The regulated secretion of hormones, neuropeptides and neurotransmitters by endocrine and neural cells occurs by a similar process in which secretory vesicles/granules exocytotically fuse with the plasma membrane in response to cytoplasmic Ca2+ elevations. In spite of its key importance, there is little understanding of the biochemical reactions that underlie Ca2+-regulated exocytosis. We initiated a molecular dissection of the regulated secretory pathway by developing a functional semi-intact neuroendocrine cell system in which a requirement for cytosolic proteins was discovered. A novel 145kD brain cytosolic protein (termed CAPS) was purified on the basis of its ability to reconstitute Ca2+-activated secretion in permeable GH3 pituitary and PC12 adrenal cells. Several biochemical properties of CAPS are Ca2+-regulated, and the protein is required at a Ca2+dependent execution point in the regulated secretory pathway. The overall goal of this project is to fully characterize CAPS, and to identify the Ca2+-regulated biochemical events for which it is required.
The specific aims are: 1. To isolate and fully characterize a cDNA encoding the novel CAPS protein. The full amino acid sequence of CAPS is anticipated to help clarify its Ca2+-dependent mechanism of action. Mutant CAPS proteins will be produced to determine whether multiple activities of CAPS are mediated by separate domains within the protein. 2. To identify cellular constituents that interact with CAPS. Ca2+-dependent CAPS binding to l8kD and 30kD cytoskeletal proteins, and to phosphoinositides, have been detected. The 18kD and 3OkD proteins will be characterized to elucidate potential site(s) of action for CAPS in regulating secretion. A purified functional complex of secretory granules and plasma membrane will be used to identify biochemical events that mediate the actions of CAPS, and that accompany regulated fusion. 3. To determine whether CAPS mediates an essential step in the regulated secretory pathway of intact neuroendocrine cells. The biochemical properties of CAPS and its restricted expression in neural, endocrine and exocrine secretory cells suggest that it is a component of the exocytotic apparatus. Microinjection studies (with neutralizing antibodies or dominant negative mutant CAPS proteins) or patch clamp capacitance studies (with CAPS and CAPS antibody) will be conducted to assess the role of CAPS in intact secretory cells. Completion of these specific aims will elucidate the structure and mechanism of action of CAPS, and provide new insights into the exocytotic apparatus and the process of regulated membrane fusion.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK040428-09
Application #
2391413
Study Section
Biochemical Endocrinology Study Section (BCE)
Program Officer
Haft, Carol R
Project Start
1988-09-01
Project End
1998-03-31
Budget Start
1997-06-10
Budget End
1998-03-31
Support Year
9
Fiscal Year
1997
Total Cost
Indirect Cost
Name
University of Wisconsin Madison
Department
Zoology
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
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
James, Declan J; Martin, Thomas F J (2013) CAPS and Munc13: CATCHRs that SNARE Vesicles. Front Endocrinol (Lausanne) 4:187
Martin, Thomas F J (2012) Role of PI(4,5)P(2) in Vesicle Exocytosis and Membrane Fusion. Subcell Biochem 59:111-30

Showing the most recent 10 out of 44 publications