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. 1980 Nov;66(5):1081–1093. doi: 10.1172/JCI109937

Binding, internalization, and lysosomal association of 125I-glucagon in isolated rat hepatocytes. A quantitative electron microscope autoradiographic study.

P Barazzone, P Gorden, J L Carpentier, L Orci, P Freychet, B Canivet
PMCID: PMC371546  PMID: 7430344

Abstract

When 125I-glucagon is incubated with freshly isolated rat hepatocytes and studied by quantitative electron microscope autoradiography, the labeled material localizes to the plasma membrane of the cell at early times of incubation of 20 degrees C; at later times of incubation at 20 degrees C, there is little further translocation of the labeled ligand. When incubations are carried out at 37 degrees C, the labeled material is progressively internalized by the cell after a brief delay. When the internalized radioactivity is further analyzed, it is found to associate preferentially with lysosome-like structures. When the cell-associated radioactivity is extracted, there is degradation of the ligand in incubations carried out at 37 degrees C. The events involved in the interaction of 125I-glucagon with the hepatocyte are similar to those previously described for labeled insulin in this cell. The process of binding, internalization, and lysosomal association appears to be a general process related to many polypeptide hormones and growth factors, and may represent the mechanism by which the specific binding of the ligand to the cell surface mediates the degradation of the ligand and the loss of its surface receptor.

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Selected References

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

  1. Amsterdam A., Nimrod A., Lamprecht S. A., Burstein Y., Lindner H. R. Internalization and degradation of receptor-bound hCG in granulosa cell cultures. Am J Physiol. 1979 Feb;236(2):E129–E138. doi: 10.1152/ajpendo.1979.236.2.E129. [DOI] [PubMed] [Google Scholar]
  2. Anderson R. G., Brown M. S., Goldstein J. L. Role of the coated endocytic vesicle in the uptake of receptor-bound low density lipoprotein in human fibroblasts. Cell. 1977 Mar;10(3):351–364. doi: 10.1016/0092-8674(77)90022-8. [DOI] [PubMed] [Google Scholar]
  3. Bergeron J. J., Levine G., Sikstrom R., O'Shaughnessy D., Kopriwa B., Nadler N. J., Posner B. I. Polypeptide hormone binding sites in vivo: initial localization of 125I-labeled insulin to hepatocyte plasmalemma as visualized by electron microscope radioautography. Proc Natl Acad Sci U S A. 1977 Nov;74(11):5051–5055. doi: 10.1073/pnas.74.11.5051. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bergeron J. J., Sikstrom R., Hand A. R., Posner B. I. Binding and uptake of 125I-insulin into rat liver hepatocytes and endothelium. An in vivo radioautographic study. J Cell Biol. 1979 Feb;80(2):427–443. doi: 10.1083/jcb.80.2.427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bhathena S. J., Voyles N. R., Smith S., Recant L. Decreased glucagon receptors in diabetic rat hepatocytes. Evidence for regulation of glucagon receptors by hyperglucagonemia. J Clin Invest. 1978 Jun;61(6):1488–1497. doi: 10.1172/JCI109069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. CARO L. G., VAN TUBERGEN R. P., KOLB J. A. High-resolution autoradiography. I. Methods. J Cell Biol. 1962 Nov;15:173–188. doi: 10.1083/jcb.15.2.173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Carpentier J. L., Gorden P., Amherdt M., Van Obberghen E., Kahn C. R., Orci L. 125I-insulin binding to cultured human lymphocytes. Initial localization and fate of hormone determined by quantitative electron microscopic autoradiography. J Clin Invest. 1978 Apr;61(4):1057–1070. doi: 10.1172/JCI109005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Carpentier J. L., Gorden P., Barazzone P., Freychet P., Le Cam A., Orci L. Intracellular localization of 125I-labeled insulin in hepatocytes from intact rat liver. Proc Natl Acad Sci U S A. 1979 Jun;76(6):2803–2807. doi: 10.1073/pnas.76.6.2803. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Carpentier J. L., Gorden P., Freychet P., Le Cam A., Orci L. Lysosomal association of internalized 125I-insulin in isolated rat hepatocytes. Direct demonstration by quantitative electron microscopic autoradiography. J Clin Invest. 1979 Jun;63(6):1249–1261. doi: 10.1172/JCI109420. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Carpentier J. L., Gorden P., Freychet P., LeCam A., Orci L. Relationship of binding to internatlization of 125I-insulin in isolated rat hepatocytes. Diabetologia. 1979 Dec;17(6):379–384. doi: 10.1007/BF01236273. [DOI] [PubMed] [Google Scholar]
  11. Carpentier J. L., Gorden P., Goldstein J. L., Anderson R. G., Brown M. S., Orci L. Binding and internalization of 125I-LDL in normal and mutant human fibroblasts. A quantitative autoradiographic study. Exp Cell Res. 1979 Jun;121(1):135–142. doi: 10.1016/0014-4827(79)90453-1. [DOI] [PubMed] [Google Scholar]
  12. Chen T. T., Abel J. H., Jr, McClellan M. C., Sawyer H. R., Diekman M. A., Niswender G. D. Localization of gonadotropic hormones in lysosomes of ovine luteal cells. Cytobiologie. 1977 Apr;14(3):412–420. [PubMed] [Google Scholar]
  13. Drochmans P., Wanson J. C., Mosselmans R. Isolation and subfractionation on ficoll gradients of adult rat hepatocytes. Size, morphology, and biochemical characteristics of cell fractions. J Cell Biol. 1975 Jul;66(1):1–22. doi: 10.1083/jcb.66.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Exton J. H., Park C. R. The role of cyclic AMP in the control of liver metabolism. Adv Enzyme Regul. 1968;6:391–407. doi: 10.1016/0065-2571(68)90024-1. [DOI] [PubMed] [Google Scholar]
  15. Fouchereau-Peron M., Rançon F., Freychet P., Rosselin G. Effect of feeding and fasting on the early steps of glucagon action in isolated rat liver cells. Endocrinology. 1976 Mar;98(3):755–760. doi: 10.1210/endo-98-3-755. [DOI] [PubMed] [Google Scholar]
  16. Freychet P., Kahn R., Roth J., Neville D. M., Jr Insulin interactions with liver plasma membranes. Independence of binding of the hormone and its degradation. J Biol Chem. 1972 Jun 25;247(12):3953–3961. [PubMed] [Google Scholar]
  17. Gavin J. R., 3rd, Roth J., Neville D. M., Jr, de Meyts P., Buell D. N. Insulin-dependent regulation of insulin receptor concentrations: a direct demonstration in cell culture. Proc Natl Acad Sci U S A. 1974 Jan;71(1):84–88. doi: 10.1073/pnas.71.1.84. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gorden P., Carpentier J. L., Cohen S., Orci L. Epidermal growth factor: morphological demonstration of binding, internalization, and lysosomal association in human fibroblasts. Proc Natl Acad Sci U S A. 1978 Oct;75(10):5025–5029. doi: 10.1073/pnas.75.10.5025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gorden P., Carpentier J. L., Cohen S., Orci L. Interaction du facteur de croissance épidermique avec des fibroblastes humains en culture : une étude autoradiographique à l'échelon ultrastructural. C R Acad Sci Hebd Seances Acad Sci D. 1978 May 22;286(20):1471–1474. [PubMed] [Google Scholar]
  20. Gorden P., Carpentier J. L., Freychet P., LeCam A., Orci L. Intracellular translocation of iodine-125-labeled insulin: direct demonstration in isolated hepatocytes. Science. 1978 May 19;200(4343):782–785. doi: 10.1126/science.644321. [DOI] [PubMed] [Google Scholar]
  21. Gordon P., Carpentier J. L., Van Obberghen E., Barazzone P., Roth J., Orci L. Insulin-induced receptor loss in the cultured human lymphocyte: quantitative morphological perturbations in the cell and plasma membrane. J Cell Sci. 1979 Oct;39:77–88. doi: 10.1242/jcs.39.1.77. [DOI] [PubMed] [Google Scholar]
  22. Haigler H. T., McKanna J. A., Cohen S. Direct visualization of the binding and internalization of a ferritin conjugate of epidermal growth factor in human carcinoma cells A-431. J Cell Biol. 1979 May;81(2):382–395. doi: 10.1083/jcb.81.2.382. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Haigler H. T., Willingham M. C., Pastan I. Inhibitors of 125I-epidermal growth factor internalization. Biochem Biophys Res Commun. 1980 May 30;94(2):630–637. doi: 10.1016/0006-291x(80)91279-6. [DOI] [PubMed] [Google Scholar]
  24. Le Cam A., Freychet P. Effect of catecholamines on amino acid transport in isolated rat hepatocytes. Endocrinology. 1978 Feb;102(2):379–385. doi: 10.1210/endo-102-2-379. [DOI] [PubMed] [Google Scholar]
  25. Le Cam A., Freychet P. Effect of glucocorticoids on amino acid transport in isolated rat hepatocytes. Mol Cell Endocrinol. 1977 Dec;9(2):205–214. doi: 10.1016/0303-7207(77)90121-6. [DOI] [PubMed] [Google Scholar]
  26. Le Cam A., Freychet P. Effect of insulin on amino acid transport in isolated rat hepatocytes. Diabetologia. 1978 Aug;15(2):117–123. doi: 10.1007/BF00422256. [DOI] [PubMed] [Google Scholar]
  27. Le Cam A., Freychet P. Glucagon stimulates the A system for neutral amino acid transport in isolated hepatocytes of adult rat. Biochem Biophys Res Commun. 1976 Oct 4;72(3):893–901. doi: 10.1016/s0006-291x(76)80216-1. [DOI] [PubMed] [Google Scholar]
  28. Le Cam A., Guillouzo A., Freychet P. Ultrastructual and biochemical studies of isolated adult rat hepatocytes prepared under hypoxic conditions. Cryopreservation of hepatocytes. Exp Cell Res. 1976 Mar 15;98(2):382–395. doi: 10.1016/0014-4827(76)90448-1. [DOI] [PubMed] [Google Scholar]
  29. Pickup J. C., Keen H. Continuous subcutaneous insulin infusion: a developing tool in diabetes research. Diabetologia. 1980 Jan;18(1):1–4. doi: 10.1007/BF01228294. [DOI] [PubMed] [Google Scholar]
  30. Salpeter M. M., Fertuck H. C., Salpeter E. E. Resolution in electron microscope autoradiography. III. Iodine-125, the effect of heavy metal staining, and a reassessment of critical parameters. J Cell Biol. 1977 Jan;72(1):161–173. doi: 10.1083/jcb.72.1.161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Seglen P. O. Preparation of isolated rat liver cells. Methods Cell Biol. 1976;13:29–83. doi: 10.1016/s0091-679x(08)61797-5. [DOI] [PubMed] [Google Scholar]
  32. Silverstein S. C., Steinman R. M., Cohn Z. A. Endocytosis. Annu Rev Biochem. 1977;46:669–722. doi: 10.1146/annurev.bi.46.070177.003321. [DOI] [PubMed] [Google Scholar]
  33. Srikant C. B., Freeman D., McCorkle K., Unger R. H. Binding and biologic activity of glucagon in liver cell membranes of chronically hyperglucagonemic rats. J Biol Chem. 1977 Nov 10;252(21):7434–7438. [PubMed] [Google Scholar]
  34. Stäubli W., Schweizer W., Suter J., Weibel E. R. The proliferative response of hepatic peroxidomes of neonatal rats to treatment with SU-13 437 (nafenopin). J Cell Biol. 1977 Sep;74(3):665–689. doi: 10.1083/jcb.74.3.665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Terris S., Steiner D. F. Binding and degradation of 125I-insulin by rat hepatocytes. J Biol Chem. 1975 Nov 10;250(21):8389–8398. [PubMed] [Google Scholar]

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