Information transfer between neurons and the normal functioning of the nervous system is dependent upon the regulated release of neurotransmitter at synapses. A number of psychiatric and neurological conditions are typified by an imbalance of particular neurotransmitters. In addition, many abused and therapeutic drugs that act on the nervous system act at the level of altering synaptic transmission. The long-term objective of the proposed research is to understand molecular mechanisms which regulate neurotransmitter or neurohormone release, as it may ultimately lead to enhanced clinical treatments as well as improved drug design. Small Ras-like GTP binding proteins are key regulators of many cellular processes including morphogenesis, cytoskeletal dynamics, membrane trafficking, transformation, and protein kinase cascades. The general hypothesis to be tested is that the monomeric GTPase Rac1 is essential to secretory responsiveness in that it coordinates events critical for secretory granule availability, priming and SNARE protein interactions. A combination of molecular, biochemical and patch-clamp techniques will be used to determine the sites of Rac1 action within functionally separable stages of the secretory cycle, and to elucidate effector pathways through which it directly exerts effects on secretory responsiveness. The investigations will be performed on bovine chromaffin cells, which present an extensively studied, physiologically relevant, neuroendocrine cell model.
The specific aims are: 1) To elucidate the physiological role of Rac1 on regulation of Ca2+- dependent exocytosis and to specifically determine effects on Ca2+ sensitivity, recruitment, and refilling of the readily releasable secretory granule pool, 2) To characterize the properties of Rac1 activation in response to secretory stimuli. We will also determine if IQGAP1, a Rac effector protein that binds Ca2+/calmodulin and F-actin, provides Ca2+-dependent regulation of Rac1, 3) To determine the contribution of the predominant Rac1 effector pathway, i.e. the p21 activated kinases (PAK), to Rac1 effects on secretory responsiveness. In addition, we will establish the role of p35/cyclin dependent kinase 5, an interacting protein kinase that regulates PAK activation, on Rac1 and PAK1 regulation of secretory responsiveness, and 4) To determine the role of Rac1 on phosphatidylinositol 4-phosphate 5- kinase (PIP5K) activity in resting and stimulated chromaffin cells. PIP5K is an essential co-factor for priming of secretory granules in neuroendocrine systems and directly interacts with Rac1. The work proposed attempts to provide a greater understanding of cytosolic and membrane delimited signaling pathways that exert an important regulatory influence on Ca2+- dependent secretory responsiveness.
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