The Progestin Receptor Interactome in the Female Mouse Hypothalamus: Interactions with Synaptic Proteins Are Isoform Specific and Ligand Dependent

Abstract

Progestins bind to the progestin receptor (PR) isoforms, PR-A and PR-B, in brain to influence development, female reproduction, anxiety, and stress. Hormone-activated PRs associate with multiple proteins to form functional complexes. In the present study, proteins from female mouse hypothalamus that associate with PR were isolated using affinity pull-down assays with glutathione S-transferase–tagged mouse PR-A and PR-B. Using complementary proteomics approaches, reverse phase protein array (RPPA) and mass spectrometry, we identified hypothalamic proteins that interact with PR in a ligand-dependent and isoform-specific manner and were confirmed by Western blot. Synaptic proteins, including synapsin-I and synapsin-II, interacted with agonist-bound PR isoforms, suggesting that both isoforms function in synaptic plasticity. In further support, synaptogyrin-III and synapsin-III associated with PR-A and PR-B, respectively. PR also interacted with kinases, including c-Src, mTOR, and MAPK1, confirming phosphorylation as an integral process in rapid effects of PR in the brain. Consistent with a role in transcriptional regulation, PR associated with transcription factors and coactivators in a ligand-specific and isoform-dependent manner. Interestingly, both PR isoforms associated with a key regulator of energy homeostasis, FoxO1, suggesting a novel role for PR in energy metabolism. Because many identified proteins in this PR interactome are synaptic proteins, we tested the hypothesis that progestins function in synaptic plasticity. Indeed, progesterone enhanced synaptic density, by increasing synapsin-I–positive synapses, in rat primary cortical neuronal cultures. This novel combination of RPPA and mass spectrometry allowed identification of PR action in synaptic remodeling and energy homeostasis and reveals unique roles for progestins in brain function and disease.

Dopamine‐induced interactions of female mouse hypothalamic proteins with progestin receptor‐A in the absence of hormone

“…Extending our earlier findings that showed that synaptic proteins interacted with ligand-activated PR, 16 in the present study, we identified a large number of synaptic proteins that interacted with PR-A in a ligand-independent but DA-dependent manner ( Table 2).…”

“…The recombinant proteins containing full-length mouse PR-A or PR-B fused to a GST tag were expressed in Spodoptera frugiperda (Sf9; RRID:CVCL_0549) insect cells at the Baculovirus/Monoclonal Antibody Facility of the Baylor College of Medicine as described previously. 16,58 Insect cell cultures for GST-PR-A or GST-PR-B were grown in the absence of PR ligand. The Sf9 cell pellets containing PR were homogenised in buffer containing 10 mmol L -1 Tris, 10%…”

“…Pull-down assays were done using GST-PR-A or GST-PR-B with either SKF-, or Veh-treated hypothalamic lysates (Figure 1), as described previously. 16 In brief, glutathione sepharose 4B packed resin (50 µL, 0.05 mg mL -1 ; Amersham Biosciences, Little Chalfont, UK) was pretreated with ovalbumin (1 mg mL -1 ; Thermo Fisher Scientific, Waltham, MA, USA) for 15 minutes in siliconised centrifuge tubes on an end-over-end rotator at 4°C. Resin was rinsed three times with TG buffer (20 mmol L -1 Tris-HCl, 10% glycerol) containing 1 mol L -1 urea and 100 mmol L -1 NaCl (TG-NaCl-U, pH 8.0).…”

“…Tryptic peptides from pull-down assays with unliganded GST-PR-A or GST-PR-B, were prepared as described previously. 16 Samples were block randomised and analysed in triplicates 59 and peptide F I G U R E 1 Experimental outline. Timeline of procedures and treatments of mice prior to collection of the hypothalamic tissue and in vitro proteinprotein pull-down assays with glutathione S-transferase (GST)-tagged mouse progestin receptors (PR) and hypothalamic lysates treated with (+) or without (−) SKF.…”

Human menstrual cycle variation in subcortical functional brain connectivity: a multimodal analysis approach

“…In animal research, there is strong evidence for estrogen-dependent synaptic remodeling in the hippocampus (Woolley and McEwen 1993;Yankova et al 2001;Qiu et al 2013) and prefrontal cortex (Khan et al 2013). Likewise, progesterone has also been shown to increase dendritic spine number and density in cortical neuron cultures (Sanchez et al 2013;Acharya et al 2017). Although this suggests similar mechanism of action for both sex hormones, animal data strongly indicate that the effects are context dependent and progesterone might facilitate some of these estrogenic actions, while modulating or opposing others (McEwen and Milner 2017).…”