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. 1994 Mar;3(3):451–458. doi: 10.1002/pro.5560030310

Circular dichroism and crosslinking studies of the interaction between four neurotrophins and the extracellular domain of the low-affinity neurotrophin receptor.

D E Timm 1, A H Ross 1, K E Neet 1
PMCID: PMC2142711  PMID: 8019416

Abstract

Interactions between the purified recombinant receptor extracellular domain (RED) of the human low-affinity neurotrophin receptor (LANR) and recombinant human brain-derived neurotrophic factor, neurotrophin-3 (NT-3) and neuotrophin-4/5 have been studied by chemical crosslinking and circular dichroism. Conformational changes subsequent to binding have been shown by these procedures. First, relative affinities of the neurotrophins for RED were determined by binding competition assays in which radioiodinated nerve growth factor (NGF) from mouse submaxillary gland was crosslinked to RED in the presence of varying amounts of unlabeled neurotrophin competitors. RED bound each of the 3 recombinant human neurotrophins with affinities that were indistinguishable from authentic mouse NGF. These results are the first measurement of binding of the neurotrophin family to their common receptor using purified components. In order to study the effect of binding on the conformation of the proteins, CD measurements were made before and after mixing neurotrophins and RED, as had previously been done with NGF and RED (Timm DE, Vissavajjhala P, Ross AH, Neet KE, 1992, Protein Sci 1:1023-1031). Similar changes in CD spectra occurred upon combination of each of the neurotrophins and RED, with negative changes near 220-225 nm and positive changes near 190-200 nm; however, significant differences existed among the various neurotrophin-RED difference spectra. The NT-3/RED complex showed the largest spectral change and NGF the smallest. Thus, specific conformational changes in secondary structure of neurotrophin, RED, or both accompany the binding of each neurotrophin to the extracellular domain of the LANR.

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Selected References

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  1. Baldwin A. N., Bitler C. M., Welcher A. A., Shooter E. M. Studies on the structure and binding properties of the cysteine-rich domain of rat low affinity nerve growth factor receptor (p75NGFR). J Biol Chem. 1992 Apr 25;267(12):8352–8359. [PubMed] [Google Scholar]
  2. Banner D. W., D'Arcy A., Janes W., Gentz R., Schoenfeld H. J., Broger C., Loetscher H., Lesslauer W. Crystal structure of the soluble human 55 kd TNF receptor-human TNF beta complex: implications for TNF receptor activation. Cell. 1993 May 7;73(3):431–445. doi: 10.1016/0092-8674(93)90132-a. [DOI] [PubMed] [Google Scholar]
  3. Brahms S., Brahms J. Determination of protein secondary structure in solution by vacuum ultraviolet circular dichroism. J Mol Biol. 1980 Apr;138(2):149–178. doi: 10.1016/0022-2836(80)90282-x. [DOI] [PubMed] [Google Scholar]
  4. Chao M. V. Neurotrophin receptors: a window into neuronal differentiation. Neuron. 1992 Oct;9(4):583–593. doi: 10.1016/0896-6273(92)90023-7. [DOI] [PubMed] [Google Scholar]
  5. Compton L. A., Johnson W. C., Jr Analysis of protein circular dichroism spectra for secondary structure using a simple matrix multiplication. Anal Biochem. 1986 May 15;155(1):155–167. doi: 10.1016/0003-2697(86)90241-1. [DOI] [PubMed] [Google Scholar]
  6. Ebendal T. Function and evolution in the NGF family and its receptors. J Neurosci Res. 1992 Aug;32(4):461–470. doi: 10.1002/jnr.490320402. [DOI] [PubMed] [Google Scholar]
  7. Fandl J. P., Tobkes N. J., McDonald N. Q., Hendrickson W. A., Ryan T. E., Nigam S., Acheson A., Cudny H., Panayotatos N. Characterization and crystallization of recombinant human neurotrophin-4. J Biol Chem. 1994 Jan 7;269(1):755–759. [PubMed] [Google Scholar]
  8. Greenfield C., Hiles I., Waterfield M. D., Federwisch M., Wollmer A., Blundell T. L., McDonald N. Epidermal growth factor binding induces a conformational change in the external domain of its receptor. EMBO J. 1989 Dec 20;8(13):4115–4123. doi: 10.1002/j.1460-2075.1989.tb08596.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hallbök F., Ibáez C. F., Persson H. Evolutionary studies of the nerve growth factor family reveal a novel member abundantly expressed in Xenopus ovary. Neuron. 1991 May;6(5):845–858. doi: 10.1016/0896-6273(91)90180-8. [DOI] [PubMed] [Google Scholar]
  10. Hempstead B. L., Schleifer L. S., Chao M. V. Expression of functional nerve growth factor receptors after gene transfer. Science. 1989 Jan 20;243(4889):373–375. doi: 10.1126/science.2536190. [DOI] [PubMed] [Google Scholar]
  11. Ibáez C. F., Ebendal T., Barbany G., Murray-Rust J., Blundell T. L., Persson H. Disruption of the low affinity receptor-binding site in NGF allows neuronal survival and differentiation by binding to the trk gene product. Cell. 1992 Apr 17;69(2):329–341. doi: 10.1016/0092-8674(92)90413-7. [DOI] [PubMed] [Google Scholar]
  12. Ip N. Y., Ibáez C. F., Nye S. H., McClain J., Jones P. F., Gies D. R., Belluscio L., Le Beau M. M., Espinosa R., 3rd, Squinto S. P. Mammalian neurotrophin-4: structure, chromosomal localization, tissue distribution, and receptor specificity. Proc Natl Acad Sci U S A. 1992 Apr 1;89(7):3060–3064. doi: 10.1073/pnas.89.7.3060. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ip N. Y., Stitt T. N., Tapley P., Klein R., Glass D. J., Fandl J., Greene L. A., Barbacid M., Yancopoulos G. D. Similarities and differences in the way neurotrophins interact with the Trk receptors in neuronal and nonneuronal cells. Neuron. 1993 Feb;10(2):137–149. doi: 10.1016/0896-6273(93)90306-c. [DOI] [PubMed] [Google Scholar]
  14. Johnson E. M., Jr, Taniuchi M., Clark H. B., Springer J. E., Koh S., Tayrien M. W., Loy R. Demonstration of the retrograde transport of nerve growth factor receptor in the peripheral and central nervous system. J Neurosci. 1987 Mar;7(3):923–929. doi: 10.1523/JNEUROSCI.07-03-00923.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Klein R., Jing S. Q., Nanduri V., O'Rourke E., Barbacid M. The trk proto-oncogene encodes a receptor for nerve growth factor. Cell. 1991 Apr 5;65(1):189–197. doi: 10.1016/0092-8674(91)90419-y. [DOI] [PubMed] [Google Scholar]
  16. Lax I., Mitra A. K., Ravera C., Hurwitz D. R., Rubinstein M., Ullrich A., Stroud R. M., Schlessinger J. Epidermal growth factor (EGF) induces oligomerization of soluble, extracellular, ligand-binding domain of EGF receptor. A low resolution projection structure of the ligand-binding domain. J Biol Chem. 1991 Jul 25;266(21):13828–13833. [PubMed] [Google Scholar]
  17. Lee K. F., Li E., Huber L. J., Landis S. C., Sharpe A. H., Chao M. V., Jaenisch R. Targeted mutation of the gene encoding the low affinity NGF receptor p75 leads to deficits in the peripheral sensory nervous system. Cell. 1992 May 29;69(5):737–749. doi: 10.1016/0092-8674(92)90286-l. [DOI] [PubMed] [Google Scholar]
  18. Levi-Montalcini R., Booker B. EXCESSIVE GROWTH OF THE SYMPATHETIC GANGLIA EVOKED BY A PROTEIN ISOLATED FROM MOUSE SALIVARY GLANDS. Proc Natl Acad Sci U S A. 1960 Mar;46(3):373–384. doi: 10.1073/pnas.46.3.373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Levi-Montalcini R. The nerve growth factor 35 years later. Science. 1987 Sep 4;237(4819):1154–1162. doi: 10.1126/science.3306916. [DOI] [PubMed] [Google Scholar]
  20. Marano N., Dietzschold B., Earley J. J., Jr, Schatteman G., Thompson S., Grob P., Ross A. H., Bothwell M., Atkinson B. F., Koprowski H. Purification and amino terminal sequencing of human melanoma nerve growth factor receptor. J Neurochem. 1987 Jan;48(1):225–232. doi: 10.1111/j.1471-4159.1987.tb13151.x. [DOI] [PubMed] [Google Scholar]
  21. Matsushima H., Bogenmann E. Nerve growth factor (NGF) induces neuronal differentiation in neuroblastoma cells transfected with the NGF receptor cDNA. Mol Cell Biol. 1990 Sep;10(9):5015–5020. doi: 10.1128/mcb.10.9.5015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. McDonald N. Q., Lapatto R., Murray-Rust J., Gunning J., Wlodawer A., Blundell T. L. New protein fold revealed by a 2.3-A resolution crystal structure of nerve growth factor. Nature. 1991 Dec 5;354(6352):411–414. doi: 10.1038/354411a0. [DOI] [PubMed] [Google Scholar]
  23. Meakin S. O., Shooter E. M. The nerve growth factor family of receptors. Trends Neurosci. 1992 Sep;15(9):323–331. doi: 10.1016/0166-2236(92)90047-c. [DOI] [PubMed] [Google Scholar]
  24. Narhi L. O., Rosenfeld R., Talvenheimo J., Prestrelski S. J., Arakawa T., Lary J. W., Kolvenbach C. G., Hecht R., Boone T., Miller J. A. Comparison of the biophysical characteristics of human brain-derived neurotrophic factor, neurotrophin-3, and nerve growth factor. J Biol Chem. 1993 Jun 25;268(18):13309–13317. [PubMed] [Google Scholar]
  25. Ohmichi M., Decker S. J., Saltiel A. R. Nerve growth factor induces the association of a 130-Kd phosphoprotein with its receptor in PC-12 pheochromocytoma cells. Cell Regul. 1991 Sep;2(9):691–697. doi: 10.1091/mbc.2.9.691. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Palmer M. R., Eriksdotter-Nilsson M., Henschen A., Ebendal T., Olson L. Nerve growth factor-induced excitation of selected neurons in the brain which is blocked by a low-affinity receptor antibody. Exp Brain Res. 1993;93(2):226–230. doi: 10.1007/BF00228389. [DOI] [PubMed] [Google Scholar]
  27. Perczel A., Hollósi M., Tusnády G., Fasman G. D. Convex constraint analysis: a natural deconvolution of circular dichroism curves of proteins. Protein Eng. 1991 Aug;4(6):669–679. doi: 10.1093/protein/4.6.669. [DOI] [PubMed] [Google Scholar]
  28. Perczel A., Park K., Fasman G. D. Deconvolution of the circular dichroism spectra of proteins: the circular dichroism spectra of the antiparallel beta-sheet in proteins. Proteins. 1992 May;13(1):57–69. doi: 10.1002/prot.340130106. [DOI] [PubMed] [Google Scholar]
  29. Pleasure S. J., Reddy U. R., Venkatakrishnan G., Roy A. K., Chen J., Ross A. H., Trojanowski J. Q., Pleasure D. E., Lee V. M. Introduction of nerve growth factor (NGF) receptors into a medulloblastoma cell line results in expression of high- and low-affinity NGF receptors but not NGF-mediated differentiation. Proc Natl Acad Sci U S A. 1990 Nov;87(21):8496–8500. doi: 10.1073/pnas.87.21.8496. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Rabizadeh S., Oh J., Zhong L. T., Yang J., Bitler C. M., Butcher L. L., Bredesen D. E. Induction of apoptosis by the low-affinity NGF receptor. Science. 1993 Jul 16;261(5119):345–348. doi: 10.1126/science.8332899. [DOI] [PubMed] [Google Scholar]
  31. Radziejewski C., Robinson R. C., DiStefano P. S., Taylor J. W. Dimeric structure and conformational stability of brain-derived neurotrophic factor and neurotrophin-3. Biochemistry. 1992 May 12;31(18):4431–4436. doi: 10.1021/bi00133a007. [DOI] [PubMed] [Google Scholar]
  32. Rodriguez-Tébar A., Dechant G., Barde Y. A. Binding of brain-derived neurotrophic factor to the nerve growth factor receptor. Neuron. 1990 Apr;4(4):487–492. doi: 10.1016/0896-6273(90)90107-q. [DOI] [PubMed] [Google Scholar]
  33. Rodríguez-Tébar A., Dechant G., Götz R., Barde Y. A. Binding of neurotrophin-3 to its neuronal receptors and interactions with nerve growth factor and brain-derived neurotrophic factor. EMBO J. 1992 Mar;11(3):917–922. doi: 10.1002/j.1460-2075.1992.tb05130.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Schaefer E. M., Erickson H. P., Federwisch M., Wollmer A., Ellis L. Structural organization of the human insulin receptor ectodomain. J Biol Chem. 1992 Nov 15;267(32):23393–23402. [PubMed] [Google Scholar]
  35. Schägger H., von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. doi: 10.1016/0003-2697(87)90587-2. [DOI] [PubMed] [Google Scholar]
  36. Smith A. P., Varon S., Shooter E. M. Multiple forms of the nerve growth factor protein and its subunits. Biochemistry. 1968 Sep;7(9):3259–3268. doi: 10.1021/bi00849a032. [DOI] [PubMed] [Google Scholar]
  37. Smith C. A., Davis T., Anderson D., Solam L., Beckmann M. P., Jerzy R., Dower S. K., Cosman D., Goodwin R. G. A receptor for tumor necrosis factor defines an unusual family of cellular and viral proteins. Science. 1990 May 25;248(4958):1019–1023. doi: 10.1126/science.2160731. [DOI] [PubMed] [Google Scholar]
  38. Soppet D., Escandon E., Maragos J., Middlemas D. S., Reid S. W., Blair J., Burton L. E., Stanton B. R., Kaplan D. R., Hunter T. The neurotrophic factors brain-derived neurotrophic factor and neurotrophin-3 are ligands for the trkB tyrosine kinase receptor. Cell. 1991 May 31;65(5):895–903. doi: 10.1016/0092-8674(91)90396-g. [DOI] [PubMed] [Google Scholar]
  39. Squinto S. P., Stitt T. N., Aldrich T. H., Davis S., Bianco S. M., Radziejewski C., Glass D. J., Masiakowski P., Furth M. E., Valenzuela D. M. trkB encodes a functional receptor for brain-derived neurotrophic factor and neurotrophin-3 but not nerve growth factor. Cell. 1991 May 31;65(5):885–893. doi: 10.1016/0092-8674(91)90395-F. [DOI] [PubMed] [Google Scholar]
  40. Taniuchi M., Clark H. B., Schweitzer J. B., Johnson E. M., Jr Expression of nerve growth factor receptors by Schwann cells of axotomized peripheral nerves: ultrastructural location, suppression by axonal contact, and binding properties. J Neurosci. 1988 Feb;8(2):664–681. doi: 10.1523/JNEUROSCI.08-02-00664.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. 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]
  42. Timm D. E., Neet K. E. Equilibrium denaturation studies of mouse beta-nerve growth factor. Protein Sci. 1992 Feb;1(2):236–244. doi: 10.1002/pro.5560010205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Timm D. E., Vissavajjhala P., Ross A. H., Neet K. E. Spectroscopic and chemical studies of the interaction between nerve growth factor (NGF) and the extracellular domain of the low affinity NGF receptor. Protein Sci. 1992 Aug;1(8):1023–1031. doi: 10.1002/pro.5560010808. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Vale R. D., Shooter E. M. Assaying binding of nerve growth factor to cell surface receptors. Methods Enzymol. 1985;109:21–39. doi: 10.1016/0076-6879(85)09073-5. [DOI] [PubMed] [Google Scholar]
  45. Vissavajjhala P., Leszyk J. D., Lin-Goerke J., Ross A. H. Structural domains of the extracellular domain of human nerve growth factor receptor detected by partial proteolysis. Arch Biochem Biophys. 1992 Apr;294(1):244–252. doi: 10.1016/0003-9861(92)90164-r. [DOI] [PubMed] [Google Scholar]
  46. Vissavajjhala P., Ross A. H. Purification and characterization of the recombinant extracellular domain of human nerve growth factor receptor expressed in a baculovirus system. J Biol Chem. 1990 Mar 15;265(8):4746–4752. [PubMed] [Google Scholar]
  47. Volonté C., Ross A. H., Greene L. A. Association of a purine-analogue-sensitive protein kinase activity with p75 nerve growth factor receptors. Mol Biol Cell. 1993 Jan;4(1):71–78. doi: 10.1091/mbc.4.1.71. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Williams J. W., Morrison J. F. The kinetics of reversible tight-binding inhibition. Methods Enzymol. 1979;63:437–467. doi: 10.1016/0076-6879(79)63019-7. [DOI] [PubMed] [Google Scholar]
  49. Woodruff N. R., Neet K. E. Beta nerve growth factor binding to PC12 cells. Association kinetics and cooperative interactions. Biochemistry. 1986 Dec 2;25(24):7956–7966. doi: 10.1021/bi00372a026. [DOI] [PubMed] [Google Scholar]
  50. Yan H., Chao M. V. Disruption of cysteine-rich repeats of the p75 nerve growth factor receptor leads to loss of ligand binding. J Biol Chem. 1991 Jun 25;266(18):12099–12104. [PubMed] [Google Scholar]
  51. Yarden Y., Ullrich A. Growth factor receptor tyrosine kinases. Annu Rev Biochem. 1988;57:443–478. doi: 10.1146/annurev.bi.57.070188.002303. [DOI] [PubMed] [Google Scholar]
  52. Zupan A. A., Osborne P. A., Smith C. E., Siegel N. R., Leimgruber R. M., Johnson E. M., Jr Identification, purification, and characterization of truncated forms of the human nerve growth factor receptor. J Biol Chem. 1989 Jul 15;264(20):11714–11720. [PubMed] [Google Scholar]

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