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Sara Olson Original Research Proposal Spring 2007

Activation of Canonical Transient Receptor Potential 3 (TRPC3) by Protein Kinase Src: Implications for Nicotine Addiction. Sara Olson Original Research Proposal Spring 2007. Nicotine Addiction. Nicotine-related diseases are the leading preventable cause of death in industrialized countries

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Sara Olson Original Research Proposal Spring 2007

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  1. Activation of Canonical Transient Receptor Potential 3 (TRPC3) by Protein Kinase Src: Implications for Nicotine Addiction Sara Olson Original Research Proposal Spring 2007

  2. Nicotine Addiction • Nicotine-related diseases are the leading preventable cause of death in industrialized countries • An estimated 45 million Americans are currently addicted to tobacco

  3. Dopamine travels to command interneurons, TRPC3 modulates body’s response Physiology of nicotine addiction Dopamine, released by receptors in the NA Nicotine Nucleus Accumbens Ventral Tegmental Area PKSrc interacts with TRPC3 to bring about its activation

  4. - Canonical Transient Receptor Potential 3 • Implicated in nicotine addiction • Ca2+ cation channel • Member of highly conserved superfamily • Activation requires DAG at the membrane and PKSrc intracellularly diacylglycerol (DAG)

  5. TRPC3 Activation by PKSrc • Tyrosine kinase • Phosphorylates TRPC3 at Y226, interacts further through SH2 domain • Activation by PKSrc often involves an “Adaptor” protein • Most commonly, XB130 is this adaptor Protein Kinase Src (PKSrc) – Ribbon Diagram based on Crystal Structure

  6. How does PKSrc activate TRPC3? I hypothesize that, following phosphorylation, PKSrc and TRPC3 have a continued interaction that involves XB130. To test this hypothesis I propose the following: • Specific aim 1: Determine whether XB130 is recruited to the site of activation. • Specific aim 2: Determine the strength of the interaction between TRPC3 and PKSrc including any effect by XB130. Specific aim 3: Determine which region in PKSrc’s SH2 domain and TRPC3’s intracellular domain are responsible for the physical interaction.

  7. Western Blots of IP with respective Abs Specific Aim 1: Determine if XB130 is recruited to site of activation • Crosslinking, immunoprecipitation, Western blot and MALDI-TOF MS Pre-Immune IP anti-XB130 anti-PKSrc anti-TRPC3 Trypsin and Chymotrypsin Digestions of excised bands SDS-PAGE stained with Coomassie MALDI-TOF MS peptide fingerprinting

  8. Bound TRPC3i Free TRPC3i Scatchard Plot: Slope = -1/Kd X-int = Bmax Bound/Free TRPC3i Bound TRPC3i Specific aim 2: Determination of binding Affinity • Equilibrium Dialysis • Regenerated Cellulose Membrane (1-50 kD) • TRPC3i = 47 kD (phosphorylated and not) • PKSrc = 59 kD • XB130 = 130 kD (present and absent)

  9. Specific Aim 3: Determine the regions responsible for interaction • Phage Display Wash column, remove unbound phage Create affinity column, apply phage Library of phage displaying peptides Repeat to enrich results Elute bound phage Alignment of recovered sequences Harvest resultant phage, sequence DNA for inserted peptide sequence Transduce into E. coli

  10. Specific Aim 3: Expected Phage Display Results Sequence alignments of all phage recovered after 5X enrichment of results should show which residues consistently bound to column Controls: Once binding region is clarified, mutate the residues involved and apply mutant peptide-displaying phage to column to confirm requirement of region in binding.

  11. Conclusions • Specific aim 1: XB130 will immunoprecipitate, showing its involvement in the activation of TRPC3 • Specific aim 2: XB130 will increase the tightness of binding of PKSrc to TRPC3 • Specific aim 3: I expect to narrow the area of interaction in the SH2 domain of PKSrc and the intracellular region of TRPC3 to specific regions

  12. Future Directions • Development of antagonists to PKSrc with respect to its activation of TRPC3 will help move toward applications in nicotine cessation therapies • Work should be directed toward detailing the nature of interactions between PKSrc and XB130 if it immunoprecipitates with TRPC3.

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