HSV-1 vector-mediated gene transfer of the human nerve growth factor receptor p75hNGFR defines high-affinity NGF binding.
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.
Department of Cell Biology and Anatomy, Cornell University Medical College, New York, New York 10021.
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Battleman
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Geller
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Chao
D016475:3T3 Cells; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000378:metabolism*; D004247:DNA / Q000235:genetics; D004622:Embryo, Mammalian; D005347:Fibroblasts / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D051379:Mice; D009414:Nerve Growth Factors / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism*; D016716:PC12 Cells; D011505:Protein-Tyrosine Kinases / Q000235:genetics; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins / Q000235:genetics; D011519:Proto-Oncogenes; D051381:Rats; D017207:Rats, Sprague-Dawley; D020917:Receptor, trkA; D017475:Receptors, Nerve Growth Factor / Q000235:genetics / Q000378:metabolism*; D012091:Repetitive Sequences, Nucleic Acid; D018139:Simplexvirus / Q000235:genetics*; D014162:Transfection / Q000379:methods*
C000715470:MAS1 protein, human; D009414:Nerve Growth Factors; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins; D017475:Receptors, Nerve Growth Factor; D004247:DNA; D011505:Protein-Tyrosine Kinases; D020917:Receptor, trkA