The focus of this thesis is to characterise the interactions between GLUT4-related SNARE proteins – syntaxin4, SNAP23 and VAMP2 – and a regulatory protein, Munc18c. GLUT4 is the primary insulin-regulated glucose transporter and is presentin fat and muscle cells. GLUT4 is held in intracellular pools of vesicles until it is transported to the cell surface upon insulin stimulation. Insulin initiates a cellular signalling cascade via the insulin receptor on the cell membrane, which in turn stimulates GLUT4 vesicles to move to the cell surface where they fuse to the plasmamembrane via SNARE proteins. SNAREs are membrane-anchored proteins present on both vesicle and target membranes that form a tight complex which brings themembranes together for fusion. Fusion of vesicles to the target membrane releases the vesicular cargo.SNARE-mediated membrane fusion is a conserved mechanism that controls many other vesicle fusion processes such as neurotransmitter release and yeast vesicular trafficking. However, the regulation of the SNARE mechanism is not fully understood. SNAREs can interact with many other proteins that could act as regulatory factors,and studies have focused primarily on a group of effector proteins called Sec1p/Munc18 (SM) proteins. SM proteins were discovered and characterised because they bind to one type of SNARE protein, syntaxin. The SM protein that interacts with the GLUT4-related SNARE, syntaxin4, is Munc18c.The aim of this thesis was to investigate Munc18c interactions with SNARE proteins, principally syntaxin4, using biochemical techniques with purified recombinant proteins. This work was carried out in several stages including: 1) development of methods to produce and purify GLUT4-related SNARE proteins, SNARE complexes and Munc18c, 2) development of an assay to quantify Munc18c interactions with binding partners using surface plasmon resonance, 3) investigation into interactions between Munc18c and SNARE ternary complex, 4) characterising Munc18c interactions with syntaxin4, and 5) developing a method to produce selenomethionine-containing Munc18c in a baculovirus system to be used in structural studies. The methods and outcomes of these experiments are described inthis thesis. There were two major outcomes from this work. Firstly, Munc18c interacts with SNARE ternary complex, and secondly, Munc18c requires only the N-terminal 29residues of syntaxin4 for an interaction to occur. These results were determined using pulldown assays with purified proteins, as well as other chromatographic methods to show that protein complexes were formed. The steps taken to develop these binding assays are also discussed. Initial crystallisation conditions forMunc18c-HIS and a peptide consisting of syntaxin4 residues 1-20 have been identified using crystallisation screens. The interactions determined for Munc18c binding to Sx4 are in direct contrast to those of neuronal SM protein, Munc18a, and its interaction with neuronal SNARE proteins - Munc18a does not bind to its ternary complex and binds to the entire cytoplasmic domain of Sx1a. Rather, the Munc18c:Sx4 interactions are similar to that for the yeast SM protein, Sly1p, which can interact with both its SNARE ternary complex and with its syntaxin via the Nterminal residues. Another interesting outcome of this research was that syntaxin4 binds to metals (cobalt and nickel). This finding represents the first reported for a syntaxin interacting with metals. Preliminary results indicate that un-tagged syntaxin4 can bind to cobalt resin, and to nickel immobilised on a chip. This interesting and novel property of syntaxin4 binding was serendipitously discovered while investigating conditions for the Munc18c assay. Overall, I have shown that Munc18c, the SM protein involved in GLUT4 trafficking, interacts with SNARE proteins in a different manner to its mammalian counterpart inneurons, Munc18a, and is more like Sly1p, a yeast ER-Golgi SM protein. Munc18c interacts with SNARE complexes and only the N-terminal residues of syntaxin4.These interactions demonstrate that the regulatory mechanism for SNARE-mediated fusion is conserved between yeast and mammals. This finding has several implications for the role of Munc18c in the exocytosis of GLUT4-containing vesicles. Munc18c could act at several stages in the fusion process via syntaxin4 binding.These interactions could involve binding to other proteins (such as synip or tomosyn), conformational switching of syntaxin4 or interaction with metal ions to induce conformational changes in the proteins. Finally, these studies of GLUT4 exocytosis contribute to our understanding of glucose transport disorders such as Type 2 diabetes and could one day pave the way for the design of therapeutic agents.

[Truncated abstract] Neuregulins (NRG’s) are pleiotropic growth factors that participate in a wide range of biological processes. The family of membrane-bound growth factors bind to and activate ErbB receptors on adjacent target cells, mediating multiple biological processes. NRG-1, NRG-2 and NRG-3 are all highly expressed in the nervous system, where it has been shown that NRG-1 is important for neuronal development, migration, synapse formation and glial cell proliferation. Little is known, however, on the specific roles of NRG-2 and NRG-3, although it is apparent that despite similar expression patterns and overlapping receptor specificity, NRG-2 and NRG-3 do not compensate for the loss of NRG-1 and mediate their own distinct activities. … Subcellular localisation experiments showed that this domain is important for trafficking of the fulllength protein to various intracellular compartments in an activity dependent manner. In addition, the ICD is required to elicit a cell death response in cultured cells and provoke an elevated α-amino-3-hydroxyl-5-methylisoxazole-4-propionate (AMPA) response in organotypic neuronal cultures following transient expression of NRG-3. A yeast two-hybrid screen identified 14-3-3ζ and PICK1 as two proteins that interacte with the human NRG-3 ICD. These interactions were confirmed both in vitro and in vivo, and were further characterised at a molecular level. This study demonstrates the ability of NRG-3 to mediate signal transduction through a biologically active ICD; a conclusion supported by identifying cytoplasmic proteins that interact with the ICD. These observations point to an additional layer of complexity where bi-directional signalling contributes to the full repertoire of NRG-3 functions.

Thesis (Ph. D. in Molecular Physiology and Biophysics)--Vanderbilt University, May 2006. / Title from title screen. Includes bibliographical references.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2006. / Additional advisors: Vithal K. Ghanta, Denise R. Shaw, Stephen A. Watts, Bradley K. Yoder. Description based on contents viewed Feb. 20, 2009; title from PDF t.p. Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 83-95).

Thesis (Ph.D.) -- Mississippi State University. Department of Biological Sciences. / Title from title screen. Includes bibliographical references.

Thesis (M.S.) -- Mississippi State University. Department of Chemistry. / Title from title screen. Includes bibliographical references.

Thesis (Ph. D.)--West Virginia University, 2009. / Title from document title page. Document formatted into pages; contains xiv, 118 p. : ill. (some col.). Includes abstract. Includes bibliographical references.

Thesis (M.S.)--Duquesne University, 2004. / Title from document title page. Abstract included in electronic submission form. Includes bibliographical references (p. 76-88) and abstract.

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (p. 187-198). Also available in print.