Faculty Bibliography

A series of recombinant herpes simplex virus (HSV-1) vectors have been constructed that encode either the full-length cDNA of the human p75 NGF receptor (p75hNGFR) or truncated forms of the receptor. Infection of cultured fibroblast cells with viral stocks results in abundant expression of all three cDNAs, as detected by affinity cross-linking, immunoblot analysis, and equilibrium binding. Furthermore, viral infection of primary neuronal cultures gives easily detectable p75 expression by immunofluorescence and affinity cross-linking. When p75 was introduced by viral infection into fibroblast cells expressing the trk proto-oncogene, a new binding site was created, consistent with high-affinity NGF binding. This site is not created by the coexpression of truncated forms of p75 that lack either the extracellular ligand binding domain or the cytoplasmic domain of the receptor, suggesting that both of these regions of the receptor are required for the formation of the high-affinity NGF binding site. Hence, these HSV-1 vectors give rise to appropriate NGF receptor binding after viral infection. The application of these HSV-1 constructs to primary neuronal culture and in vivo models of p75NGFR function is discussed

Nerve growth factor (NGF) represents a family of structurally related trophic factors, including brain-derived neurotrophin factor (BDNF), neurotrophin-3 (NT-3), NT-4, and NT-5. These neurotrophin factors interact with two classes of receptors, the trk receptor tyrosine kinase family, and the low affinity p75 neurotrophin receptor. To study potential ligand-receptor interactions, recombinant trk fusion proteins have been constructed, and pan-trk polyclonal antisera directed against the cytoplasmic tyrosine kinase domain have been generated. The recombinant proteins were assessed for in vitro kinase activity and for the ability of K-252a to inhibit phosphorylation. Antibodies made against the fusion protein recognize all trk family members, and are effective in immunoprecipitation of affinity-crosslinked receptors. Comparative crosslinking indicates that NGF can recognize all trk receptor members, illustrating the large number of potential ligand-receptor interactions between neurotrophins and their receptors

We have generated mice carrying a mutation of the gene encoding the low affinity NGF receptor p75NGFR by targeted mutation in embryonic stem cells. Mice homozygous for the mutation were viable and fertile. Immunohistochemical analyses of the footpad skin of mutant mice revealed markedly decreased sensory innervation by calcitonin gene-related peptide- and substance P-immunoreactive fibers. The defective innervation was correlated with loss of heat sensitivity and associated with the development of ulcers in the distal extremities. Complicated by secondary bacterial infection, the ulcers progressed to toenail and hair loss. Crossing a human transgene encoding p75NGFR into the mutant animals rescued the absent heat sensitivity and the occurrence of skin ulcers and increased the density of neuropeptide-immunoreactive sensory innervation of footpad skin. The mutation in the gene encoding p75NGFR did not decrease the size of sympathetic ganglia or the density of sympathetic innervation of the iris or salivary gland. Our results suggest that p75NGFR has an important role in the development and function of sensory neurons

The rat pheochromocytoma PC12 cell line differentiates into a sympathetic neuronal phenotype upon treatment with either nerve growth factor (NGF) or basic fibroblast growth factor. The alkaloid-like compound K-252a has been demonstrated to be a specific inhibitor of NGF-induced biological responses in PC12 cells (Koizumi, S., Contreras, M. L., Matsuda, Y., Hama, T., Lazarovici, P., and Guroff, G. (1988) J. Neurosci. Res. 8, 715-721). NGF interacts with the protein product of the proto-oncogene trk and rapidly stimulates the tyrosine phosphorylation of both p140prototrk and a number of cellular substrates. Here we show that these phosphorylation events are directly inhibited in PC12 cells by K252a in a dose-dependent manner, indicating that the site of action of this inhibitor is at the NGF receptor level. K-252a inhibits p140prototrk activity in vitro, demonstrating that K-252a has a direct effect on the p140prototrk tyrosine kinase. Though many of the biochemical responses to NGF in PC12 cells are mimicked by basic fibroblast growth factor and epidermal growth factor, K-252a has no effect on the action of these growth factors in PC12 cells, demonstrating that the initial biological events initiated by NGF are distinctive during neuronal differentiation

A cDNA encoding the full-length 75-kD human nerve growth factor receptor was transfected into MDCK cells and its product was found to be expressed predominantly (80%) on the apical membrane, as a result of vectorial targeting from an intracellular site. Apical hNGFR bound NGF with low affinity and internalized it inefficiently (6% of surface bound NGF per hour). Several mutant hNGFRs were analyzed, after transfection in MDCK cells, for polarized surface expression, ligand binding, and endocytosis. Deletionof juxta-membrane attachment sites for a cluster of O-linked sugars did not alter apical localization. A mutant receptor lacking the entire cytoplasmic tail (except for the five proximal amino acids) was also expressed on the apical membrane, suggesting that information for apical sorting was contained in the ectoplasmic or transmembrane domains. However, a 58 amino acid deletion in the hNGFR tail that moved a cytoplasmic tyrosine (Tyr 308) closer to the membrane into a more charged environment resulted in a basolateral distribution of the mutant receptor and reversed vectorial (basolateral) targeting. The basolateral mutant receptor also internalized 125I-NGF rapidly (90% of surface bound NGF per hour), exhibited a larger intracellular fraction and displayed a considerably shortened half-life (approximately 3 h). We suggest that hNGFR with the internal cytoplasmic deletion expresses a basolateral targeting signal, related to endocytic signals, that is dominant over apical targeting information in the ecto/transmembrane domains. These results apparently contradict a current model that postulates that basolateral targeting is a default mechanism.