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

1. 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

2. Dopamine Projections

3. The Dopamine Terminal

4. DAT Belongs to a Family of Na+/Cl--dependent Neurotransmitter Transporters • Plasma Membrane • DAT, NET, SERT • GABA Transporter • Glycine Transporters • Orphan Transporters

5. Assembly Targeting . . . . . . . . . . . . . Trafficking . . . . . . . . . . . . . . . . . . . Cell Biology of DAT

6. 1 2 . . . . . . . . . . . . . 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 Trafficking Internalization? . . .

7. 1 2 . . . . . . . . . . . . . 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 . . .

8. 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)

9. Does 6His-tagged DAT interact with HA-tagged DAT HA 6His ? HA 6His Ni++

10. Interaction of epitope-tagged DAT proteins

11. 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

12. DATY335A is not functional but expressed at the cell surface DATY335A acts as dominant-negative mutant on wt-DAT function

13. 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

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

15. 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

16. 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

17. TM2 is required for DAT assembly

18. 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

19. 1 2 . . . . . . . . . . . . . 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 . . .

20. Identification of DAT interacting proteins using the Yeast 2-H system + brain cDNA library (20 million clones)

21. 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

22. 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 . .

23. 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 * *

24. 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

25. DAT and PICK1 form “clusters” in HEK 293 cells PICK1 DAT PICK1 + DAT PICK1 + DAT PICK1 -DAT - PICK1 overlay

26. Functional Interaction between DAT and PICK1 in HEK293 cells

27. DAT and PICK1 co-localize in dopamine neurons -DAT -DAT surface -DAT -DAT -PICK1 -PICK1 -VMAT2 -synapsin overlay

28. The PICK1 binding site in DAT is critical for the targeting of the transporter to neuronal processes -HA -PICK1 overlay HA-DAT HA-DATW617

29. DAT and PICK1 form a protein complex in brain DAT +/+ DAT -/- DAT+/+ DAT -/- DAT PICK1 IP:  -PICK1 IP:  -DAT IB:  -DAT IB:  -PICK1

30. 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

31. 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

32. 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

33. 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

34. 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

35. Future Studies Identification of the protein network associated with monoamine transporters Relevance of these mechanisms to drug addiction

36. 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

38. 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

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

40. N-linked Glycosylation is not essential for oligomerization

41. 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.