IDENTIFICATION OF NOVEL PROTEIN CONSTITUENTS OF THE DOPAMINE D2 RECEPTOR SIGNALPLEX: A STUDY OF TRPC1/D2R INTERACTION AND ITS ROLE IN SIGNALPLEX TRAFFICKING
Open Access
- Author:
- Hannan, Meredith A
- Graduate Program:
- Genetics
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- May 25, 2007
- Committee Members:
- Robert G Levenson, Committee Chair/Co-Chair
Ralph Lauren Keil, Committee Member
Blaise Peterson, Committee Member
Janet Robishaw, Ph D, Committee Member - Keywords:
- Signalplex
Transient Receptor Potential
Dopamine D2 Receptor
Schizophrenia - Abstract:
- Dopamine exerts its effects through the activation of a family of dopamine receptors (DRs) and their subsequent coupling to downstream effector molecules. Dysfunction of dopaminergic neurotransmission is responsible for a myriad of diseases including Parkinson’s disease, Tourette’s syndrome, attention deficit hyperactivity disorder (ADHD), drug abuse, and schizophrenia. Schizophrenia is a devastating mental illness that affects approximately one percent of the population worldwide. It attacks its victims upon their emergence into adulthood and the workforce and leaves them severely disabled and dependent on life-long pharmacological treatment for. The majority of therapeutic antipsychotic drugs act as antagonists at D2R sites in the brain, but D2Rs are not genetically altered in schizophrenic individuals. We hypothesize that pathological dysregulation of dopaminergic signaling may stem from alterations in expression or function of proteins which interact with and regulate DR-mediated signaling, otherwise known as DRIPs (dopamine receptor interacting proteins) and DRAPs (dopamine receptor associated proteins). Work presented in this thesis further investigates protein constituents of the D2R-signalplex and characterizes their effects on the regulation of dopaminergic signaling. A systematic survey of the literature has culminated in the collection of information concerning approximately fifty DRIPs and DRAPs. These proteins comprise the D2R-signalplex and affect various aspects of the life cycle of D2Rs, including regulation, trafficking, degradation, and recycling. Efforts were undertaken to investigate whether interacting partners of these known DRIPs could themselves bind the D2R. Isolation and characterization of the cohort of proteins that comprise the D2R signalplex will provide insight into the mechanisms of receptor function in both healthy and diseased states. TRPC1 was identified as a novel DRIP via yeast two-hybrid strategy. Here we validate the interaction of TRPC1/D2R in both heterologous and native protein expression systems. TRPC1, a nonselective, nonvoltage gated cation channel, colocalizes with D2R in cell culture systems and primate cortical neurons. TRPC proteins can function as scaffolding molecules as well as ion channels, and the TRPC1/D2R interaction affects trafficking of the D2R signaling complex to the plasma membrane. TRPC1 provides a direct link between D2R and intracellular calcium stores and may play a functional role in the pathogenesis of neuropsychiatric diseases. Interactions between individual DRIPs NCS-1 and TRPC5 were further investigated. NCS-1 and TRPC5 were confirmed to interact through biochemical assays and native protein expression systems. NCS-1 regulates currents through TRPC5 channels, and both proteins were identified to function in a common pathway that operates to control the process of neurite extension. An interruption of this interaction would have significant neurodevelopmental consequences, and may contribute to neurological dysfunction in pathologic states. zDHHC4, a newly discovered palmitoyltransferase, has been confirmed to interact with the D2R in a number of protein-interaction systems. Evidence is presented that suggests that zDHHC4/D2R interaction has different functional consequences for the two D2R isoforms, D2S and D2L. The zDHHC4/D2L interaction promotes the internalization of the receptor, whereas the zDHHC4/D2S interaction appears to have no effect on the trafficking of this isoform. This suggests that zDHHC4 differentially palmitoylates the D2L on cysteine residues not present in the D2S. zDHHC4 may contribute to the differences in D2S/D2L trafficking observed in neurons.