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Subject: search.filters.subject.Nerve growth factor

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    Characterisation of ERK distribution and activity in rat pheochromocytoma cells : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Molecular Biology at Massey University
    (Massey University, 1998) MacCormick, Matthew Edgar
    Nerve growth factor (NGF) binds to the NGF receptor, TrkA, at the tips of nerve cell axons, sending a signal that prevents programmed cell death and causes survival, growth, and differentiation of the nerve cell. Both NGF and TrkA have been demonstrated to be retrogradely transported from axon tips to nerve cell bodies, however the mechanism of this transport, and its function, is strongly debated. Using a recently developed cell fractionation protocol in conjunction with in vitro reactions using an ATP regenerating system, our lab has isolated small vesicles containing NGF bound to activated TrkA. These vesicles may provide a vehicle for retrograde transport of the NGF signal and initiation of signal transduction in the cell body. ERK1 is a serine/threonine kinase that is activated by NGF-activated TrkA. Prolonged ERK1 activity is characteristic of cells stimulated by NGF. The purpose of the experiments in this thesis was to characterise the intracellular distribution and activity of ERK1 before and after NGF stimulation, in rat pheochromocytoma (PC12) cells, which are a good model for nerve cells. We have found that ERK1 activity is redistributed between cell compartments after NGF stimulation of PC12 cells. ERK1 activity increased in sedimentable fractions that emerged from mechanically permeabilised cells after NGF treatment and in vitro reactions with ATP. Importantly, the results from glycerol velocity gradient experiments showed that ERK1 was not associated with membranes. Instead ERK1 was found in a rapidly sedimenting particle whose sedimentation was not affected by detergent solubilisation. These results suggest that ERK1 is recruited into a protein complex, after activation, which may be an important step in signal transduction. Formation of this complex is likely to be downstream of signalling vesicles containing NGF bound TrkA.
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    Association of NGF receptors with membrane rafts in PC12 cells : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University
    (Massey University, 2001) Clements, Shona Marie
    Nerve Growth Factor (NGF) signal transduction is involved in the survival, differentiation and maintenance of neurons through the receptors TrkA and p75NTR. These receptors activate downstream protein kinase cascades that regulate cell survival. NGF binding to TrkA promotes cell survival, however NGF binding to the low-affinity receptor, p75NTR can lead to cell death in the absence of TrkA. Therefore the interaction of these two receptors and their downstream pathways are very important for determining cell survival. Recent studies have shown that many receptors and their associated downstream proteins have been found in membrane rafts, areas of the plasma membrane enriched in sphingolipids and cholesterol. To investigate the presence of the NGF receptors and downstream signalling proteins in these rafts, we have devised a method of cellular fractionation and detergent extraction quite different from those used previously. Mechanical permeabilisation separated the cytosolic components of PC12 cells. Non-ionic detergent extraction was used to solubilise the majority of the plasma membranes, leaving the detergent-insoluble membranes and cytoskeleton. Equilibrium flotation gradients were used to separate the membrane rafts from other detergent-insoluble material such as the cytoskeleton. Using these methods, we found that not only are TrkA and p75NTR present in rafts, but also the downstream signalling protein ERK1 and the cytoskeletal protein, tubulin. In addition to plasma membrane rafts, we have isolated detergent-insoluble intracellular membranes from the endoplasmic reticulum and Golgi. NGF binding, in vitro reactions with an ATP regenerating system and the addition of ganglioside GM1 to the cells, have been found to have a large effect on the raft association of both TrkA and p75NTR. These results indicate an important role for membrane rafts in NGF signalling through its receptors TrkA and p75NTR, and suggest a model in which signalling centres form around rafts and microtubules.