# Presentation: 128064

## 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

## Dopamine Projections

## The Dopamine Terminal

## DAT Belongs to a Family of Na+/Cl--dependent Neurotransmitter Transporters

- Plasma Membrane
-  DAT, NET, SERT
-  GABA Transporter
-  Glycine Transporters
-  Orphan Transporters

## Cell Biology of DAT

## .

- 1. Assembly and Transport to PM
-         Oligomerization

- 2. Proper Subcellular Targeting
-        Interacting protein PICK1

- 4. Plasma Membrane Trafficking
-        Internalization?

- 3. Modulation of Function and Signaling
-           Interacting protein Hic5

## .

- 1. Assembly and Transport to PM
-         Oligomerization

- 2. Proper Subcellular Targeting
-        Interacting protein-PICK1

- 4. Plasma Membrane Recycling
-        Clathrin coated vesicles

- 3. Modulation of Function and Signaling
-           Interacting protein Hic5

## 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)

## Ni++

- 6His

- HA

- 6His

- HA

**?**

- Ni++

- Does 6His-tagged DAT interact with HA-tagged DAT

## Interaction of epitope-tagged DAT proteins

## Topology of the human DAT

## DATY335A is not functional but expressed at the cell surface

- DATY335A acts as dominant-negative mutant on wt-DAT function

## Topology of the human DAT

## Amino terminal truncations of DAT function as 

- Amino terminal truncations of DAT function as
- dominant-negative mutants

## Topology of the human DAT

**Leucine repeat**
**       ****TM2**

**Leucine repeat**
**       ****TM9**

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

## TM2 is required for DAT assembly

## .

- 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*

## .

- 1. Assembly and Transport to PM
-         Oligomerization

- 2. Proper Subcellular Targeting
-        Interacting protein-PICK1

- 4. Plasma Membrane Recycling
-        Clathrin coated vesicles

- 3. Modulation of Function and Signaling
-           Interacting protein Hic5

## Identification of DAT interacting proteins using 

- Identification of DAT interacting proteins using
- the Yeast 2-H system

- 	brain cDNA library
- 	(20 million clones)

**+**

## 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

- 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

- .

- .

- .

**KD**

**PDZ domain                       coiled coil**

**N**

- C

**20                                   110     139              166                                           415**

## The Interaction between DAT and PICK1 requires the 

- 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			    -

- Class II PDZ binding site

- PDZ domain

## Interaction between full-length DAT and PICK1

## DAT and PICK1 form “clusters” in HEK 293 cells

- PICK1

- -DAT

- - PICK1

- overlay

- PICK1	                 DAT                 PICK1 + DAT        PICK1 + DAT

## Functional Interaction between DAT and PICK1 in HEK293 cells

## DAT and PICK1 co-localize in dopamine neurons 

******-DAT**

******-DAT surface**

******-DAT**

******-DAT**

******-PICK1**

******-PICK1**

******-VMAT2**

******-synapsin**

**overlay**

- DAT and PICK1 co-localize in dopamine neurons

##      -HA              -PICK1              overlay

-      -HA              -PICK1              overlay

-  HA-DAT

- HA-DATW617

- The PICK1 binding site in DAT is critical for the targeting
- of the transporter to neuronal processes

## DAT and PICK1 form a protein complex in brain

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

**IP: ******** -PICK1****	****	****	****   IP: ******** -DAT**
**IB: ******** -DAT****	****	****	****   IB: ******** -PICK1**

**DAT**

-                                               **PICK1**

## 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
*                                                    *

## 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

- IB: DAT

- Cell Surface

- Total

## Function of monoamine transporters

**PICK1**

**Hic-5**

- targeting, clustering

- signaling,
- multi-protein
- complex?

**AP2**

**calmodulin**

**torsinA**

**snap25**

**shank**

**syntenin**

- PDZ domain-containing proteins
- Adaptor proteins
- Neurotransmitter release proteins
- Uncharacterized proteins

- using the protein-protein interaction approach

## A proteomic approach to identify the entire 

- DAT +/+ (DAT -/-)

- Proteomic Approach to Identify DAT interacting proteins

- -   synaptosomal
-      preparation
-    Immuno
-     precipitation

-           **Mass Spectrometry                                  2D-gel separation**

- A proteomic approach to identify the entire
- network of proteins associated with DAT

## Future Studies

- Identification of the protein network associated with monoamine transporters
- Relevance of these mechanisms to drug addiction

##                   thanks to....

- _Marc G. Caron_			Jeff Staudinger
- Ava Sweeney
- Ana Marin		 		Susan Amara
- Veronica Sandoval
- Wei-Dong Yao 			Susan Ingram
- 	 					Sheila Thomas

- Duke University Medical Center

- Washington State University

- Kansas University

- NIDA/NIH

- Harvard Medical School

- University of Pittsburgh

## Slide 37

## The Neurotransmitter Cycle

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

- HA-

## N-linked Glycosylation is not essential for oligomerization

## 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.

## Slide 42