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. 1986 Mar 1;102(3):878–888. doi: 10.1083/jcb.102.3.878

Receptor-bound somatostatin and epidermal growth factor are processed differently in GH4C1 rat pituitary cells

PMCID: PMC2114115  PMID: 2869047

Abstract

GH4C1 cells, a clonal strain of rat pituitary tumor cells, have high- affinity, functional receptors for the inhibitory hypothalamic peptide somatostatin (SRIF) and for epidermal growth factor (EGF). In this study we have examined the events that follow the initial binding of SRIF to its specific plasma membrane receptors in GH4C1 cells and have compared the processing of receptor-bound SRIF with that of EGF. When cells were incubated with [125I-Tyr1]SRIF at temperatures ranging from 4 to 37 degrees C, greater than 80% of the specifically bound peptide was removed by extraction with 0.2 M acetic acid, 0.5 M NaCl, pH 2.5. In contrast, the subcellular distribution of receptor-bound 125I-EGF was temperature dependent. Whereas greater than 95% of specifically bound 125I-EGF was removed by acid treatment after a 4 degrees C binding incubation, less than 10% was removed when the binding reaction was performed at 22 or 37 degrees C. In pulse-chase experiments, receptor-bound 125I-EGF was transferred from an acid-sensitive to an acid-resistant compartment with a half-time of 2 min at 37 degrees C. In contrast, the small amount of [125I-Tyr1]SRIF that was resistant to acid treatment did not increase during a 2-h chase incubation at 37 degrees C. Chromatographic analysis of the radioactivity released from cells during dissociation incubations at 37 degrees C showed that greater than 90% of prebound 125I-EGF was released as 125I-tyrosine, whereas prebound [125I-Tyr1]SRIF was released as a mixture of intact peptide (55%) and 125I-tyrosine (45%). Neither chloroquine (0.1 mM), ammonium chloride (20 mM), nor leupeptin (0.1 mg/ml) increased the amount of [125I-Tyr1]SRIF bound to cells at 37 degrees C. Furthermore, chloroquine and leupeptin did not alter the rate of dissociation or degradation of prebound [125I-Tyr1]SRIF. In contrast, these inhibitors increased the amount of cell-associated 125I-EGF during 37 degrees C binding incubations and decreased the subsequent rate of release of 125I-tyrosine. The results presented indicate that, as in other cell types, EGF underwent rapid receptor-mediated endocytosis in GH4C1 cells and was subsequently degraded in lysosomes. In contrast, SRIF remained at the cell surface for several hours although it elicits its biological effects within minutes. Furthermore, a constant fraction of the receptor-bound [125I-Tyr1]SRIF was degraded at the cell surface before dissociation. Therefore, after initial binding of [125I- Tyr1]SRIF and 125I-EGF to their specific membrane receptors, these peptides are processed very differently in GH4C1 cells.

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Selected References

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  1. Blanchard S. G., Chang K. J., Cuatrecasas P. Characterization of the association of tritiated enkephalin with neuroblastoma cells under conditions optimal for receptor down regulation. J Biol Chem. 1983 Jan 25;258(2):1092–1097. [PubMed] [Google Scholar]
  2. Böhlen P., Castillo F., Ling N., Guillemin R. Purification of peptides: an efficient procedure for the separation of peptides from amino acids and salt. Int J Pept Protein Res. 1980 Oct;16(4):306–310. doi: 10.1111/j.1399-3011.1980.tb02591.x. [DOI] [PubMed] [Google Scholar]
  3. Carpenter G., Cohen S. 125I-labeled human epidermal growth factor. Binding, internalization, and degradation in human fibroblasts. J Cell Biol. 1976 Oct;71(1):159–171. doi: 10.1083/jcb.71.1.159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Carpentier J. L., van Obberghen E., Gordon P., Orci L. Binding, membrane redistribution, internalization and lysosomal association of [125I]anti-insulin receptor antibody in IM-9-cultured human lymphocyte. A comparison with [125I]insulin. Exp Cell Res. 1981 Jul;134(1):81–92. doi: 10.1016/0014-4827(81)90465-1. [DOI] [PubMed] [Google Scholar]
  5. Ciechanover A., Schwartz A. L., Lodish H. F. The asialoglycoprotein receptor internalizes and recycles independently of the transferrin and insulin receptors. Cell. 1983 Jan;32(1):267–275. doi: 10.1016/0092-8674(83)90517-2. [DOI] [PubMed] [Google Scholar]
  6. Corin R. E., Bancroft F. C., Sonenberg M., Donner D. B. Binding and degradation of 125I-labeled insulin by a clonal line of rat pituitary tumor cells. Biochim Biophys Acta. 1983 Jul 14;762(4):503–511. doi: 10.1016/0167-4889(83)90053-8. [DOI] [PubMed] [Google Scholar]
  7. Dautry-Varsat A., Ciechanover A., Lodish H. F. pH and the recycling of transferrin during receptor-mediated endocytosis. Proc Natl Acad Sci U S A. 1983 Apr;80(8):2258–2262. doi: 10.1073/pnas.80.8.2258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dorflinger L. J., Schonbrunn A. Somatostatin inhibits vasoactive intestinal peptide-stimulated cyclic adenosine monophosphate accumulation in GH pituitary cells. Endocrinology. 1983 Nov;113(5):1541–1550. doi: 10.1210/endo-113-5-1541. [DOI] [PubMed] [Google Scholar]
  9. Duckworth W. C., Kitabchi A. E. Insulin metabolism and degradation. Endocr Rev. 1981 Spring;2(2):210–233. doi: 10.1210/edrv-2-2-210. [DOI] [PubMed] [Google Scholar]
  10. Friedman T. C., Orlowski M., Wilk S. Peptide-degrading enzymatic activities in GH3 cells and rat anterior pituitary homogenates. Endocrinology. 1984 Apr;114(4):1407–1412. doi: 10.1210/endo-114-4-1407. [DOI] [PubMed] [Google Scholar]
  11. Gliemann J., Sonne O. Binding and receptor-mediated degradation of insulin in adipocytes. J Biol Chem. 1978 Nov 10;253(21):7857–7863. [PubMed] [Google Scholar]
  12. Gorden P., Carpentier J. L., Fan J. Y., Orci L. Receptor mediated endocytosis of polypeptide hormones: mechanism and significance. Metabolism. 1982 Jul;31(7):664–669. doi: 10.1016/0026-0495(82)90196-2. [DOI] [PubMed] [Google Scholar]
  13. Haigler H. T., Maxfield F. R., Willingham M. C., Pastan I. Dansylcadaverine inhibits internalization of 125I-epidermal growth factor in BALB 3T3 cells. J Biol Chem. 1980 Feb 25;255(4):1239–1241. [PubMed] [Google Scholar]
  14. Halpern J., Hinkle P. M. Binding and internalization of epidermal growth factor by rat pituitary tumor cells. Mol Cell Endocrinol. 1983 Dec;33(2-3):183–196. doi: 10.1016/0303-7207(83)90166-1. [DOI] [PubMed] [Google Scholar]
  15. Hinkle P. M., Kinsella P. A. Rapid temperature-dependent transformation of the thyrotropin-releasing hormone-receptor complex in rat pituitary tumor cells. J Biol Chem. 1982 May 25;257(10):5462–5470. [PubMed] [Google Scholar]
  16. Kahn C. R., Baird K. L., Jarrett D. B., Flier J. S. Direct demonstration that receptor crosslinking or aggregation is important in insulin action. Proc Natl Acad Sci U S A. 1978 Sep;75(9):4209–4213. doi: 10.1073/pnas.75.9.4209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Koch B. D., Schonbrunn A. The somatostatin receptor is directly coupled to adenylate cyclase in GH4C1 pituitary cell membranes. Endocrinology. 1984 May;114(5):1784–1790. doi: 10.1210/endo-114-5-1784. [DOI] [PubMed] [Google Scholar]
  18. Law P. Y., Hom D. S., Loh H. H. Down-regulation of opiate receptor in neuroblastoma x glioma NG108-15 hybrid cells. Chloroquine promotes accumulation of tritiated enkephalin in the lysosomes. J Biol Chem. 1984 Apr 10;259(7):4096–4104. [PubMed] [Google Scholar]
  19. Mock E. J., Niswender G. D. Differences in the rates of internalization of 125I-labeled human chorionic gonadotropin, luteinizing hormone, and epidermal growth factor by ovine luteal cells. Endocrinology. 1983 Jul;113(1):259–264. doi: 10.1210/endo-113-1-259. [DOI] [PubMed] [Google Scholar]
  20. O'Connor-McCourt M., Hollenberg M. D. Receptors, acceptors, and the action of polypeptide hormones: illustrative studies with epidermal growth factor (urogastrone). Can J Biochem Cell Biol. 1983 Jul;61(7):670–682. doi: 10.1139/o83-085. [DOI] [PubMed] [Google Scholar]
  21. Olefsky J. M., Kobayashi M., Chang H. Interactions between insulin and its receptors after the initial binding event. Functional heterogeneity and relationships to insulin degradation. Diabetes. 1979 May;28(5):460–471. doi: 10.2337/diab.28.5.460. [DOI] [PubMed] [Google Scholar]
  22. Pastan I. H., Willingham M. C. Receptor-mediated endocytosis of hormones in cultured cells. Annu Rev Physiol. 1981;43:239–250. doi: 10.1146/annurev.ph.43.030181.001323. [DOI] [PubMed] [Google Scholar]
  23. Peterson G. L. A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal Biochem. 1977 Dec;83(2):346–356. doi: 10.1016/0003-2697(77)90043-4. [DOI] [PubMed] [Google Scholar]
  24. Reichlin S. Somatostatin (second of two parts). N Engl J Med. 1983 Dec 22;309(25):1556–1563. doi: 10.1056/NEJM198312223092506. [DOI] [PubMed] [Google Scholar]
  25. Reyl-Desmars F., Lewin M. J. Evidence for an intracellular somatostatin receptor in pancreas: a comparative study with reference to gastric mucosa. Biochem Biophys Res Commun. 1982 Dec 31;109(4):1324–1331. doi: 10.1016/0006-291x(82)91922-2. [DOI] [PubMed] [Google Scholar]
  26. Reyl F. J., Lewin M. J. Intracellular receptor for somatostatin in gastric mucosal cells: decomposition and reconstitution of somatostatin-stimulated phosphoprotein phosphatases. Proc Natl Acad Sci U S A. 1982 Feb;79(4):978–982. doi: 10.1073/pnas.79.4.978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Schlegel W., Wuarin F., Wollheim C. B., Zahnd G. R. Somatostatin lowers the cytosolic free Ca2+ concentration in clonal rat pituitary cells (GH3 cells). Cell Calcium. 1984 Jun;5(3):223–236. doi: 10.1016/0143-4160(84)90038-1. [DOI] [PubMed] [Google Scholar]
  28. Schonbrunn A., Dorflinger L. J., Koch B. D. Mechanisms of somatostatin action in pituitary cells. Adv Exp Med Biol. 1985;188:305–324. doi: 10.1007/978-1-4615-7886-4_17. [DOI] [PubMed] [Google Scholar]
  29. Schonbrunn A., Krasnoff M., Westendorf J. M., Tashjian A. H., Jr Epidermal growth factor and thyrotropin-releasing hormone act similarly on a clonal pituitary cell strain. Modulation of hormone production and inhbition of cell proliferation. J Cell Biol. 1980 Jun;85(3):786–797. doi: 10.1083/jcb.85.3.786. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Schonbrunn A., Rorstad O. P., Westendorf J. M., Martin J. B. Somatostatin analogs: correlation between receptor binding affinity and biological potency in GH pituitary cells. Endocrinology. 1983 Nov;113(5):1559–1567. doi: 10.1210/endo-113-5-1559. [DOI] [PubMed] [Google Scholar]
  31. Schonbrunn A., Tashjian H., Jr Characterization of functional receptors for somatostatin in rat pituitary cells in culture. J Biol Chem. 1978 Sep 25;253(18):6473–6483. [PubMed] [Google Scholar]
  32. Schreiber A. B., Lax I., Yarden Y., Eshhar Z., Schlessinger J. Monoclonal antibodies against receptor for epidermal growth factor induce early and delayed effects of epidermal growth factor. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7535–7539. doi: 10.1073/pnas.78.12.7535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Schreiber A. B., Libermann T. A., Lax I., Yarden Y., Schlessinger J. Biological role of epidermal growth factor-receptor clustering. Investigation with monoclonal anti-receptor antibodies. J Biol Chem. 1983 Jan 25;258(2):846–853. [PubMed] [Google Scholar]
  34. Sonne O., Gliemann J. The mechanism of receptor-mediated degradation of insulin in isolated rat adipocytes: indirect evidence for a non-lysosomal pathway. Mol Cell Endocrinol. 1983 Aug;31(2-3):315–331. doi: 10.1016/0303-7207(83)90157-0. [DOI] [PubMed] [Google Scholar]
  35. Stadel J. M., Strulovici B., Nambi P., Lavin T. N., Briggs M. M., Caron M. G., Lefkowitz R. J. Desensitization of the beta-adrenergic receptor of frog erythrocytes. Recovery and characterization of the down-regulated receptors in sequestered vesicles. J Biol Chem. 1983 Mar 10;258(5):3032–3038. [PubMed] [Google Scholar]
  36. Tashjian A. H., Jr Clonal strains of hormone-producing pituitary cells. Methods Enzymol. 1979;58:527–535. doi: 10.1016/s0076-6879(79)58167-1. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. Westendorf J. M., Schonbrunn A. Characterization of bombesin receptors in a rat pituitary cell line. J Biol Chem. 1983 Jun 25;258(12):7527–7535. [PubMed] [Google Scholar]

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