Browsing by Subject "palmitoylation"

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    Palmitoylation of Caveolin-1 and its importance for structural and functional plasticity
    (2018-11) Eisinger, Katherine
    This dissertation examines the regulation and function of caveolin-1 (Cav1). Cav1 is an integral membrane protein that creates functional microdomains of neuronal proteins within lipid rafts. Cav1 regulates a variety of signaling pathways, including mGluR-activated G protein cascades, and is involved in membrane trafficking of proteins such as estrogen and dopamine receptors. The function of Cav1 is regulated by palmitoylation, a reversible post-translational addition of a 16-carbon lipid chain that is involved in trafficking and compartmentalizing target proteins. This regulatory mechanism is important not only for Cav1, but also for membrane association of estrogen receptors. Within the nervous system, palmitoylation of estrogen receptor alpha (ERα) is necessary for surface membrane localization and mediation of downstream signaling through the activation of metabotropic glutamate receptors (mGluRs). Mutation of the single palmitoylation site on ERα prevents its physical association with Cav1, which in turn is required for the formation of the estrogen receptor/mGluR signaling complex. Interestingly, siRNA knockdown of either of two palmitoyl acyltransferases, DHHC7 or DHHC21, also eliminates this signaling mechanism. As ERα has only one palmitoylation site, I hypothesized that one of these DHHCs palmitoylates another essential protein in this signaling complex, namely Cav1. I investigated this using an acyl-biotin exchange assay in HEK293 cells in conjunction with DHHC overexpression, and found that DHHC7 increased Cav1 palmitoylation. Mutation of the palmitoylation sites on Cav1 eliminated this effect, but did not disrupt the ability of the DHHC enzyme to associate with the protein. In contrast, siRNA knockdown of DHHC7 alone was not sufficient to decrease Cav1 palmitoylation, but rather required simultaneous knockdown of DHHC21. These findings raise questions about the overall influence of palmitoylation on the membrane-initiated estrogen signaling pathway, and highlight the importance of considering the influence of palmitoylation on other Cav1-dependent processes. Additionally, recent studies have shown that altering Cav1 expression influences neuronal plasticity and related behaviors in contexts ranging from learning and memory to chronic injury. Given this relationship between Cav1 and experience-dependent plasticity, I hypothesized that Cav1 expression would also be involved in drug-induced changes in neuronal signaling. I utilized a locomotor sensitization paradigm to test this hypothesis. Animals receiving repeated cocaine displayed behavioral sensitization and greater expression of Cav1 mRNA in the nucleus accumbens when compared to saline-treated controls. Overexpression of Cav1 in the nucleus accumbens enhanced cocaine-induced locomotor responses to cocaine, while Cav1 KO animals did not sensitize. Cultured neurons from the nucleus accumbens, a brain region critical for the development of sensitization, had enhanced dendritic complexity in Cav1 KO mice and altered responses to cocaine. Finally, I report that Cav1 palmitoylation is required for its normal function. Together, these findings suggest that (1) Cav1 KO mice may be structurally saturated such that normal drug-induced plasticity is prevented, (2) Cav1 palmitoylation plays an important role in facilitating the proper activity of signaling molecules associated with Cav1, and (3) understanding Cav1 function will be necessary for fully understanding the development of addiction.