Skip to main content
SpringerLink
Log in

Molecular analysis of the function of the neuronal growth-associated protein GAP-43 by genetic intervention

  • Basic Molecular Aspects of Synaptic Plasticity
  • Published:
Molecular Neurobiology Aims and scope Submit manuscript

Abstract

GAP-43 is a presynaptic membrane phosphoprotein that has been implicated in both the development and the modulation of neural connections. The availability of cDNA clones for GAP-43 makes it possible to examine with greater precision its role in neuronal outgrowth and physiology. We used Northern blots andin situ hybridization with GAP-43 antisense RNA probes to show that GAP-43 is expressed selectively in associative regions of the adult brain. Immunocytochemical analyses showed alterations in the pattern of GAP-43 expression in the hippocampus during reactive synaptogenesis following lesions of the perforant pathway. Genetic intervention methodology was used to analyze the molecular nature of GAP-43 involvement in synaptic plasticity. GAP-43-transfected PC12 cells displayed an enhanced response to nerve growth factor, suggesting that GAP-43 may be directly involved in neurite extension and in the modulation of the neuronal response to extrinsic trophic factors. Studies of PC12 cell transfectants, in which the synthesis of GAP-43 was blocked by expression of GAP-43 antisense RNA, showed that evoked dopamine release was significantly attenuated in these cells. The use of gene transfer into neurons with the HSV-1 vector is presented as a method of analyzing the interaction of GAP-43 with signal transduction systems during neurotransmitter release.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Akers R. F. and Routtenberg A. (1985) Protein kinase C phosphorylates a 47 Mr protein directly related to synaptic plasticity.Brain Res. 334 147–151.

    Article  PubMed  CAS  Google Scholar 

  • Alexander K. A., Cimler B. M., Meier K. E., and Storm D. R. (1987) Regulation of calmodulin binding to P-57.J. Biol. Chem. 262, 6108–6113.

    PubMed  CAS  Google Scholar 

  • Alexander K. A., Wakim B. T., Doyle G. S., Walsh K. A., and Storm D. R. (1988) Identification and characterization of the calmodulin binding domain of neuromodulin, a neurospecific calmodulin-binding protein.J. Biol. Chem. 263, 7044–7049.

    Google Scholar 

  • Aloya V. J., Zwiers H., and Gispen W. H. (1983) Phosphorylation of B-50 protein by calcium-activated, phospholipid-dependent protein kinase and B-50 protein kinase.J. Neurochem. 41, 649–653.

    Article  Google Scholar 

  • Andreasen T. J., Luetje C. W., Heiderman W., and Storm D. R. (1983) Purification of a novel calmodulin binding protein from bovine cerebral cortex membranes.Biochemistry 2, 4615–4618.

    Article  Google Scholar 

  • Baetge E. E. and Hammang J. P. (1991) Neurite outgrowth in PC12 cells deficient in GAP-43.Neuron 6, 21–30.

    Article  PubMed  CAS  Google Scholar 

  • Basi G. S., Jacobson R. D., Virag I., Schilling J., and Skene J. H. P. (1987) Primary structure and transcriptional regulation of GAP-43, a protein associated with nerve growth.Cell 49, 785–791.

    Article  PubMed  CAS  Google Scholar 

  • Bendotti C., Servadio A., and Samanin R. (1991) Distribution of GAP-43 mRNA in the brain stem of adult rats as evidenced by in situ hybridization: localization within monoaminergic neurons.J. Neurosci. 11, 600–607.

    PubMed  CAS  Google Scholar 

  • Benowitz L. I. and Routtenberg A. (1987) A membrane phosphoprotein associated with neural development, axonal regeneration, phospholipid metabolism, and synaptic plasticity.Trends Neurosci. 10, 527–532.

    Article  CAS  Google Scholar 

  • Benowitz L. I., Rodriguez W. R., and Neve R. L. (1990) The pattern of GAP-43 immunostaining changes in the rat hippocampal formation during reactive synaptogenesis.Mol. Brain Res. 8, 17–23.

    Article  PubMed  CAS  Google Scholar 

  • Benowitz L. I., Shashoua V. E., and Yoon M. G. (1981) Specific changes in rapidly transported proteins during regeneration of the goldfish optic nerve.J. Neurosci. 1, 300–307.

    PubMed  CAS  Google Scholar 

  • Benowitz L. I., Apostolides P. J., Perrone-Bizozero N. I., Finklestein S. P., and Zwiers H. (1988) Anatomical distribution of the growth-associated protein GAP-43/B-50 in the adult rat brain.J. Neurosci. 8, 339–352.

    PubMed  CAS  Google Scholar 

  • Cimler B. M., Gielbelhaus D. H., Wakim B. T., Storm D. R., and Moon R. T. (1987) Characterization of murine cDNAs encoding P-57, a neurospecific calmodulin-binding protein.J. Biol. Chem. 262, 12,158–12,163.

    CAS  Google Scholar 

  • De Graan P. N. E., Oestreicher A. B., De Wit M., Kroef M., Schrama L. H., and Gispen W. H. (1990) Evidence for the binding of calmodulin to endogenous B-50 (GAP-43) in native synaptosomal plasma membranes.J. Neurochem. 55, 2139–2141.

    Article  PubMed  Google Scholar 

  • De Graan P. N. E., Van Hoof C. O. M., Tilly B. C., Oestreicher A. B., Schotman P., and Gispen W. H. (1985) Phosphoprotein B-50 in nerve growth cones from fetal rat brain.Neurosci. Lett. 61, 235–241.

    Article  PubMed  Google Scholar 

  • Dekker L. V., De Graan P. N. E., Pijnappel P., Oestreicher A. B., and Gispen W. H. (1991) Noradrenaline release from streptolysin O-permeated rat cortical synaptosomes: effects of calcium, phorbol esters, protein kinase inhibitors, and antibodies to the neuron-specific protein kinase C substrate B-50 (GAP-43).J. Neurochem. 56, 1146–1153.

    Article  PubMed  CAS  Google Scholar 

  • Dekker L. V., De Graan P. N. E., Versteeg D. H. G., Oestreicher A. B., and Gispen W. H. (1989) Phosphorylation of B-50 (GAP-43) is correlated with neurotransmitter release in rat hippocampal slices.J. Neurochem. 52, 24–30.

    Article  PubMed  CAS  Google Scholar 

  • Fidel S. A., Dawes L. R., Neve K. A., and Neve R. L. (1990) Effects of manipulation of GAP-43 expression on morphology in PC12 cells and hippocampal neurons.Soc. Neurosci. Abst. 16, 811.

    Google Scholar 

  • Freese A., Geller A. I., and Neve R. L. (1990) HSV-1 vector mediated neuronal gene delivery: strategies for molecular neuroscience and neurology.Biochem. Pharmacol. 40, 2189–2199.

    Article  PubMed  CAS  Google Scholar 

  • Geller A. I. and Breakefield X. (1988) A defective HSV-1 vector expressesEscherichia coli β-galactosidase in peripheral neurons.Science 241, 1667–1669.

    Article  PubMed  CAS  Google Scholar 

  • Geller A. I. and Freese A. (1990) Infection of cultured central nervous system neurons with a defective herpes simplex virus 1 vector results in stable expression ofEscherichia coli β-galactosidase.Proc. Natl. Acad. Sci. USA 87, 1149–1153.

    Article  PubMed  CAS  Google Scholar 

  • Geller A. I., Bryan J., Ashe O., During M. J., and Neve R. L. (1991) Expression of an unregulated protein kinase C in cortical neurons, from a HSV-1 vector, causes a long term, activity dependent increase in neurotransmitter release.Soc. Neurosci. Abst. 17, 603.

    Google Scholar 

  • Geller A. I., During M. J., and Neve R. L. (1991b) Molecular analysis of neuronal physiology by gene transfer into neurons with herpes simplex virus vectors.Trends Neurosci. 14, 428–432.

    Article  PubMed  CAS  Google Scholar 

  • Geller A. I., During M. J., Freese A., and Neve R. L. A. HSV-1 vector expressing an unregulated adenylate cyclase stably increases neurotransmitter release from cultured rat sympathetic neurons.Proc. Natl. Acad. Sci. USA, in press.

  • Gispen W. H., Leunissen J. L. M., Oestreicher A. B., Verkleij A. J., and Zwiers H. (1985) Presynaptic localization of B-50 phosphoprotein: the ACTH-sensitive protein kinase substrate involved in rat brain polyphosphoinositide metabolism.Brain Res. 328, 381–385.

    Article  PubMed  CAS  Google Scholar 

  • Ivins K. J., Fidel S. A., Neve K. A., and Neve R. L. (1991) Antisense GAP-43 mRNA blocks evoked release of dopamine from PC12 cells.Soc. Neurosci. Abst. 17, 576.

    Google Scholar 

  • Jolles J., Zwiers H., van Dongen C., Schotman P., Wirtz K. A. W., and Gispen W. J. (1980) Modulation of brain polyphosphoinositide metabolism by ACTH-sensitive protein phosphorylation.Nature (Lond.) 286, 623–625.

    Article  CAS  Google Scholar 

  • Kataoka T., Broek D., and Wigler M. (1985) DNA sequence and characterization of theS. cerevisiae gene encoding adenylate cyclase.Cell 43, 493–505.

    Article  PubMed  CAS  Google Scholar 

  • Katz F., Ellis L., and Pfenninger K. H. (1985) Nerve growth cones isolated from fetal rat brain. III. Calcium-dependent protein phosphorylation.J. Neurosci. 5, 1402–1411.

    PubMed  CAS  Google Scholar 

  • Kosik K. S., Orecchio L. D., Bruns G. A. P., MacDonald G. P., Cox D. R., and Neve R. L. (1988) Human GAP-43: its deduced amino acid sequence and chromosomal localization in mouse and human.Neuron 1, 127–132.

    Article  PubMed  CAS  Google Scholar 

  • Liu Y. and Storm D. R. (1990) Regulation of free calmodulin levels by neuromodulin: neuron growth and regeneration.Trends Pharmacol. Sci. 11, 107–111.

    Article  PubMed  CAS  Google Scholar 

  • Liu Y. and Storm D. R. (1991) Targeting of neuromodulin (GAP-43) fusion proteins to growth cones in cultured rat embryonic neurons.Neuron 6, 411–420.

    Article  PubMed  CAS  Google Scholar 

  • Lovinger D. M., Akers R. F., Nelson R. B., Barnes C. A., McNaughton B. L., and Routtenberg A. (1985) A selective increase in phosphorylation of protein F1, a protein kinase C substrate, directly related to three day growth of long term synaptic enhancement.Brain Res. 343, 137–143.

    Article  PubMed  CAS  Google Scholar 

  • Lynch G., Gall C., Rose G., and Cotman C. (1976) Changes in the distribution of the dentate gyrus association system following unilateral or bilateral entorhinal lesion in the adult rat.Brain Res. 110, 57–71.

    Article  PubMed  CAS  Google Scholar 

  • Meiri K. F., Pfenninger K. H., and Willard M. B. (1986) Growth-associated protein, GAP-43, a polypeptide that is induced when neurons extend axons, is a component of growth cones and corresponds to pp46, a major polypeptide of a subcellular fraction enriched in growth cones.Proc. Natl. Acad. Sci. USA 83, 3537–3541.

    Article  PubMed  CAS  Google Scholar 

  • Nelson R. and Routtenberg A. (1985) Characterization of protein F1 (47 kDA, 4.5 pl): a kinase C substrate directly related to neural plasticity.Exp. Neurol. 89, 213–244.

    Article  PubMed  CAS  Google Scholar 

  • Neve R. L., Perrone-Bizzozero N. I., Finklestein S. P., Zwiers H., Bird E., Kumit D. M., and Benowitz L. I. (1987) The neuronal growth-associated protein GAP-43 (B-50, F1): neuronal specificity, developmental regulation and regional distribution of the human and rat mRNAs.Mol. Brain Res. 2, 177–183.

    Article  CAS  Google Scholar 

  • Neve R. L., Dawes L. R., and Geller A. I. (1990) Fusion of the aminoterminal 10 amino acids of GAP-43 to β-galactosidase targets the chimeric protein to neuronal processes.Soc. Neurosci. Abst. 16, 50.

    Google Scholar 

  • Neve R. L., Finch E. A., Bird E. D., and Benowitz L. I. (1988) The growth-associated protein GAP-43 (B-50, F1) is expressed selectively in associative regions of the adult human brain.Proc. Natl. Acad. Sci. USA 85, 3638–3642.

    Article  PubMed  CAS  Google Scholar 

  • Ng S.-C., de la Monte S. M., Conboy G. L., Karns L. R., and Fishman M. C. (1988) Cloning of human GAP-43: growth association and ischemic resurgence.Neuron 1, 133–139.

    Article  PubMed  CAS  Google Scholar 

  • Oestreicher A. B. and Gispen W. H. (1986) Comparison of the immunocytochemical distribution of the phosphoprotein B-50 in the cerebellum and hippocampus of immature and adult rat brain.Brain Res. 375, 267–279.

    Article  PubMed  CAS  Google Scholar 

  • Pfenninger K. H. (1986) Of nerve growth cones, leukocytes and memory: second messenger systems and growth-related proteins.Trends Neurosci. 9, 562–565.

    Article  CAS  Google Scholar 

  • Pisano M. R., Hegazy M. G., Reimann E. M., and Dokas L. A. (1988) Phosphorylation of 21 protein B-50 (GAP-43) from adult rat brain cortex by casein kinase 11.Biochem. Biophys. Res. Comm. 155, 1207–1212.

    Article  PubMed  CAS  Google Scholar 

  • Rosenthal A., Chan S. Y., Henzel W., Haskell C., Kuang W.-J., Chen J. N., Wilcox A., Ullrich D. V., Goeddel E., and Routtenberg A. (1987) Primary structure and mRNA localization of protein F1, a growth-related protein kinase C substrate associated with synaptic plasticity.EMBO J. 6, 3641–3646.

    PubMed  CAS  Google Scholar 

  • Schuh S. M., Spencer S. A., and Willard M. (1989) Phosphorylation sites on the growth-associated protein GAP-43.Soc. Neurosci. Abst. 15, 573.

    Google Scholar 

  • Skene J. H. P. and Virag I. (1989) Post-translational membrane attachment and dynamic fatty acylation of neuronal growth cone protein, GAP-43.J. Cell Biol. 108, 613–625.

    Article  PubMed  CAS  Google Scholar 

  • Skene J. H. P. and Willard M. J. (1981a) Axonally transported proteins associated with growth in rabbit central and peripheral nervous system.Cell Biol. 89, 96–103.

    Article  CAS  Google Scholar 

  • Skene J. H. P. and Willard M. J. (1981b) Changes in axonally transported proteins during axon regeneration in toad retinal ganglion cells.Cell Biol. 89, 86–95.

    Article  CAS  Google Scholar 

  • Skene J. H. P., Jacobson R. D., Snipes G. J., McGuire C. B., Norden J., and Freeman J. A. (1986) A protein induced during nerve growth (GAP-43) is a major component of growth cone membranes.Science 233, 783–785.

    Article  PubMed  CAS  Google Scholar 

  • Steward O., Cotman C., and Lynch G. S. (1974) Growth of a new fiber projection in the brain of adult rats: reinnervation of the dentate gyrus by the contralateral entorhinal cortex following ipsilateral entorhinal lesions.Exp. Brain Res. 20, 466.

    Article  Google Scholar 

  • Strittmatter S. M., Valenzuela D., Kennedy T. E., Neer E. J., and Fishman M. C. (1990) G0 is a major growth cone protein subject to regulation by GAP-43.Nature 344, 836–841.

    Article  PubMed  CAS  Google Scholar 

  • Yankner B. A., Benowitz L. I., Villa-Komaroff L., and Neve R. L. (1990) Transfection of PC12 cells with the human GAP-43 gene: effects on neurite out-growth and regeneration.Mol. Brain Res. 7, 39–44.

    Article  PubMed  CAS  Google Scholar 

  • Zuber M. W., Strittmatter S. M., and Fishman M. C. (1989) A membrane-targeting signal in the amino terminus of the neuronal protein GAP-43.Nature 341, 345–348.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Psychobiology, University of California, 92717, Irvine, CA

    Rachael L. Neve & Kathryn J. Ivins

  2. Department of Surgery, The Children's Hospital, 02115, Boston, MA

    Larry I. Benowitz

  3. Department of Neurosurgery, Yale University School of Medicine, 06510, New Haven, CT

    Matthew J. During

  4. Department of Endocrinology, The Children's Hospital, 02115, Boston, MA

    Alfred I. Geller

Authors
  1. Rachael L. Neve
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kathryn J. Ivins
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Larry I. Benowitz
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Matthew J. During
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Alfred I. Geller
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Neve, R.L., Ivins, K.J., Benowitz, L.I. et al. Molecular analysis of the function of the neuronal growth-associated protein GAP-43 by genetic intervention. Mol Neurobiol 5, 131–141 (1991). https://doi.org/10.1007/BF02935542

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02935542

Index Entries

Search

Navigation