The objective of this proposal is development of the candidate's skills in basic research relating broadly to development and molecular neurobiology and specifically to the mechanism of action of nerve growth factor. This will be achieved by course work, discussions, and supervised investigation of the role of oncogenes in the mechanism of action of the receptors for nerve growth factor. Previous studies indicate that some effects of nerve growth factor (NGF) on neural cell may be influenced by oncogenes or proto-oncogenes. My preliminary studies demonstrate that a clonal cell line (PCNA) expressing high levels of the low-affinity form of the NGF receptor can be transfected successfully with a mamalian expression vector carrying a specific oncogene and that these recipient cells demonstrate an increase in NGF receptor binding activity. I hypothesize that oncogenes may influence NGF-mediated responses by changes in the binding characteristics of NGF receptors and that the cytoplasmic component of the NGF receptor complex which functions in transmembrane signaling may be homologous with or induced by a protooncogene. I will pursue these hypotheses by (1) establishing and isolating subclones of specific oncogenes or proto-oncogenes ligated into a mammalian expression vector, (2) investigating whether any of a number of oncogenes is the effector protein of the S-NGFR by transfecting the oncogenes into the PCNA cell line and looking (by NGF receptor binding) for expression of S-NGFR, (3) confirming and quantitating the effects of oncogene expression, (4) attempting to purify the S-NGFR from PC12 cells (or successfully transfected PCNA cells which express both receptors) for biochemical characterization, and (5) correlating in vitro findings with in vivo observations in animal models of neuronal development and injury. These studies are directed towards achieving a better understanding of the biological mechanisms underlying normal nervous system development, with the hope and expectation that a better understanding of the molecular processes in developmental neurobiology will ultimately provide better understanding of prevention and treatment of nervous system injuries in neonates.
