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.
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