Activation of Canonical Transient Receptor Potential 3 TRPC3 by Protein Kinase Src: Implications for

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Activation of Canonical Transient Receptor
Potential 3 (TRPC3) by Protein Kinase Src
Implications for Nicotine Addiction

• Sara Olson
• Original Research Proposal
• Spring 2007

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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

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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
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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)
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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
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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 PKSrcs
SH2 domain and TRPC3s intracellular domain are
responsible for the physical interaction.
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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
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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)

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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
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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.
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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

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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.