CHARACTERIZATION OF THE D2 DOPAMINE RECEPTOR/NEURONAL CALCIUM SENSOR-1 INTERACTION
Open Access
- Author:
- Woll, Matthew Patrick
- Graduate Program:
- Pharmacology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- September 09, 2009
- Committee Members:
- Robert G Levenson, Dissertation Advisor/Co-Advisor
Robert G Levenson, Committee Chair/Co-Chair
John Ellis, Committee Member
Melvin Lee Billingsley, Committee Member
Victor Alan Canfield, Committee Member
Blaise Peterson, Committee Member - Keywords:
- Fluorescence Polarization
D2 Dopamine Receptor
NCS-1 - Abstract:
- Abnormalities in D2 dopamine receptor (D2R) neurotransmission are implicated in a number of neurological and psychiatric conditions including bipolar disorder, depression, and schizophrenia. Studies suggest that the D2R both affects and is affected by intracellular Ca2+ concentrations, and these actions are most likely through interactions with components of the D2R signalplex. A disruption in the interaction between the D2R and proteins within the signalplex may promote the onset of these disease states. Neuronal calcium sensor-1 (NCS-1) directly interacts with the D2R and is a proven modulator of neurosecretion by regulation of Ca2+ signaling. The result of the D2R/NCS-1 interaction is attenuation of agonist-induced internalization of the D2R by inhibition of G-protein coupled receptor kinase 2 (GRK2) phosphorylation of the D2R. This inhibition of D2R internalization promotes continued signaling of the D2R, which may have disease related consequences. The hypothesis of this dissertation is that the interaction between D2R and NCS-1 is dependent on ion binding and structural features of NCS-1. This dissertation presents a method for production and purification of recombinant NCS-1 to be used in biochemical experiments, enabling the characterization of NCS-1 and the D2R/NCS-1 interaction. Ca2+ binding affects on the functional properties of NCS-1 are explored and reveal a Ca2+ requirement for the formation of a NCS-1 homo-dimer. Implications for a Ca2+/Mg2+ regulation of the D2R/NCS-1 interaction and the involvement of the NCS-1 homo-dimer are suggested from results of a developed fluorescence anisotropy (FA) assay. A model for a D2R/NCS-1 monomer-dimer complex formation was generated to demonstrate a novel mechanism of how Ca2+ and Mg2+ influence NCS-1 regulation of D2R activity.