Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1990 May;9(5):1477–1483. doi: 10.1002/j.1460-2075.1990.tb08265.x

Structure-function studies of nerve growth factor: functional importance of highly conserved amino acid residues.

C F Ibáñez 1, F Hallböök 1, T Ebendal 1, H Persson 1
PMCID: PMC551838  PMID: 2328722

Abstract

Selected amino acid residues in chicken nerve growth factor (NGF) were replaced by site-directed mutagenesis. Mutated NGF sequences were transiently expressed in COS cells and the yield of NGF protein in conditioned medium was quantified by Western blotting. Binding of each mutant to NGF receptors on PC12 cells was evaluated in a competition assay. The biological activity was determined by measuring stimulation of neurite outgrowth from chick sympathetic ganglia. The residues homologous to the proposed receptor binding site of insulin (Ser18, Met19, Val21, Asp23) were substituted by Ala. Replacement of Ser18, Met19 and Asp23 did not affect NGF activity. Modification of Val21 notably reduced both receptor binding and biological activity, suggesting that this residue is important to retain a fully active NGF. The highly conserved Tyr51 and Arg99 were converted into Phe and Lys respectively, without changing the biological properties of the molecule. However, binding and biological activity were greatly impaired after the simultaneous replacement of both Arg99 and Arg102 by Gly. The three conserved Trp residues at positions 20, 75 and 98 were substituted by Phe. The Trp mutated proteins retained 15-60% of receptor binding and 40-80% of biological activity, indicating that the Trp residues are not essential for NGF activity. However, replacement of Trp20 significantly reduced the amount of NGF in the medium, suggesting that this residue may be important for protein stability.

Full text

PDF
1477

Images in this article

1480Fig. 4.
on p.1480

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Angeletti R. H., Bradshaw R. A. Nerve growth factor from mouse submaxillary gland: amino acid sequence. Proc Natl Acad Sci U S A. 1971 Oct;68(10):2417–2420. doi: 10.1073/pnas.68.10.2417. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Angeletti R. H., Hermodson M. A., Bradshaw R. A. Amino acid sequences of mouse 2.5S nerve growth factor. II. Isolation and characterization of the thermolytic and peptic peptides and the complete covalent structure. Biochemistry. 1973 Jan 2;12(1):100–115. doi: 10.1021/bi00725a018. [DOI] [PubMed] [Google Scholar]
  3. Banerjee S. P., Snyder S. H., Cuatrecasas P., Greene L. A. Binding of nerve growth factor receptor in sympathetic ganglia. Proc Natl Acad Sci U S A. 1973 Sep;70(9):2519–2523. doi: 10.1073/pnas.70.9.2519. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bartus R. T., Dean R. L., 3rd, Beer B., Lippa A. S. The cholinergic hypothesis of geriatric memory dysfunction. Science. 1982 Jul 30;217(4558):408–414. doi: 10.1126/science.7046051. [DOI] [PubMed] [Google Scholar]
  5. Cohen P., Sutter A., Landreth G., Zimmermann A., Shooter E. M. Oxidation of tryptophan-21 alters the biological activity and receptor binding characteristics of mouse nerve growth factor. J Biol Chem. 1980 Apr 10;255(7):2949–2954. [PubMed] [Google Scholar]
  6. Cunningham B. C., Wells J. A. High-resolution epitope mapping of hGH-receptor interactions by alanine-scanning mutagenesis. Science. 1989 Jun 2;244(4908):1081–1085. doi: 10.1126/science.2471267. [DOI] [PubMed] [Google Scholar]
  7. Ebendal T., Larhammar D., Persson H. Structure and expression of the chicken beta nerve growth factor gene. EMBO J. 1986 Jul;5(7):1483–1487. doi: 10.1002/j.1460-2075.1986.tb04386.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Ebendal T. NGF in CNS: experimental data and clinical implications. Prog Growth Factor Res. 1989;1(3):143–159. doi: 10.1016/0955-2235(89)90008-2. [DOI] [PubMed] [Google Scholar]
  9. Ebendal T., Persson H., Larhammar D., Lundströmer K., Olson L. Characterization of antibodies to synthetic nerve growth factor (NGF) and proNGF peptides. J Neurosci Res. 1989 Mar;22(3):223–240. doi: 10.1002/jnr.490220302. [DOI] [PubMed] [Google Scholar]
  10. Frazier W. A., Angeletti R. H., Bradshaw R. A. Nerve growth factor and insulin. Science. 1972 May 5;176(4034):482–488. doi: 10.1126/science.176.4034.482. [DOI] [PubMed] [Google Scholar]
  11. Frazier W. A., Hogue-Angeletti R. A., Sherman R., Bradshaw R. A. Topography of mouse 2.5S nerve growth factor. Reactivity of tyrosine and tryptophan. Biochemistry. 1973 Aug 14;12(17):3281–3293. doi: 10.1021/bi00741a021. [DOI] [PubMed] [Google Scholar]
  12. Gluzman Y. SV40-transformed simian cells support the replication of early SV40 mutants. Cell. 1981 Jan;23(1):175–182. doi: 10.1016/0092-8674(81)90282-8. [DOI] [PubMed] [Google Scholar]
  13. Greene L. A., Tischler A. S. Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc Natl Acad Sci U S A. 1976 Jul;73(7):2424–2428. doi: 10.1073/pnas.73.7.2424. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. HUNTER W. M., GREENWOOD F. C. Preparation of iodine-131 labelled human growth hormone of high specific activity. Nature. 1962 May 5;194:495–496. doi: 10.1038/194495a0. [DOI] [PubMed] [Google Scholar]
  15. Hallbök F., Ebendal T., Persson H. Production and characterization of biologically active recombinant beta nerve growth factor. Mol Cell Biol. 1988 Jan;8(1):452–456. doi: 10.1128/mcb.8.1.452. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hefti F., Hartikka J., Salvatierra A., Weiner W. J., Mash D. C. Localization of nerve growth factor receptors in cholinergic neurons of the human basal forebrain. Neurosci Lett. 1986 Aug 15;69(1):37–41. doi: 10.1016/0304-3940(86)90410-6. [DOI] [PubMed] [Google Scholar]
  17. Hefti F. Is Alzheimer disease caused by lack of nerve growth factor? Ann Neurol. 1983 Jan;13(1):109–110. doi: 10.1002/ana.410130127. [DOI] [PubMed] [Google Scholar]
  18. Herrup K., Shooter E. M. Properties of the beta nerve growth factor receptor of avian dorsal root ganglia. Proc Natl Acad Sci U S A. 1973 Dec;70(12):3884–3888. doi: 10.1073/pnas.70.12.3884. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  20. Leibrock J., Lottspeich F., Hohn A., Hofer M., Hengerer B., Masiakowski P., Thoenen H., Barde Y. A. Molecular cloning and expression of brain-derived neurotrophic factor. Nature. 1989 Sep 14;341(6238):149–152. doi: 10.1038/341149a0. [DOI] [PubMed] [Google Scholar]
  21. Levi-Montalcini R., Angeletti P. U. Nerve growth factor. Physiol Rev. 1968 Jul;48(3):534–569. doi: 10.1152/physrev.1968.48.3.534. [DOI] [PubMed] [Google Scholar]
  22. Luthman H., Magnusson G. High efficiency polyoma DNA transfection of chloroquine treated cells. Nucleic Acids Res. 1983 Mar 11;11(5):1295–1308. doi: 10.1093/nar/11.5.1295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Meier R., Becker-André M., Götz R., Heumann R., Shaw A., Thoenen H. Molecular cloning of bovine and chick nerve growth factor (NGF): delineation of conserved and unconserved domains and their relationship to the biological activity and antigenicity of NGF. EMBO J. 1986 Jul;5(7):1489–1493. doi: 10.1002/j.1460-2075.1986.tb04387.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Merrell R., Pulliam M. W., Randono L., Boyd L. F., Bradshaw R. A., Glaser L. Temporal changes in tectal cell surface specificity induced by nerve growth factor. Proc Natl Acad Sci U S A. 1975 Nov;72(11):4270–4274. doi: 10.1073/pnas.72.11.4270. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Pettmann B., Manthorpe M., Powell J. A., Varon S. Biological activities of nerve growth factor bound to nitrocellulose paper by Western blotting. J Neurosci. 1988 Oct;8(10):3624–3632. doi: 10.1523/JNEUROSCI.08-10-03624.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Pielak G. J., Mauk A. G., Smith M. Site-directed mutagenesis of cytochrome c shows that an invariant Phe is not essential for function. Nature. 1985 Jan 10;313(5998):152–154. doi: 10.1038/313152a0. [DOI] [PubMed] [Google Scholar]
  27. Pullen R. A., Lindsay D. G., Wood S. P., Tickle I. J., Blundell T. L., Wollmer A., Krail G., Brandenburg D., Zahn H., Gliemann J. Receptor-binding region of insulin. Nature. 1976 Feb 5;259(5542):369–373. doi: 10.1038/259369a0. [DOI] [PubMed] [Google Scholar]
  28. Pulliam M. W., Boyd L. F., Baglan N. C., Bradshaw R. A. Specific binding of covalently cross-linked mouse nerve growth factor to responsive peripheral neurons. Biochem Biophys Res Commun. 1975 Dec 15;67(4):1281–1289. doi: 10.1016/0006-291x(75)90165-5. [DOI] [PubMed] [Google Scholar]
  29. Richardson P. M., Issa V. M., Riopelle R. J. Distribution of neuronal receptors for nerve growth factor in the rat. J Neurosci. 1986 Aug;6(8):2312–2321. doi: 10.1523/JNEUROSCI.06-08-02312.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sabesan M. N., Harper E. T. Are aromatic residues essential at the "active sites" of peptide hormones? J Theor Biol. 1980 Apr 7;83(3):457–467. doi: 10.1016/0022-5193(80)90052-1. [DOI] [PubMed] [Google Scholar]
  31. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Schwarz M. A., Fisher D., Bradshaw R. A., Isackson P. J. Isolation and sequence of a cDNA clone of beta-nerve growth factor from the guinea pig prostate gland. J Neurochem. 1989 Apr;52(4):1203–1209. doi: 10.1111/j.1471-4159.1989.tb01867.x. [DOI] [PubMed] [Google Scholar]
  33. Scott J., Selby M., Urdea M., Quiroga M., Bell G. I., Rutter W. J. Isolation and nucleotide sequence of a cDNA encoding the precursor of mouse nerve growth factor. Nature. 1983 Apr 7;302(5908):538–540. doi: 10.1038/302538a0. [DOI] [PubMed] [Google Scholar]
  34. Selby M. J., Edwards R. H., Rutter W. J. Cobra nerve growth factor: structure and evolutionary comparison. J Neurosci Res. 1987;18(2):293–298. doi: 10.1002/jnr.490180205. [DOI] [PubMed] [Google Scholar]
  35. Sutter A., Riopelle R. J., Harris-Warrick R. M., Shooter E. M. Nerve growth factor receptors. Characterization of two distinct classes of binding sites on chick embryo sensory ganglia cells. J Biol Chem. 1979 Jul 10;254(13):5972–5982. [PubMed] [Google Scholar]
  36. Taniuchi M., Schweitzer J. B., Johnson E. M., Jr Nerve growth factor receptor molecules in rat brain. Proc Natl Acad Sci U S A. 1986 Mar;83(6):1950–1954. doi: 10.1073/pnas.83.6.1950. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Thoenen H., Bandtlow C., Heumann R. The physiological function of nerve growth factor in the central nervous system: comparison with the periphery. Rev Physiol Biochem Pharmacol. 1987;109:145–178. doi: 10.1007/BFb0031026. [DOI] [PubMed] [Google Scholar]
  38. Thoenen H., Barde Y. A. Physiology of nerve growth factor. Physiol Rev. 1980 Oct;60(4):1284–1335. doi: 10.1152/physrev.1980.60.4.1284. [DOI] [PubMed] [Google Scholar]
  39. Ullrich A., Gray A., Berman C., Dull T. J. Human beta-nerve growth factor gene sequence highly homologous to that of mouse. Nature. 1983 Jun 30;303(5920):821–825. doi: 10.1038/303821a0. [DOI] [PubMed] [Google Scholar]
  40. Valenzuela D., Weber H., Weissmann C. Is sequence conservation in interferons due to selection for functional proteins? Nature. 1985 Feb 21;313(6004):698–700. doi: 10.1038/313698a0. [DOI] [PubMed] [Google Scholar]
  41. Van Ostade X., Tavernier J., Fiers W. Two conserved tryptophan residues of tumor necrosis factor and lymphotoxin are not involved in the biological activity. FEBS Lett. 1988 Oct 10;238(2):347–352. doi: 10.1016/0014-5793(88)80510-6. [DOI] [PubMed] [Google Scholar]
  42. Whitehouse P. J., Price D. L., Struble R. G., Clark A. W., Coyle J. T., Delon M. R. Alzheimer's disease and senile dementia: loss of neurons in the basal forebrain. Science. 1982 Mar 5;215(4537):1237–1239. doi: 10.1126/science.7058341. [DOI] [PubMed] [Google Scholar]
  43. Whitehouse P. J., Price D. L., Struble R. G., Clark A. W., Coyle J. T., Delon M. R. Alzheimer's disease and senile dementia: loss of neurons in the basal forebrain. Science. 1982 Mar 5;215(4537):1237–1239. doi: 10.1126/science.7058341. [DOI] [PubMed] [Google Scholar]
  44. Whittemore S. R., Friedman P. L., Larhammar D., Persson H., Gonzalez-Carvajal M., Holets V. R. Rat beta-nerve growth factor sequence and site of synthesis in the adult hippocampus. J Neurosci Res. 1988 Aug;20(4):403–410. doi: 10.1002/jnr.490200402. [DOI] [PubMed] [Google Scholar]
  45. Whittemore S. R., Seiger A. The expression, localization and functional significance of beta-nerve growth factor in the central nervous system. Brain Res. 1987 Nov;434(4):439–464. doi: 10.1016/0165-0173(87)90008-7. [DOI] [PubMed] [Google Scholar]
  46. Yang Y. C., Ciarletta A. B., Temple P. A., Chung M. P., Kovacic S., Witek-Giannotti J. S., Leary A. C., Kriz R., Donahue R. E., Wong G. G. Human IL-3 (multi-CSF): identification by expression cloning of a novel hematopoietic growth factor related to murine IL-3. Cell. 1986 Oct 10;47(1):3–10. doi: 10.1016/0092-8674(86)90360-0. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES