β-Arrestin Regulates Estradiol Membrane-Initiated Signaling in Hypothalamic Neurons

March -Arrestin Regulates Estradiol Membrane- Initiated Signaling in Hypothalamic Neurons Angela M. Wong 0 1 Matthew C. Abrams 0 1 Paul E. Micevych 0 1 0 Department of Neurobiology David Geffen School of Medicine at UCLA and Laboratory of Neuroendocrinology of the Brain Research Institute, at University of California Los Angeles , Los Angeles, California , United States of America 1 Academic Editor: Ferdinando Auricchio, University of Naples 2, ITALY Estradiol (E2) action in the nervous system is the result of both direct nuclear and membrane-initiated signaling (EMS). E2 regulates membrane estrogen receptor- (ER) levels through opposing mechanisms of EMS-mediated trafficking and internalization. While arrestin-mediated mER internalization has been described in the cortex, a role of arrestin in EMS, which underlies multiple physiological processes, remains undefined. In the arcuate nucleus of the hypothalamus (ARH), membrane-initiated E2 signaling modulates lordosis behavior, a measure of female sexually receptivity. To better understand EMS and regulation of ER membrane levels, we examined the role of -arrestin, a molecule associated with internalization following agonist stimulation. In the present study, we used an immortalized neuronal cell line derived from embryonic hypothalamic neurons, the N-38 line, to examine whether -arrestins mediate internalization of mER. -arrestin-1 (Arrb1) was found in the ARH and in N-38 neurons. In vitro, E2 increased trafficking and internalization of full-length ER and ER4, an alternatively spliced isoform of ER, which predominates in the membrane. Treatment with E2 also increased phosphorylation of extracellular-signal regulated kinases 1/2 (ERK1/2) in N-38 neurons. Arrb1 siRNA knockdown prevented E2-induced ER4 internalization and ERK1/2 phosphorylation. In vivo, microinfusions of Arrb1 antisense oligodeoxynucleotides (ODN) into female rat ARH knocked down Arrb1 and prevented estradiol benzoate-induced lordosis behavior compared with nonsense scrambled ODN (lordosis quotient: 3 2.1 vs. 85.0 6.0; p < 0.0001). These results indicate a role for Arrb1 in both EMS and internalization of mER, which are required for the E2-induction of female sexual receptivity. - Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The authors have declared that no competing interests exist. Estrogens act at cell membrane receptors to activate intracellular signaling, which is implicated in many brain functions including the regulation of female sexual receptivity [1, 2]. In estradiol (E2) membrane-initiated signaling (EMS), estrogen receptor- (ER) transactivates metabotropic glutamate receptors (mGluRs; [35]) regulating signaling pathways [612]. Plasma membrane ER (mER) levels are determined by a balance of trafficking to the membrane, requiring ER palmitoylation and interaction with caveolin-1 (CAV1; [13, 14]), and internalization, requiring -arrestin-1 (Arrb1; [15]). To study EMS, we used previously characterized immortalized hypothalamic neurons (N-38s). These cells expressed neuropeptide Y, full length ER and ER4, a splice variant lacking exon 4. ER4 is enriched in the plasma membranes of cultured neurons and astrocytes, and its mRNA is widely distributed in the CNS [1619]. Trafficking of ER and ER4 to the membrane and internalization is regulated in parallel by E2 [17, 18]. As in other neuronal hypothalamic cells that have membrane ERs, N-38 neurons respond to E2 treatment by increasing free cytoplasmic calcium levels ([Ca2+]i), and by activating extracellular signal-regulated kinases 1/2 and protein kinase C (PKC). Internalization is an important aspect of membrane receptor dynamics and limits cellular responses initiated by agonist stimulation of G protein-coupled receptors (GPCRs). Receptor stimulation leads to GPCR kinase (GRK) activation, which results in the phosphorylation of receptors including mER [15, 20]. With other GPCRs, -arrestins bind to phosphorylated receptors, uncouple G proteins and link receptors to clathrin-dependent internalization pathways [2124]. A more recently discovered function of -arrestins is to organize members of the ERK1/2 (aka mitogen-activated protein kinase, MAPK) pathway [2427]. While it is unknown whether -arrestins are involved in organizing signaling molecules for mERs, E2 activates ERK1/2 [3, 28], potentially through a -arrestin-mediated mechanism. Thus, -arrestins may be crucial not only for limiting E2 signaling via mER internalization, but may be involved in the initial EMS through an ERK1/2 pathway. This rapid, membrane-initiated action of E2 is important for activating the limbic-hypothalamic lordosis-regulating circuit, in which stimulating -endorphin release activates -opioid receptors (MOR) in the medial preoptic nucleus ([29], reviewed in [30, 31]). Within this lordosis-regulating circuit, E2 activates NPY-expressing neurons in the ARH, which we modeled with NPY mRNA expressing N-38 neurons in the present studies. Following E2 activation of N-38 neurons, calcium levels increase [17] and ERK1/2 is phosphorylated [15]. In vivo, EMS activates a transiently inhibitory circuit that is ultimately necessary for the full display of lordosis behavior [2, 29, 32]. We hypothesize that Arrb1 knockdown would abrogate EMS and consequently lordosis behavior. Thus, in this study, we examined the role of Arrb1 regulation of E2-induced ER internalization and subsequent ERK1/2 signaling in N-38 neurons. Arrb1 siRNA was used to reduce Arrb1 protein levels. In addition, we tested whether Arrb1 knockdown in vivo regulated sexual receptivity using Arrb1 antisense oligodeoxynucleotides (asODN) infused into the ARH prior to estradiol benzoate (EB) priming. N-38 neurons were obtained from CELLutions Biosystems (Burlington, ON, Canada). Cultures were prepared from a frozen stock of N-38 neuronal cells and maintained in DMEM supplemented with 4.5 mg/ml glucose, 10% FBS, 1% penicillin/streptomycin, 0.15% sodium bicarbonate at 37C, 5% CO2. Cells were plated in T75 flasks at 1,000,000 cells/flask 16 h prior to transfections. Total RNA was isolated using TRIzol reagent (Life Technologies; Carlsbad, CA), according to the manufacturers protocol using 1 mL of TRIzol/100 mm plate. RNA from cells was extracted using chloroform. RNA pellets were washed with 100% isopropanol followed by 75% ethanol in DEPC-treated water. Pellets were allowed to dry for 10 minutes at room temperature and were resuspended in DEPC-treated water. RNA concentration and quality were assessed using a spectrophotometer (NanoDrop 1000, Thermo Fisher Scientific; Waltham, MA). 12 g total RNA were used to synthesize cDNA with the SuperScript III Reverse Transcriptase kit (Invitrogen; Carlsbad, CA) using Oligo(dT)20 primers. The RT reaction was performed at 50C for 50 minutes, followed by a 5 minute termination at 85C. cDNA was used immediately for RT-PCR or stored at 20C for 1 mo (...truncated)