131115-Revised Thesis-Formats-2

FUNCTIONAL PROTEOMIC ANALYSIS OF THE DOPAMINE TRANSPORTER INTERACTOME by Sarah Michelle Rogstad B.S., Harvey Mudd College, 2008 A thesis submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirements for the degree of Doctor of Philosophy Pharmacology Program 2013 This thesis for the Doctor of Philosophy degree by Sarah Michelle Rogstad has been approved for the Pharmacology Program by J. David Port, Chair Robert C. Murphy Tatiana G. Kutateladze Nancy R. Zahniser Alexander Sorkin Christine C. Wu, Advisor Date 11/18/13 ii Rogstad, Sarah Michelle (Ph.D., Pharmacology) Functional Proteomic Analysis of the Dopamine Transporter Interactome Thesis directed by Professor Christine C. Wu. ABSTRACT Dopamine transporter (DAT) is a twelve-transmembrane domain integral membrane protein. It functions by clearing the neurotransmitter dopamine (DA) from the synapse in order to terminate synaptic transmission. DAT has been associated with a variety of diseases and disorders including ADHD, Parkinson’s disease, and schizophrenia and is affected by drugs of abuse such as cocaine and amphetamines. This dissertation focused on examination of the DAT interactome under multiple conditions in both cell and mouse models through immunoprecipitation (IP) coupled with mass spectrometry (MS). The DAT interactome was analyzed using cell models that stably expressed three dually tagged DAT mutants and parental cells. The tags allowed for highly specific IPs using tag-specific antibodies with the parental cell IPs as a negative control. Analysis of these mutants, which had varying expression levels, indicated that IP-MS studies require normalization prior to comparative analysis. Thus, a normalization method was developed for selected reaction monitoring (SRM) studies based on antibody peptide peak areas. This method was implemented with supplementary cell MS studies. Additionally, knock-in mice expressed DAT with an HA tag to allow for similar IP-MS studies using an HA antibody. Such studies were conducted on untreated striatal homogenates as well as on striatal synaptosomes treated with amphetamine. Antibody based normalization was applied in these studies as well. iii Both cell and mouse studies identified, through data-dependent acquisition based MS analyses, a variety of putative DAT associated proteins under both normal conditions and mutated or drug treated conditions. Relative quantitation of these proteins was conducted at the peptide level using SRM analysis for verification and further assessment purposes. The normalization method developed throughout this dissertation is recommended for future IP-MS studies as it accounts for intersample variability and results in more comparable data across experiments. The form and content of this abstract are approved. I recommend its publication. Approved: Christine C. Wu iv To my husband, Zachary Rogstad, and my parents, Hugh and Jane Moore v TABLE OF CONTENTS CHAPTER Page I. THE DOPAMINE TRANSPORTER 1 Sturcture and Function 1 Expression and Trafficking 5 Polymorphisms and Modifications 7 Dopaminergic Disorders 9 Drugs of Abuse 10 Known DAT Interactome 15 Summary 18 II. MASS SPECTROMETRY BASED PROTEOMICS 20 Sample Preparation 21 Chromatography 24 Ionization 26 Mass Spectrometers 28 Summary 34 III. ANALYSIS OF THE MEMBRANE TOPOLOGY OF LEUCINE TRANSPORTER 36 Introduction 36 Experimental Procedures 40 Results and Discussion 43 Summary 48 vi IV. RELATIVE NORMALIZATION OF DAT TO IMMUNOGLOBULINS 49 Introduction 49 Experimental Procedures 52 Results and Discussion 59 Summary 65 V. ANALYSIS OF THE DAT INTERACTOME IN PORCINE AORTIC ENDOTHELIAL CELLS 69 Introduction 69 Experimental Procedures 73 Results and Discussion 75 Summary 84 VI. ANALYSIS OF THE DAT INTERACTOME IN MICE WITH AMPHETAMINE AND PMA TREATMENTS 87 Introduction 87 Experimental Procedures 90 Results and Discussion 95 Summary 106 VII. CONCLUSIONS AND FUTURE DIRECTIONS 109 Introduction 109 Summary of Findings 110 Future Directions 114 Concluding Remarks 1145 REFERENCES 117 vii APPENDIX A. IgG SRM Transition Information 130 B. IgG Normalization Calculations and Statistics 135 C. Mouse IP SRM Transition Information 141 D. Mouse IP Sum Normalization 146 viii LIST OF TABLES Table Page 1.1 Known DAT Interactome 16 4.1 Western Blot Densitometry 61 4.2 Antibody Profiling Characterization 62 5.1 Most Abundant Proteins Across DAT IP Samples 80 5.2 Proteins with Most Changed Abundances between FL and ΔN IPs 81 5.3 Proteins with Most Changed Abundances between FL and ΔC IPs 82 6.1 Interactome Candidates for SRM Analyses 96 ix LIST OF FIGURES Figure Page 1.1 Chemical Structure of the Biogenic Amines 2 1.2 Conformations of LeuT 3 1.3 LeuT-DAT Protein Sequence Alignment 4 1.4 DAT Trafficking 6 1.5 Putative DAT Membrane Topology and Post-translational Modifications 8 1.6 Chemical Structures of DAT-interacting Drugs 12 1.7 Effects of Drugs on DAT Trafficking 13 2.1 MS Sample Preparation 22 2.2 Electrospray Ionization 27 2.3 Tandem MS 29 3.1 Membrane Shaving Schematic 37 3.2 Structures of Leu and pL 39 3.3 3H-Leu Uptake Assay 44 3.4 Identified LeuT Peptides 46 3.5 LeuT Representative SRM Chromatography 47 4.1 IP Schematic and Densitometry 50 4.2 Characterization of Mouse IgG 58 4.3 Characterization of Rabbit IgG 59 4.4 Mouse IgG SRM 63 4.5 Rabbit IgG SRM 64 4.6 Relative Normalization of YFP-DAT AUCs to Mouse IgG AUCs 66 x 4.7 Relative Normalization of YFP-DAT AUCs to Rabbit IgG AUCs 67 5.1 YFP-HA-DAT Constructs and Expression 71 5.2 DAT Sequence Coverage 76 5.3 IgG and DAT Peptide Sums 78 5.4 Normalized DAT Peptide Levels 79 5.5 Normalized peptide Abundance Levels of Putative DAT Interactome Members 84 6.1 Peptide Levels of Quantitation 99 6.2 IgG Normalization of IgG Peptides 100 6.3 IgG Normalization on DAT Peptides 102 6.4 DAT Time Courses 103 6.5 DAT Interactor Time Courses 104 xi CHAPTER I THE DOPAMINE TRANSPORTER Dopamine transporter (DAT) is a twelve transmembrane domain (TMD) integral membrane protein (IMP)1. As a member of the solute carrier 6 (SLC6) gene family of neurotransmitter transporters, DAT functions by clearing extracellular dopamine (DA), from the synapse in order to terminate its synaptic transmission2,3. Dysfunction of DAT is known to be associated with a variety of diseases and disorders, such as attention deficit hoperactivity disorder (ADHD)4, Parkinson’s disease5, and schizophrenia5. Drugs of abuse such as amphetamine and cocaine also affect DAT. This results in a decrease in DAT function and a subsequent increase in DA signaling6,7. Thus, DAT is highly significant clinically. Furthermore, increasing our comprehension of DAT function and processing is critical for furthering our understanding of how these disorders and drugs affect people and how to treat them. This chapter outlines DAT’s structure and function, expression and trafficking, known polymorphisms and modifications, associated disorders, alterations with drug treatments, and known interacting proteins. It also serves to illustrate the complex relationship between these attributes and relate these issues to the following dissertation, which uses mass spectrometric methods to analyze DAT and the DAT interactome. Structure and Function The SLC6, or neurotransmitter sodium symporter (NSS) family, is a group of 12 TMD IMPs, including DAT as well as norepinephrine, serotonin, glycine, and GABA 1 transporters3. In addition to their 12 TMDs, intracellular amino and carboxyl termini and glycosylation of the second extracellular loop structurally characterize this family of transporters8. Functionally, these transporters co-transport their specific substrate with two sodium ions and one chloride ion using the pre-existing neuronal sodium gradient for energy9. These substrates are known as the biogenic amines and are highly similar in structure (Figure 1.1). Currently, none of the mammalian NSS family members have a solved crystal structure. This conundrum largely results from the fact that they contain 12 TMDs. These domains lie within the lipid bilayer and largely consist of non-polar residues making them highly hydrophobic and difficult to crystalize. However, the crystal structure of one bacterial homolog of the family has been solved in a variety of different bound states. NH2 NH2 OH NH2 HO HO HO HO N H OH Dopamine Serotonin Norepinephrine Figure 1.1 – Chemical Structures of the Biogenic Amines. The biogenic amines transported by the NSS family are dopamine, serotonin, and norepinephrine. All three contain a six-membered aromatic ring with at least one attached hydroxyl group as well as a primary amine group attached to the ring by a carbon chain. The leucine transporter (LeuT) from Aquifex aeolicus was first crystalized while bound to its substrate leucine and two sodium ions in 20059,10. Yamashita et al found that leucine was bound to LeuT between TMDs 1 and 6. This structure revealed a conformation that resembled a “shallow shot glass”, which did not allow for solvent 2 accessibility to the substrate. This “occluded state” indicated that the transporter likely forms outward and inward facing conformations as well, thus allowing for movement of the substrate through the transporter across the plasma membrane. Since 2005, LeuT has been re-crystallized with multiple additional substrates. Several antidepressants that act as non-competitive inhibitors were found to lock the transporter into its occluded state11,12. a) LeuT b) 1b 6a 8 1b 6a 10 10 6b c) 1a 3 8 1a 3 6b 1b LeuT 3 6a 10 LeuT LeuT 8 d) e) 1a 6b 8 Non-Comp 1b 1b 6a 3 6a Comp 10 10 LeuT 3 8 1a 1a 6b 6b Figure 1.2 – Conformations of LeuT. The a) outward facing conformation and b) inward facing conformation were most recently solved13. The original solved LeuT conformation c) is known as the substrate occluded conformation10. Two additional states have been solved with d) competitive14 and e) non-competitive inhibitors11,12. These states are known as outward-locked and locked-occluded states, respectively.

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