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NIDA Proteomics Center
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Investigators
> James A. Bibb
Phosphoproteomic Studies of NMDA
Receptor-Cdk5 Interactions
James A. Bibb, Department of Psychiatry, The University of Texas Southwestern
Medical Center It is now
clear that addiction results from usurpation of biochemical and molecular
mechanisms that underlie normal learning processes in the reward circuitry of
the brain. These involve both glutamatergic and dopaminergic neurotransmission
mediated by the second messengers Ca2+ and cAMP, respectively. Our
research has revealed that the neuronal protein kinase Cdk5 plays a critical
synaptic role in learning, and regulates behavioral responses to cocaine. Cdk5
associates with NMDA receptor complexes. It regulates the constituency of these
postsynaptic complexes and is itself regulated by NMDA receptors in a Ca2+-dependent
manner. The downstream effectors of this regulation appear to be multiple
components of the dopamine/cAMP/PKA signaling cascade. The key to understanding
Cdk5’s role in addition is to better understand the regulatory relationship that
exists between it and NMDA receptors, and how chronic exposure to cocaine
affects this relationship. Therefore the goal of this research is to combine the
powerful approach of neuroproteomics with transgenic technology to explore this
important interaction. We propose to (1) characterize the effects of Cdk5
conditional knockout and transgenic overexpression of the Cdk5 activating
cofactor p25 on the NMDA receptor complex in adult mouse brain. We will then (2)
assess the effect that chronic cocaine exposure upon this profile and the
contribution of Cdk5 to these effects. NMDA receptor complexes will be isolated
from control, Cdk5 knockout, and p25 overexpressing mice and analyzed with
regard to the spectrum of NMDA receptor constituents and phosphoproteins using
an array of proteomic technologies including IMAC, DIGE, nLC-MS/MS, and
LTQ-Orbitrap MS. Bioinformatic analysis will be conducted to define
phosphorylation sites. The effects of chronic cocaine exposure will be assessed
using similar samples from drug versus saline treated control, Cdk5 conditional
knockout, and p25 overexpressing mice. From these studies we delineate how Cdk5
regulates NMDA receptor complexes and gain basic knowledge of the precise
biochemical mechanisms of addiction. These findings of these important analyses
will be pursued as part of an ongoing and aggressive addiction research program. |
