The Biosynthesis of Neurotrophin Heterodimers by Transfected Mammalian Cells

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Prompted by the recent discovery that neurotrophins, which are known to be biologically active as noncovalently linked homodimers, can also be induced to form biologically active heterodimers in vitro, we have investigated the biosynthesis of neurotrophin heterodimers by transfected mammalian cells. When COS cells were cotransfected with expression plasmids for nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), or neurotrophin-3 (NT-3), the appropriate heterodimers were detected in the conditioned medium by immunoprecipitation and, in the case of NGF.NT-3, using a two-site enzyme-linked immunosorbent assay. Heterodimer formation occurred predominantly intracellularly and did not require precursor cleavage, because heterodimers containing pro-NGF and pro-BDNF were detected in the conditioned medium. When rat C6 glioma cells or mouse AtT-20 neuroendocrine cells were cotransfected with expression plasmids for NGF and NT-3, NGF.NT-3 heterodimer was detected at levels comparable with those of homodimeric NGF and NT-3, indicating that heterodimer formation can occur at significant levels in a variety of cell types. These data provide evidence that NGF, BDNF, and NT-3 are capable of forming heterodimers when coexpressed in mammalian cells and suggest that such heterodimers are likely to be formed in vivo when a single cell expresses multiple neurotrophins.