Synaptic transmission is mediated by release of fast-acting transmitters at synapses via synaptic vesicle (SV) exocytosis. Synaptic modulation is, in contrast, mediated by release from dense-core vesicles (DCVs) of modulatory transmitters that act pre- and postsynaptically to modify synaptic transmission. The understanding of the molecular mechanisms that underlie rapid Ca2+-dependent SV exocytosis has increased over the past decade and the same molecular constituents are required for DCV exocytosis. However, differences in the physiological regulation of SV and DCV exocytosis suggest that there are also molecular mechanisms distinct to each. We discovered that CAPS (ca2+-dependent activator protein for secretion) resides on DCVs but not SVs and is required for DCV but not SV exocytosis. In our continuation studies, we will determine the molecular mechanism through which CAPS acts to facilitate Ca2-dependent DCV fusion with the plasma membrane. This will be accomplished by identifying domains on CAPS that mediate its interactions with plasma and DCV membranes (Aim 1). These studies will define the basis of the DCV-selectivity of CAPS function. To further elucidate the mechanism of CAPS action, the functional importance of its interactions with proteins such as syntaxin, rabphilin and Muncl8 will be determined (Aim 2). These studies will provide a molecular explanation of how CAPS regulates the fusion machinery to facilitate DCV exocytosis. To further define domains of CAPS required for function, we will characterize loss-of-function CAPS mutants as well as determine CAPS structure by X-ray crystallographic studies (Aim 3). To relate the molecular interactions of CAPS to its role in DCV exocytosis, we will study fusion pore dynamics in cells with modified CAPS function (Aim 4). Completion of these studies will provide insight on the regulation of the fusion machinery and on molecular differences between DCV and SV exocytosis. The results may find application in the therapy of nervous system and endocrine disorders that involve hypo- or hypersecretion of monoamine transmitters or peptide hormones.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
2R01DK040428-14
Application #
6475197
Study Section
Special Emphasis Panel (ZRG1-SSS-P (01))
Program Officer
Haft, Carol R
Project Start
1998-06-10
Project End
2007-03-31
Budget Start
2002-04-12
Budget End
2003-03-31
Support Year
14
Fiscal Year
2002
Total Cost
$337,272
Indirect Cost
Name
University of Wisconsin Madison
Department
Biochemistry
Type
Schools of Earth Sciences/Natur
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

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