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20## Annual Report of the Division of Intramural Research, NICHD National Institutes of Health Eunice Kennedy Shriver National Institute of Child Health and Human Development

Kevin Catt, MD, PhD, Chief

The Endocrinology and Reproduction Research Branch (ERRB) investigates the molecular and cellular mechanisms of reproductive hormone action and neuronal function, the structure-function properties and signaling pathways of peptide hormone receptors, and the roles of phosphoinositides, phosphorylation, and sulfonation in signal transduction and metabolic regulation.

Tamás Balla's Section on Molecular Signal Transduction investigates signal transduction pathways mediating the actions of hormones, growth factors, and neurotransmitters in mammalian cells, with emphasis on the role of phosphoinositide-derived messengers. PtdIns4P and PI4K enzymes regulate the lipid transport function of CERT, which is responsible for ceramide transport to the Golgi. The group developed a strategy, based on heterodimerization of the FRB domain of mTOR and FKBP12, to regulate membrane PtdIns(4,5)P2 levels by drug-inducible membrane targeting. Rapa-induced plasma membrane (PM) recruitment of a truncated type-IV 5-phosphatase, containing only the 5-phosphatase domain fused to FKBP12, rapidly decreased PM PtdIns(4,5)P2. Such a decrease was paralleled by termination of ATP-induced Ca2+ signaling and inactivation of TRPM8 channels. Rapid inducible depletion of PM PtdIns(4,5)P2 is a powerful tool for studying the regulatory roles of this phospholipid and the differential sensitivities of specific processes to PtdIns(4,5)P2 depletion.

Kevin Catt's Section on Hormonal Regulation investigates the molecular mechanisms of activation, signaling, and function of G protein-coupled receptors for angiotensin II (AT1R) and gonadotropin-releasing hormone (GnRHR) and their interactions with the EGF receptor (EGFR). Current studies are analyzing lipid rafts from hepatic C9 cells to determine the mechanisms of interaction and trans-phosphorylation of the AT1R and EGFR as well as investigating the roles of other signaling proteins in this process. Using BRET analysis with labeled receptor and G protein constructs, the laboratory is investigating the mechanism by which GnRH activates several G proteins in the GnRH neuron. Stimulation of endogenous LH/hCG receptors in GnRH neurons activates GIRK channels, causing suppression of membrane excitability and inhibition of AP firing. These effects mediate gonadotropin-induced abolition of pulsatile GnRH release and possibly the suppression of pituitary function during pregnancy.

Maria Dufau's Section on Molecular Endocrinology found that PI3K/PKCz mediates site-dependent activation of Sp1 by the HDAC inhibitor TSA and transcriptional derepression of the luteinizing hormone receptor (LHR). In this novel action of TSA, PI3K-activated PKCz phosphorylation of Sp1 releases its associated repressor p107. Studies on the human prolactin receptor (hPRLR) gene revealed that an estradiol-regulated, non-estrogen responsive element-dependent mechanism mediates the gene's transcriptional expression. In studies on long-chain acyl CoA synthetase (GRLACS) and gonadotropin-regulated testicular RNA helicase (GRTH/Ddx25)--two gonadotropin-responsive genes discovered in the Dufau laboratory, of which the latter is essential for spermatogenesis-researchers identified short GRLACS variants that regulate the long form's activity in the brain. Only cytoplasmic species of GRTH/Ddx25 were phosphorylated. In addition to its storage functions, phosphorylated GRTH associates with polyribosomes to regulate the transcriptional activity of specific subsets of expressed testicular genes.

Kuo-Ping Huang's Section on Metabolic Regulation uses Ng knockout mice to study the role of neurogranin (Ng) in the enhancement of synaptic plasticity. The mice exhibit deficits in learning and memory (L&M) of cognitive tasks and long-term potentiation (LTP). In normal and heterozygous mice, hippocampal Ng correlates highly with L&M performance. Enriched environments increase hippocampal Ng content, behavioral test performance, and expression of LTP. Ng promotes rises in neuronal free Ca2+ that enhance synaptic responses and favor induction of LTP over long-term depression. Ng may regulate neuronal signaling and enhance synaptic plasticity because, at a higher Ng concentration, the formation of Ng/CaM complexes raises [Ca2+]i at any level of Ca2+ influx. The consequent signal amplification enhances synaptic plasticity and L&M. Ng is a potent reductant and may protect neurons from damage during oxidative stress.

Stanko Stojilkovic's Section on Cellular Signaling investigates cellular signaling cascades and secretion in neuroendocrine cells, with the focus on channels and calcium signaling. Recent studies on native ATP-gated calcium-conducting purinergic receptor channels revealed that pituitary gonadotrophs express the P2X2 subtype of these receptors, which could operate as pacemaking channels and modulators of agonist-controlled electrical activity and secretion. Experiments with recombinant P2X2 channels revealed a critical role of the cytoplasmic inter-subunit interactions during agonist-dependent receptor desensitization. Studies on the metabolism and contribution of cyclic nucleotides to the control of voltage-gated calcium influx and secretion in pituitary cells included characterization of the role of cyclic nucleotide-gated channels in pacemaking and calcium-mediated secretion and the participation of multidrug-resistance proteins in the regulation of cyclic nucleotide intracellular levels.

Charles Strott's Section on Steroid Regulation investigates molecular mechanisms and biologic implications of modifying substances by sulfonation. The laboratory clones human and animal sulfotransferase genes in order to examine gene products, tissue expression, and transcriptional regulation. The group is studying human SULT2B1 isozymes that sulfonate steroids/sterols, examining their roles in physiology and disease processes. The SULT21B1a isoform produces pregnenolone sulfate, an important neurosteroid involved in learning and memory development. The SULT2B1b isoform produces cholesterol sulfate, which plays an important role in epidermal development (barrier formation), platelet aggregation (hemostasis), and sperm capacitation (reproduction). SULT2B1b also carries out a more classic role by detoxifying cholesterol metabolites such as 7-ketocholesterol, which can induce macular degeneration and formation of macrophage foam cells involved in the development of atherosclerosis.

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