Abstract: LB MON 32

Synaptic Proteins and Kinases from Female Hypothalamus Associate with Unliganded Progestin Receptors (PR) in a Dopamine-Dependent Manner

Presenter: Kalpana D Acharya


Abstract


Kalpana D Acharya*1, Sabin A Nettles1, Cheryl F Lichti2, Larry Denner2 and Marc J Tetel1

1Wellesley College, Wellesley, MA, 2Univ of TX Med Branch, Galveston, TX

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Kalpana D Acharya*1, Sabin A Nettles1, Cheryl F Lichti2, Larry Denner2 and Marc J Tetel1

1Wellesley College, Wellesley, MA, 2Univ of TX Med Branch, Galveston, TX

Progestin receptor (PR) action in brain is essential for neural development, neuroprotection, reproductive behavior, cognition, and learning and memory. While many PR-mediated effects are dependent on progestins, PR can also be activated in the absence of hormone by neurotransmitters such as dopamine. This ligand-independent activation of PR by dopamine in the female rodent hypothalamus can induce female sexual behavior. In this study, we explored the mechanisms regulating ligand-independent activation of PR in the female brain by asking which proteins from brain associate with dopamine-activated PR in the absence of hormone. Eight week old C57BL6 mice were ovariectomized and one week later subcutaneously injected with estradiol benzoate (EB, 1 µg) to induce PR. Forty-eight hours after EB, mice were infused with 100 ng of SKF, a D1 receptor agonist, or vehicle into the 3rd ventricle. Thirty minutes after the SKF infusion, mice were sacrificed and hypothalamus, a brain region rich in PR and integral for female reproduction, was collected and proteins were extracted. Pull-down assays were done using GST-tagged mouse PR-A or PR-B in the presence or absence of R5020, a PR agonist. Isolated protein complexes from SKF or vehicle-treated tissue that interacted with unliganded PR were analyzed by tandem mass spectrometry. Of the many hypothalamic proteins identified to interact in an SKF-dependent manner with unliganded PR-A, kinases and synaptic proteins dominated the PR interactome. The other major identified functional groups of proteins consisted of phosphatases, transcriptional and translational regulators, enzymes involved in cellular energy production and cytoskeletal proteins. In contrast to PR-A, unliganded PR-B did not associate with any hypothalamic proteins in an SKF-dependent manner, suggesting that PR-A contributes more than PR-B to ligand-independent activation of mouse PR which is consistent with previous behavioral studies. These novel findings provide mechanisms by which dopamine can activate PR in the absence of hormone and a comprehensive interactome of dopamine-activated PR. Additionally, these findings offer further evidence for differential mechanisms of PR-A and PR-B action. Finally, these findings provide potential candidates for therapeutic targets in abnormal ligand-independent PR activation affecting female reproductive health.

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