Increases in cytoplasmic Ca2+ are known to trigger secretory exocytosis in a wide variety of endocrine, exocrine, neural and other cells. This proposal is directed at elucidating the biochemical mechanisms through which elevations in cytoplasmic Ca2+ stimulate secretion. We will employ GH3 pituitary cells in which regulated secretion of prolactin (PRL) has been well- studied. Previous work has implicated a Ca2+-dependent (as well as a protein kinase C-mediated) pathway in the stimulation of PRL secretion by a variety of secretagogues. The proposed work will utilize a newly-developed method (cell cracking) for rendering cells permeable to macromolecules. This method allows preparation of washed """"""""cracked"""""""" GH3 cells which are structurally preserved cellular """"""""ghosts"""""""" devoid of cytoplasm. Addition of micromolar Ca2+, millimolar MgATP and a cytosolic protein to GH3 cell ghosts results in the release of PRL and other secretory proteins at 30 degrees. This system will provide a means for elucidating the biochemical events involved in Ca2+-activated secretion. The proposed work will: 1. Extend the biochemical and structural characterization of cracked cells; 2. Purify and characterize the cytosolic protein required for Ca2- activated PRL release; 3. Directly examine several hypothesized mechanisms for Ca2+ regulation of secretion (protein kinases, Ca2+-binding proteins, phospholipases) in order to facilitate identification of the cytosolic factor; 4. Determine whether the characterized cytosolic protein is required for Ca2+-regulated secretion in intact cells (Gh3, Paramecium and sea urchin eggs); and 5. Elucidate secretory granule- and membrane-associated mechanisms involving the cytosolic factor (using Gh3 cells lactotrophs and chromaffin cells). The outcome of this work will provide direct biochemical characterization of Ca2+-regulated secretion and evaluate the possibility that secretory regulation in several model systems share common features.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK040428-04
Application #
3240693
Study Section
Biochemical Endocrinology Study Section (BCE)
Project Start
1988-09-01
Project End
1993-08-31
Budget Start
1991-09-01
Budget End
1992-08-31
Support Year
4
Fiscal Year
1991
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
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:
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
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
Kabachinski, Greg; Yamaga, Masaki; Kielar-Grevstad, D Michelle et al. (2014) CAPS and Munc13 utilize distinct PIP2-linked mechanisms to promote vesicle exocytosis. Mol Biol Cell 25:508-21
James, Declan J; Martin, Thomas F J (2013) CAPS and Munc13: CATCHRs that SNARE Vesicles. Front Endocrinol (Lausanne) 4:187
Zhang, Zhao; Takeuchi, Hiroshi; Gao, Jing et al. (2013) PRIP (phospholipase C-related but catalytically inactive protein) inhibits exocytosis by direct interactions with syntaxin 1 and SNAP-25 through its C2 domain. J Biol Chem 288:7769-80
Martin, Thomas F J (2012) Role of PI(4,5)P(2) in Vesicle Exocytosis and Membrane Fusion. Subcell Biochem 59:111-30

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