This application addresses important issues for understanding peptide and biogenic amine signaling in the nervous and endocrine systems. Peptide precursors are packaged into DCVs (dense-core vesicles) in the Golgi and undergo processing at the low pH in DCVs. DCVs also load biogenic amines dependent on the pH gradient. The regulated secretion of peptides and amines occurs by DCV fusion with the plasma membrane in response to elevations in Ca2+. The machinery that mediates evoked DCV exocytosis is well-characterized consisting of complexes of the vesicle v-SNARE VAMP-2 with the plasma membrane t-SNAREs syntaxin-1 and SNAP-25. SNARE complexes are assembled during DCV docking and priming reactions that precede Ca2+-triggered membrane fusion. Previous work in this project discovered CAPS (aka CADPS) as a major priming factor and revealed its activity in promoting SNARE complex assembly. Based on major advances in the previous project period, aim 1 will characterize the precise steps in the sequential SNARE complex assembly pathway that are regulated by CAPS. These consist of t-SNARE oligomer formation and the N- terminal entry of VAMP-2 into t-SNAREs. Liposome reconstitution studies will recapitulate CAPS function in DCV docking and priming. CAPS may also function on DCVs at earlier steps in the regulated secretory pathway. We discovered that CAPS localizes to DCVs in neuroendocrine cells, and that DCV localization was required for CAPS function in exocytosis. Studies on how CAPS localizes to DCVs led to identification of DMXL2 and associated proteins as potential CAPS-binding partners on DCVs. DMXL2 is a regulator of V- ATPase, and the studies of aim 2 seek to broaden the role of CAPS to several DCV functions. We will determine whether CAPS-DMXL2 interactions are direct, and whether these interactions regulate V-ATPase activity and lumenal pH of DCVs for peptide packaging in the Golgi, for prohormone conversion, and for biogenic amine uptake. The work will provide a molecular understanding for roles of CAPS on DCVs from Golgi biogenesis to plasma membrane exocytosis. Lastly, recent work revealed the first example of CAPS dysfunction in human disease. This work identified CAPS variants in individuals with early onset bipolar disorder. The mutations are in many of the functional domains on CAPS that we previously characterized. The studies of aim 3 will determine the basis for CAPS loss-of-function in early onset bipolar disorder. This work will contribute to understanding aspects of a complex polygenic disorder that may involve altered DCV-mediated BDNF secretion and/or synaptic dysfunction. Completion of the work in this application will provide a molecular description of DCV priming, fill important gaps of knowledge about the regulated secretory pathway, and determine bases for CAPS dysfunction in disease.

Public Health Relevance

Neuropeptides and peptide hormones are secreted from nerve and endocrine cells by the regulated fusion of secretory granules with the plasma membrane. We will study key proteins that convert secretory granules into a state ready for secretion. Because mutations in these proteins (e.g., CADPS, DMXL2) result in human disease, the work will provide basic knowledge for understanding these diseases and for developing therapies for them.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Synapses, Cytoskeleton and Trafficking Study Section (SYN)
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Ainsztein, Alexandra M
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University of Wisconsin Madison
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United States
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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
Boswell, Kristin L; James, Declan J; Esquibel, Joseph M et al. (2012) Munc13-4 reconstitutes calcium-dependent SNARE-mediated membrane fusion. J Cell Biol 197:301-12