An investigation into the role of the neuronal calcium sensor NCS-1 in mediating D2 dopamine receptor signaling

Based on pharmacology, structure, and sequence homology the dopamine receptor family is classically subdivided into the D1-like (D1 and D5) and D2-like (D2, D3, and D4) receptor subfamilies. The D2-like receptor subfamily is implicated in a vast range of neurological functions including locomotor regulation and aspects of cognition and learning. In addition, D2-like receptors are the principal molecular target of typical and atypical classes of anti-psychotic drugs. We have sought out to identify and characterize D2-like receptor interacting proteins (DRIPs) using the yeast two-hybrid method. Successive yeast two-hybrid screens of a human brain cDNA library were performed using the second and carboxyl intracellular segments of the human D2 dopamine receptor (D2R) as bait. A cohort of candidate DRIPs were identified from these screens. In this thesis I describe the functionality of one such interaction between the D2R and the neuronal calcium sensor protein NCS-1.; We have validated NCS-1/D2R interaction in both heterolgous, as well as, native protein expression systems. In addition, we have localized the domains of association between D2Rs and NCS-1. Ultrastructural analysis of NCS-1/D2Rs coexpression in rodent and primate brain indicates that NCS-1 and D2Rs colocalize within sites of synaptic transmission and in close proximity to intracellular calcium stores. Collectively, our studies present a novel mechanism linking D2Rs with intracellular calcium pathways, and suggest an important role for NCS-1 in mediating dopaminergic transmission.; Additional members of the neuronal calcium sensor family are known to play a role in GPCR signaling by regulating G-protein coupled receptor kinase (GRK) activity. We tested the hypothesis that NCS-1 mediates D2R desensitization. Our studies indicate that NCS-1 interacts with both D2Rs and GRKs in a calcium sensitive manner, and that interaction between these proteins attenuates D2R desensitization. Moreover, studies of tissue obtained from the prefrontal cortex of schizophrenic and bipolar patients, reveals a two-fold increase in total NCS-1 levels in these individuals. Taken together, these studies demonstrate that NCS-1/D2R interaction mediates a calcium-dependent component of D2R signaling. This mechanism is likely to contribute to important features of dopaminergic transmission in the healthy and diseased brain.