1

IP: DAT

IB: PICK1

PICK1

PICK1

DAT

total

PICK1

DAT - + +

PICK1 + + +

total

DATW617*

IP: DAT total

IB: PICK1 PICK1

Interaction between full-length DAT and PICK1


Nida mini convention structure

DAT and PICK1 form “clusters” in HEK 293 cells

PICK1 DAT PICK1 + DAT PICK1 + DAT

PICK1

-DAT

- PICK1

overlay


Nida mini convention structure

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


Nida mini convention structure

The PICK1 binding site in DAT is critical for the targeting

of the transporter to neuronal processes

-HA -PICK1 overlay

HA-DAT

HA-DATW617


Nida mini convention structure

DAT and PICK1 form a protein complex in brain

DAT +/+ DAT -/- DAT+/+ DAT -/-

DAT

PICK1

IP:  -PICK1 IP:  -DAT

IB:  -DAT IB:  -PICK1


Nida mini convention structure

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


Nida mini convention structure

Putative DAT Interacting Proteins

PICK1

Shank PDZ domain-containing proteins

Syntenin

Hic-5

 subunit AP2 Adaptor proteins

Calmodulin

SNAP-25 Transmitter release protein

TorsinA Unknown


Regulation of dat function by calmodulin

Control

W-7

92

52.9

CaM

Seph

Input

Seph

*

*

*

*

*

92

IB: DAT

52.9

Regulation of DAT Function by Calmodulin

Cell Surface

Total

IB: DAT


Nida mini convention structure

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


Thanks to

thanks to….

Marc G. CaronJeff 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


Nida mini convention structure

pre-synaptic

neuron

vesicles containing

neurotransmitter

vesicular transporter

plasma membrane

transporter

pre-synaptic

receptors

synaptic cleft

post-synaptic

receptors

post-synaptic

neuron

The Neurotransmitter Cycle


Nida mini convention structure

The leucine-repeat in TM2 is important for DAT assembly and trafficking

HA-


Nida mini convention structure

N-linked Glycosylation is not essential for oligomerization


Nida mini convention structure

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.

Sholze 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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