Peptide hormones interact with cells at specific cell-surface receptors. Cellular activation requires transmembrane signalling and possible generation of intracellular second messengers. Hormone-regulated polyphosphoinositide (PPI) metabolism has been identified as a likely transduction mechanism. We will investigate the molecular basis of receptor-stimulated PPI metabolism in clonal pituitary cells (GH3 cells) which are responsive to thyrotropin-releasing hormone (TRH). TRH stimulates protein secretion and previous studies identified rapid hormone-triggered events involving Ca2+-dependent diglyceride-activated protein phosphorylation which may be involved in regulating secretion. TRH-stimulated PPI turnover has been linked to regulation of these pathways. The objective of our research is to establish the means by which TRH receptor occupancy can regulate PPI turnover. Three of the specific aims for this project period will be: 1. to identify the reactions of PPI metabolism subject to regulation by TRH; 2. to characterize and purify enzymes catalyzing these reactions and 3. to develop a cell-free TRH-responsive membrane/cytosol system. PPI-metabolizing enzymes have previously been studied in systems which are incapable of hormone regulation. Recently-developed techniques allow TRH-stimulated PPI turnover to be studied in permeable GH3 cells. This permeable cell system will enable us to identify low molecular weight cofactors involved and to selectively inhibit individual reactions. Once the requirements for hormonal regulation in permeable cells are established and we have identified the critical enzymes involved, a hormone-responsive broken cell system will be developed. The subcellular distributions and regulatory characteristics of PPI-metabolizing enzymes will be determined. We will then reassemble membranes and cytosolic enzymes systematically to reconstitute PPI metabolism in a TRH-responsive manner. Success in this endeavor would enable fuller biochemical elucidation including identification of coupling proteins. The fourth specific aim for this project period will be to develop probes for characterizing and purifying the TRH receptor so that receptor-associated elements can be identified. TRH is representative of a large number of physiological regulators (hormones, neurotransmitters and mitogens) whose actions may be mediated through receptor-activated PPI turnover. Elucidation of the components involved in TRH action will contribute to understanding basic cell regulatory mechanisms.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK025861-10
Application #
3227645
Study Section
Endocrinology Study Section (END)
Project Start
1979-07-01
Project End
1991-01-31
Budget Start
1989-02-01
Budget End
1990-01-31
Support Year
10
Fiscal Year
1989
Total Cost
Indirect Cost
Name
University of Wisconsin Madison
Department
Type
Schools of Arts and Sciences
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Bruinsma, Stephen; James, Declan J; Quintana Serrano, Melanie et al. (2018) Small molecules that inhibit the late stage of Munc13-4-dependent secretory granule exocytosis in mast cells. J Biol Chem 293:8217-8229
Zhang, Xingmin Aaron; Martin, Thomas F J (2018) High Throughput NPY-Venus and Serotonin Secretion Assays for Regulated Exocytosis in Neuroendocrine Cells. Bio Protoc 8:
Messenger, Scott W; Woo, Sang Su; Sun, Zhongze et al. (2018) A Ca2+-stimulated exosome release pathway in cancer cells is regulated by Munc13-4. J Cell Biol 217:2877-2890
Zhang, Xingmin; Jiang, Shan; Mitok, Kelly A et al. (2017) BAIAP3, a C2 domain-containing Munc13 protein, controls the fate of dense-core vesicles in neuroendocrine cells. J Cell Biol 216:2151-2166
Woo, Sang Su; James, Declan J; Martin, Thomas F J (2017) Munc13-4 functions as a Ca2+ sensor for homotypic secretory granule fusion to generate endosomal exocytic vacuoles. Mol Biol Cell 28:792-808
Chehab, Tarek; Santos, Nina Criado; Holthenrich, Anna et al. (2017) A novel Munc13-4/S100A10/annexin A2 complex promotes Weibel-Palade body exocytosis in endothelial cells. Mol Biol Cell 28:1688-1700
Kabachinski, Greg; Kielar-Grevstad, D Michelle; Zhang, Xingmin et al. (2016) Resident CAPS on dense-core vesicles docks and primes vesicles for fusion. Mol Biol Cell 27:654-68
Petrie, Matt; Esquibel, Joseph; Kabachinski, Greg et al. (2016) The Vesicle Priming Factor CAPS Functions as a Homodimer via C2 Domain Interactions to Promote Regulated Vesicle Exocytosis. J Biol Chem 291:21257-21270
Martin, Thomas F J (2015) PI(4,5)P?-binding effector proteins for vesicle exocytosis. Biochim Biophys Acta 1851:785-93
Yamaga, Masaki; Kielar-Grevstad, D Michelle; Martin, Thomas F J (2015) Phospholipase C?2 Activation Redirects Vesicle Trafficking by Regulating F-actin. J Biol Chem 290:29010-21

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