The Biosynthesis of Neurotrophin Heterodimers by Transfected Mammalian Cells

doi:10.1074/jbc.270.20.12297

The Biosynthesis of Neurotrophin Heterodimers by Transfected Mammalian Cells (*)

From the (1) Department of Neurobiology, Stanford University School of Medicine, Stanford, California 94305-5401

^¶To whom correspondence should be addressed. Tel.: 415-723-6638; Fax: 415-725-0388.

Abstract

Prompted by the recent discovery that neurotrophins, which are known to be biologically active as non-covalently 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 NGFNT-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, NGFNT-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.

Footnotes

↵^§ Supported by National Institutes of Health Neonatal and Developmental Biology Training Grant HD-07249.
↵* This work was supported by Grant NS 04270 from the National Institutes of Health, by Grant PRG-94-138 from the Alzheimer's Association, and by Contracts 92-15942 and 93-18647 from the State of California, Department of Health Services. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

NGF

nerve growth factor

BDNF

brain-derived neurotrophic factor

NT-3

neurotrophin-3

ELISA

enzyme-linked immunosorbent assay

PBS

phosphate-buffered saline

PAGE

polyacrylamide gel electrophoresis

BDNF-Myc

form of BDNF containing a 10-amino acid Myc epitope at its carboxyl terminus.
↵²J. V. Heymach, Jr., and E. M. Shooter, unpublished data.
↵³Theoretically, if the association between monomers is unbiased and if A and B are the relative amounts of NGF and NT-3 monomers present prior to dimerization, the relative amounts of NGF homodimer, NGFNT-3 heterodimer, and NT-3 homodimer formed would be A², 2AB, and B², respectively. Therefore, if A = B, they would be formed in a 1:2:1 ratio. However, if NGF and NT-3 (monomers) were expressed in a 9:1 ratio, the dimerized neurotrophins would consist of 81% NGF, 18% NGFNT-3, and only 1% NT-3.