? Chronic abuse of substances such as cocaine leads to structural changes in the neurons of the ventral tegmental area/nucleus accumbens. The changes in dendritic branching, spine density and spine morphology that occur in the neurons comprising this common reward pathway presumably underlie some of the behavioral changes that characterize addiction. Although it is clear that DARPP-32 (Dopamine and cAMP-Regulated PhosphoProtein, 32 kDa) and PP-1 (Protein phosphatase-1) play key roles in integrating the signals impinging on medium spiny neurons, it is not yet clear how structural changes are regulated. Rho family small GTP binding proteins and the families of GDP/GTP exchange factors and GTPase activating proteins that control their activation are key regulators of cytoskeletal dynamics. Our preliminary data reveal a substantial increase in expression of Kalirin, a Rho family GDP/GTP exchange factor, following chronic cocaine treatment of adult male rats. Exogenous expression of Kalirin in organotypic slice cultures increases spine density, while antisense-mediated reductions in Kalirin levels result in a decrease in spine density. We will first complete our exploration of the effects of chronic cocaine exposure on Kalirin. Kalirin-7 interactors will be identified through immunological and biochemical analysis of extracts prepared from control and cocaine-treated rats. The major sites at which Kalirin-7 is phosphorylated under control and cocaine-treated conditions will be identified by tandem mass spectroscopic analysis of proteolytic digests. The alterations in spine-like structures observed in medium spiny neurons following changes in Kalirin-7 expression will be evaluated using biolistic transfection of organotypic slice cultures and primary neuronal cultures. Spine dynamics will be examined using time lapse imaging and GFP-tagged Kalirin. Our preliminary data demonstrate that several well-described cocaine-responsive signaling proteins interact with Kalirin. Cdk5 phosphorylates Kalirin-7 along with DARPP-32 and Pak. Kalirin activates and forms a complex with Pak, a key regulator of actin polymerization. Protein kinase A, which phosphorylates and inactivates Pak, also phosphorylates Kalirin. PP-1, a DARPP-32 target localized to spines, also binds to Kalirin. It is our hypothesis that Kalirin-7, through its interactions with these regulators, integrates the effects of diverse signaling pathways on spine formation. ? ?
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