The Regulated Secretion and Vectorial Targeting of Neurotrophins in Neuroendocrine and Epithelial Cells*
- From the 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
The varied roles that neurotrophins play in the development and activity-dependent plasticity of the nervous system presumably require that the sites and quantity of neurotrophin release be precisely regulated. As a step toward understanding how different neurotrophins are sorted and secreted by neurons, we expressed nerve growth factor (NGF), brain-derived neurotrophic factor, and neurotrophin-3 in cell lines used as models for neuronal protein sorting. All three neurotrophins were secreted by a regulated pathway in transfected AtT-20 and PC12 neuroendocrine cells, with a 3-6-fold increase in neurotrophin release in response to 8-bromo-cAMP or depolarization, respectively. To determine if the propeptide directs the intracellular sorting of mature NGF, we examined mutants in which regions spanning the propeptide were deleted. These mutants underwent regulated release in every case in which expression could be detected. Similarly, NGF sorting was not significantly altered by mutations which specifically abolished N-glycosylation or proteolytic processing sites within the NGF precursor. Finally, we found that all three neurotrophins were secreted 65-75% basolaterally by polarized Madin-Darby canine kidney epithelial cells. These findings suggest that the determinants of regulated neurotrophin secretion lie within the mature neurotrophin moiety and that NGF, brain-derived neurotrophic factor, and neurotrophin-3 are likely to be sorted similarly and released in a regulated manner by neurons.
Footnotes
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↵* This work was supported in part by National Institutes of Health Grants NS04270, NS07158, and HD07249, the Alzheimer's Association (PRG 94-138), Deutsche Forschungsgemeinschaft (to A. K.), the Swiss National Science Foundation (to U. S.), the Stanford Medical Student Scholars program, the Glenn/American Federation For Aging Research, and a Howard Hughes Medical Student Fellowship (to J. V. H.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵1 The abbreviations used are:
- NGF
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nerve growth factor
- BDNF
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brain-derived neurotrophic factor
- NT
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neurotrophin
- MDCK
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Madin-Darby canine kidney
- DMEM
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Dulbecco's modified Eagle's medium
- ELISA
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enzyme-linked immunosorbent assay
- BSA
-
bovine serum albumin
- CHX
-
cycloheximide
- PAGE
-
polyacrylamide gel electrophoresis
- Br
-
bromo.
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↵2 A. Krüttgen, J. V. Heymach, Jr., P. Kahle, and E. M. Shooter, unpublished observations.
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↵3 J. V. Heymach, Jr., A, Krüttgen, U. Suter, and E. M. Shooter, unpublished observations.
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- Received May 24, 1996.
- Revision received July 5, 1996.
- © 1996 by The American Society for Biochemistry and Molecular Biology, Inc.
