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

Abstract

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.