Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1987 May;6(5):1197–1202. doi: 10.1002/j.1460-2075.1987.tb02354.x

The internalization of nerve growth factor by high-affinity receptors on pheochromocytoma PC12 cells.

M Hosang, E M Shooter
PMCID: PMC553919  PMID: 3038514

Abstract

The internalization and subsequent fate of the two populations of nerve growth factor (NGF) receptors on pheochromocytoma PC12 cells were explored either by identifying the relative amounts and sizes of the receptors, after incubation of cells with [125I]NGF, by cross-linking with a photoreactive heterobifunctional reagent or by following the topological distribution of the cross-linked receptors with time. The ratio of the slow, high-affinity to the fast, low-affinity NGF receptor decreased over a 5-h incubation with [125I]NGF in a process which did not involve proteolytic conversion of the slow to the fast receptor. During this period the cross-linked slow receptor moved from a trypsin-labile to a trypsin-stable site suggestive of internalization. In contrast, the cross-linked fast NGF receptor remained trypsin sensitive for at least 2 h of incubation, indicative of a constant cell surface localization. The internalized [125I]NGF in the cross-linked slow NGF receptor was not degraded, indicating that cross-linking, by preventing the acid pH-induced dissociation of the NGF-receptor complex in the endosomes, blocks normal sorting of [125I]NGF to the lysosomes. The cross-linked receptor was not recycled to the cell surface. If this reflects the properties of the unmodified receptor then another process, possibly receptor conversion, is required to replenish slow NGF receptors in the cell surface.

Full text

PDF
1197

Images in this article

1198Fig. 1.
on p.1198
1199Fig. 2.
on p.1199
1199Fig. 3.
on p.1199

Selected References

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

  1. Berhanu P., Kolterman O. G., Baron A., Tsai P., Olefsky J. M., Brandenburg D. Insulin receptors in isolated human adipocytes. Characterization by photoaffinity labeling and evidence for internalization and cellular processing. J Clin Invest. 1983 Dec;72(6):1958–1970. doi: 10.1172/JCI111160. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bernd P., Greene L. A. Association of 125I-nerve growth factor with PC12 pheochromocytoma cells. Evidence for internalization via high-affinity receptors only and for long-term regulation by nerve growth factor of both high- and low-affinity receptors. J Biol Chem. 1984 Dec 25;259(24):15509–15516. [PubMed] [Google Scholar]
  3. Bernd P., Greene L. A. Electron microscopic radioautographic localization of iodinated nerve growth factor bound to and internalized by PC12 cells. J Neurosci. 1983 Mar;3(3):631–643. doi: 10.1523/JNEUROSCI.03-03-00631.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bishop J. M. Cellular oncogenes and retroviruses. Annu Rev Biochem. 1983;52:301–354. doi: 10.1146/annurev.bi.52.070183.001505. [DOI] [PubMed] [Google Scholar]
  5. Brown M. S., Anderson R. G., Goldstein J. L. Recycling receptors: the round-trip itinerary of migrant membrane proteins. Cell. 1983 Mar;32(3):663–667. doi: 10.1016/0092-8674(83)90052-1. [DOI] [PubMed] [Google Scholar]
  6. Burton L. E., Wilson W. H., Shooter E. M. Nerve growth factor in mouse saliva. Rapid isolation procedures for and characterization of 7 S nerve growth factor. J Biol Chem. 1978 Nov 10;253(21):7807–7812. [PubMed] [Google Scholar]
  7. Campenot R. B. Development of sympathetic neurons in compartmentalized cultures. II. Local control of neurite survival by nerve growth factor. Dev Biol. 1982 Sep;93(1):13–21. doi: 10.1016/0012-1606(82)90233-0. [DOI] [PubMed] [Google Scholar]
  8. Campenot R. B. Development of sympathetic neurons in compartmentalized cultures. Il Local control of neurite growth by nerve growth factor. Dev Biol. 1982 Sep;93(1):1–12. doi: 10.1016/0012-1606(82)90232-9. [DOI] [PubMed] [Google Scholar]
  9. Chandler C. E., Herschman H. R. Binding, sequestration, and processing of epidermal growth factor and nerve growth factor by PC12 cells. J Cell Physiol. 1983 Mar;114(3):321–327. doi: 10.1002/jcp.1041140311. [DOI] [PubMed] [Google Scholar]
  10. Claude P., Hawrot E., Dunis D. A., Campenot R. B. Binding, internalization, and retrograde transport of 125I-nerve growth factor in cultured rat sympathetic neurons. J Neurosci. 1982 Apr;2(4):431–442. doi: 10.1523/JNEUROSCI.02-04-00431.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Connolly J. L., Green S. A., Greene L. A. Pit formation and rapid changes in surface morphology of sympathetic neurons in response to nerve growth factor. J Cell Biol. 1981 Jul;90(1):176–180. doi: 10.1083/jcb.90.1.176. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dumas M., Schwab M. E., Thoenen H. Retrograde axonal transport of specific macromolecules as a tool for characterizing nerve terminal membranes. J Neurobiol. 1979 Mar;10(2):179–197. doi: 10.1002/neu.480100207. [DOI] [PubMed] [Google Scholar]
  13. Ebina Y., Ellis L., Jarnagin K., Edery M., Graf L., Clauser E., Ou J. H., Masiarz F., Kan Y. W., Goldfine I. D. The human insulin receptor cDNA: the structural basis for hormone-activated transmembrane signalling. Cell. 1985 Apr;40(4):747–758. doi: 10.1016/0092-8674(85)90334-4. [DOI] [PubMed] [Google Scholar]
  14. Fehlmann M., Carpentier J. L., Van Obberghen E., Freychet P., Thamm P., Saunders D., Brandenburg D., Orci L. Internalized insulin receptors are recycled to the cell surface in rat hepatocytes. Proc Natl Acad Sci U S A. 1982 Oct;79(19):5921–5925. doi: 10.1073/pnas.79.19.5921. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Godfrey E. W., Shooter E. M. Nerve growth factor receptors on chick embryo sympathetic ganglion cells: binding characteristics and development. J Neurosci. 1986 Sep;6(9):2543–2550. doi: 10.1523/JNEUROSCI.06-09-02543.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Goldstein J. L., Anderson R. G., Brown M. S. Coated pits, coated vesicles, and receptor-mediated endocytosis. Nature. 1979 Jun 21;279(5715):679–685. doi: 10.1038/279679a0. [DOI] [PubMed] [Google Scholar]
  17. Green S. H., Rydel R. E., Connolly J. L., Greene L. A. PC12 cell mutants that possess low- but not high-affinity nerve growth factor receptors neither respond to nor internalize nerve growth factor. J Cell Biol. 1986 Mar;102(3):830–843. doi: 10.1083/jcb.102.3.830. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gunning P. W., Landreth G. E., Layer P., Ignatius M., Shooter E. M. Nerve growth factor-induced differentiation of PC12 cells: evaluation of changes in RNA and DNA metabolism. J Neurosci. 1981 Apr;1(4):368–379. doi: 10.1523/JNEUROSCI.01-04-00368.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Halegoua S., Patrick J. Nerve growth factor mediates phosphorylation of specific proteins. Cell. 1980 Nov;22(2 Pt 2):571–581. doi: 10.1016/0092-8674(80)90367-0. [DOI] [PubMed] [Google Scholar]
  20. Hendry I. A., Stöckel K., Thoenen H., Iversen L. L. The retrograde axonal transport of nerve growth factor. Brain Res. 1974 Mar 15;68(1):103–121. doi: 10.1016/0006-8993(74)90536-8. [DOI] [PubMed] [Google Scholar]
  21. Heumann R., Korsching S., Scott J., Thoenen H. Relationship between levels of nerve growth factor (NGF) and its messenger RNA in sympathetic ganglia and peripheral target tissues. EMBO J. 1984 Dec 20;3(13):3183–3189. doi: 10.1002/j.1460-2075.1984.tb02277.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Heumann R., Schwab M., Merkl R., Thoenen H. Nerve growth factor-mediated induction of choline acetyltransferase in PC12 cells: evaluation of the site of action of nerve growth factor and the involvement of lysosomal degradation products of nerve growth factor. J Neurosci. 1984 Dec;4(12):3039–3050. doi: 10.1523/JNEUROSCI.04-12-03039.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Heumann R., Schwab M., Thoenen H. A second messenger required for nerve growth factor biological activity? Nature. 1981 Aug 27;292(5826):838–840. doi: 10.1038/292838a0. [DOI] [PubMed] [Google Scholar]
  24. Hogue-Angeletti R., Stieber A., Gonatas N. K. Endocytosis of nerve growth factor by PC12 cells studied by quantitative ultrastructural autoradiography. Brain Res. 1982 Jun 3;241(1):145–156. doi: 10.1016/0006-8993(82)91237-9. [DOI] [PubMed] [Google Scholar]
  25. Hosang M., Shooter E. M. Molecular characteristics of nerve growth factor receptors on PC12 cells. J Biol Chem. 1985 Jan 10;260(1):655–662. [PubMed] [Google Scholar]
  26. Johnson E. M., Jr, Andres R. Y., Bradshaw R. A. Characterization of the retrograde transport of nerve growth factor (NGF) using high specific activity [125I] NGF. Brain Res. 1978 Jul 14;150(2):319–331. doi: 10.1016/0006-8993(78)90283-4. [DOI] [PubMed] [Google Scholar]
  27. Kahn C. R. Membrane receptors for hormones and neurotransmitters. J Cell Biol. 1976 Aug;70(2 Pt 1):261–286. doi: 10.1083/jcb.70.2.261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kamps M. P., Taylor S. S., Sefton B. M. Direct evidence that oncogenic tyrosine kinases and cyclic AMP-dependent protein kinase have homologous ATP-binding sites. Nature. 1984 Aug 16;310(5978):589–592. doi: 10.1038/310589a0. [DOI] [PubMed] [Google Scholar]
  29. King A. C., Willis R. A., Cuatrecasas P. Accumulation of epidermal growth factor within cells does not depend on receptor recycling. Biochem Biophys Res Commun. 1980 Dec 16;97(3):840–845. doi: 10.1016/0006-291x(80)91453-9. [DOI] [PubMed] [Google Scholar]
  30. Korsching S., Thoenen H. Nerve growth factor in sympathetic ganglia and corresponding target organs of the rat: correlation with density of sympathetic innervation. Proc Natl Acad Sci U S A. 1983 Jun;80(11):3513–3516. doi: 10.1073/pnas.80.11.3513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Korsching S., Thoenen H. Quantitative demonstration of the retrograde axonal transport of endogenous nerve growth factor. Neurosci Lett. 1983 Aug 19;39(1):1–4. doi: 10.1016/0304-3940(83)90155-6. [DOI] [PubMed] [Google Scholar]
  32. Korsching S., Thoenen H. Treatment with 6-hydroxydopamine and colchicine decreases nerve growth factor levels in sympathetic ganglia and increases them in the corresponding target tissues. J Neurosci. 1985 Apr;5(4):1058–1061. doi: 10.1523/JNEUROSCI.05-04-01058.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  34. Landreth G. E., Shooter E. M. Nerve growth factor receptors on PC12 cells: ligand-induced conversion from low- to high-affinity states. Proc Natl Acad Sci U S A. 1980 Aug;77(8):4751–4755. doi: 10.1073/pnas.77.8.4751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Layer P. G., Shooter E. M. Binding and degradation of nerve growth factor by PC12 pheochromocytoma cells. J Biol Chem. 1983 Mar 10;258(5):3012–3018. [PubMed] [Google Scholar]
  36. 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]
  37. Marshall S. Kinetics of insulin receptor internalization and recycling in adipocytes. Shunting of receptors to a degradative pathway by inhibitors of recycling. J Biol Chem. 1985 Apr 10;260(7):4136–4144. [PubMed] [Google Scholar]
  38. Massague J., Guillette B. J., Czech M. P., Morgan C. J., Bradshaw R. A. Identification of a nerve growth factor receptor protein in sympathetic ganglia membranes by affinity labeling. J Biol Chem. 1981 Sep 25;256(18):9419–9424. [PubMed] [Google Scholar]
  39. Mellman I., Fuchs R., Helenius A. Acidification of the endocytic and exocytic pathways. Annu Rev Biochem. 1986;55:663–700. doi: 10.1146/annurev.bi.55.070186.003311. [DOI] [PubMed] [Google Scholar]
  40. Murthy U., Basu M., Sen-Majumdar A., Das M. Perinuclear location and recycling of epidermal growth factor receptor kinase: immunofluorescent visualization using antibodies directed to kinase and extracellular domains. J Cell Biol. 1986 Aug;103(2):333–342. doi: 10.1083/jcb.103.2.333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Radeke M. J., Misko T. P., Hsu C., Herzenberg L. A., Shooter E. M. Gene transfer and molecular cloning of the rat nerve growth factor receptor. Nature. 1987 Feb 12;325(6105):593–597. doi: 10.1038/325593a0. [DOI] [PubMed] [Google Scholar]
  42. Reed B. C. Metabolism of covalent receptor-insulin complexes by 3T3-L1 adipocytes. Synthesis and use of photosensitive insulin analogs to study insulin receptor metabolism in cell culture. J Biol Chem. 1983 Apr 10;258(7):4424–4433. [PubMed] [Google Scholar]
  43. Resh M. D., Erikson R. L. Highly specific antibody to Rous sarcoma virus src gene product recognizes a novel population of pp60v-src and pp60c-src molecules. J Cell Biol. 1985 Feb;100(2):409–417. doi: 10.1083/jcb.100.2.409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Rohrer H., Schäfer T., Korsching S., Thoenen H. Internalization of nerve growth factor by pheochromocytoma PC12 cells: absence of transfer to the nucleus. J Neurosci. 1982 Jun;2(6):687–697. doi: 10.1523/JNEUROSCI.02-06-00687.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Schechter A. L., Bothwell M. A. Nerve growth factor receptors on PC12 cells: evidence for two receptor classes with differing cytoskeletal association. Cell. 1981 Jun;24(3):867–874. doi: 10.1016/0092-8674(81)90112-4. [DOI] [PubMed] [Google Scholar]
  46. Schlessinger J., Schreiber A. B., Levi A., Lax I., Libermann T., Yarden Y. Regulation of cell proliferation by epidermal growth factor. CRC Crit Rev Biochem. 1983;14(2):93–111. doi: 10.3109/10409238309102791. [DOI] [PubMed] [Google Scholar]
  47. Schnapp B. J., Vale R. D., Sheetz M. P., Reese T. S. Single microtubules from squid axoplasm support bidirectional movement of organelles. Cell. 1985 Feb;40(2):455–462. doi: 10.1016/0092-8674(85)90160-6. [DOI] [PubMed] [Google Scholar]
  48. Schwab M. E., Heumann R., Thoenen H. Communication between target organs and nerve cells: retrograde axonal transport and site of action of nerve growth factors. Cold Spring Harb Symp Quant Biol. 1982;46(Pt 1):125–134. doi: 10.1101/sqb.1982.046.01.016. [DOI] [PubMed] [Google Scholar]
  49. Schwab M., Thoenen H. Selective trans-synaptic migration of tetanus toxin after retrograde axonal transport in peripheral sympathetic nerves: a comparison with nerve growth factor. Brain Res. 1977 Feb 25;122(3):459–474. doi: 10.1016/0006-8993(77)90457-7. [DOI] [PubMed] [Google Scholar]
  50. Seeley P. J., Keith C. H., Shelanski M. L., Greene L. A. Pressure microinjection of nerve growth factor and anti-nerve growth factor into the nucleus and cytoplasm: lack of effects on neurite outgrowth from pheochromocytoma cells. J Neurosci. 1983 Jul;3(7):1488–1494. doi: 10.1523/JNEUROSCI.03-07-01488.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Shelton D. L., Reichardt L. F. Expression of the beta-nerve growth factor gene correlates with the density of sympathetic innervation in effector organs. Proc Natl Acad Sci U S A. 1984 Dec;81(24):7951–7955. doi: 10.1073/pnas.81.24.7951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Shooter E. M., Yankner B. A., Landreth G. E., Sutter A. Biosynthesis and mechanism of action of nerve growth factor. Recent Prog Horm Res. 1981;37:417–446. doi: 10.1016/b978-0-12-571137-1.50013-x. [DOI] [PubMed] [Google Scholar]
  53. 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]
  54. 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]
  55. Tycko B., Maxfield F. R. Rapid acidification of endocytic vesicles containing alpha 2-macroglobulin. Cell. 1982 Mar;28(3):643–651. doi: 10.1016/0092-8674(82)90219-7. [DOI] [PubMed] [Google Scholar]
  56. Ullrich A., Bell J. R., Chen E. Y., Herrera R., Petruzzelli L. M., Dull T. J., Gray A., Coussens L., Liao Y. C., Tsubokawa M. Human insulin receptor and its relationship to the tyrosine kinase family of oncogenes. 1985 Feb 28-Mar 6Nature. 313(6005):756–761. doi: 10.1038/313756a0. [DOI] [PubMed] [Google Scholar]
  57. Ullrich A., Coussens L., Hayflick J. S., Dull T. J., Gray A., Tam A. W., Lee J., Yarden Y., Libermann T. A., Schlessinger J. Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431 epidermoid carcinoma cells. 1984 May 31-Jun 6Nature. 309(5967):418–425. doi: 10.1038/309418a0. [DOI] [PubMed] [Google Scholar]
  58. 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]
  59. Weissman A. M., Harford J. B., Svetlik P. B., Leonard W. L., Depper J. M., Waldmann T. A., Greene W. C., Klausner R. D. Only high-affinity receptors for interleukin 2 mediate internalization of ligand. Proc Natl Acad Sci U S A. 1986 Mar;83(5):1463–1466. doi: 10.1073/pnas.83.5.1463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Yamamoto T., Davis C. G., Brown M. S., Schneider W. J., Casey M. L., Goldstein J. L., Russell D. W. The human LDL receptor: a cysteine-rich protein with multiple Alu sequences in its mRNA. Cell. 1984 Nov;39(1):27–38. doi: 10.1016/0092-8674(84)90188-0. [DOI] [PubMed] [Google Scholar]
  61. Yankner B. A., Shooter E. M. Nerve growth factor in the nucleus: interaction with receptors on the nuclear membrane. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1269–1273. doi: 10.1073/pnas.76.3.1269. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES