Nerve growth factor (NGF) is a neurotrophic factor known to affect the survival and differentiation of neuroblasts and some other neural-related cells. The effects of NGF are mediated through a 75,000-Da cell surface receptor (gp75) which has recently been characterized and cloned in our laboratory. Our basic goal in this proposal is to further characterize gp75, to identify other subunits of the NGF receptor which might be associated with the heterogeneity of receptor affinity and biological response in different cell types, and to elucidate the mechanism of signal transduction for NGF. Our first specific aim is to prepare recombinant NGF receptor and individual domains of the receptor using eukaryotic expression systems. The integrity of the recombinant protein will be judged by binding of conformation-dependent antibodies and by NGF binding. The second specific aim is to characterize the chemical and physical properties of the recombinant receptor including glycosylation, disulfide bonding pattern, secondary structure and the presence of receptor oligomers. We also will prepare an anti-recombinant NGF receptor antiserum and assess the role of the receptor itself as a second message by microinjecting the recombinant protein into NGF-responsive cells. The third specific aim is to determine the nature of the NGF receptor in PC12 cells which express both low-and high-affinity NGF-binding sites. To determine if there is a limiting second factor required for high-affinity binding, we will infect PC12 cells with an NGF receptor cDNA-bearing retrovirus to increase the number of gp75 molecules. If the human gp75 molecules encoded by the virus are included in high-affinity receptors we will prepare additional retroviruses with altered gp75 cDNAs and determine which sections of gp75 are required for high-affinity binding. We will solubilize PC12 cells under mild conditions which may stabilize the complex between gp75 and other NGF receptor subunits. We will purify gp75-associated proteins by affinity chromatography using the recombinant gp75. For the fourth specific aim, we will compare NGF receptor molecules displayed on PC12 cells, sensory neurons, Schwann cells, and melanoma cells which differ in their affinities for NGF and their responses to NGF.
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