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. 1993 Sep 2;122(6):1243–1252. doi: 10.1083/jcb.122.6.1243

Two steps of insulin receptor internalization depend on different domains of the beta-subunit [published erratum appears in J Cell Biol 1993 Nov;123(4):1047]

PMCID: PMC2119852  PMID: 8376461

Abstract

The internalization of signaling receptors such as the insulin receptor is a complex, multi-step process. The aim of the present work was to determine the various steps in internalization of the insulin receptor and to establish which receptor domains are implicated in each of these by the use of receptors possessing in vitro mutations. We find that kinase activation and autophosphorylation of all three regulatory tyrosines 1146, 1150, and 1151, but not tyrosines 1316 and 1322 in the COOH-terminal domain, are required for the ligand-specific stage of the internalization process; i.e., the surface redistribution of the receptor from microvilli where initial binding occurs to the nonvillous domain of the cell. Early intracellular steps in insulin signal transduction involving the activation of phosphatidylinositol 3'-kinase are not required for this redistribution. The second step of internalization consists in the anchoring of the receptors in clathrin- coated pits. In contrast to the first ligand specific step, this step is common to many receptors including those for transport proteins and occurs in the absence of kinase activation and receptor autophosphorylation, but requires a juxta-membrane cytoplasmic segment of the beta-subunit of the receptor including a NPXY sequence. Thus, there are two independent mechanisms controlling insulin receptor internalization which depend on different domains of the beta-subunit.

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Selected References

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  1. Ahle S., Mann A., Eichelsbacher U., Ungewickell E. Structural relationships between clathrin assembly proteins from the Golgi and the plasma membrane. EMBO J. 1988 Apr;7(4):919–929. doi: 10.1002/j.1460-2075.1988.tb02897.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Backer J. M., Kahn C. R., Cahill D. A., Ullrich A., White M. F. Receptor-mediated internalization of insulin requires a 12-amino acid sequence in the juxtamembrane region of the insulin receptor beta-subunit. J Biol Chem. 1990 Sep 25;265(27):16450–16454. [PubMed] [Google Scholar]
  3. Backer J. M., Kahn C. R., White M. F. Tyrosine phosphorylation of the insulin receptor during insulin-stimulated internalization in rat hepatoma cells. J Biol Chem. 1989 Jan 25;264(3):1694–1701. [PubMed] [Google Scholar]
  4. Backer J. M., Kahn C. R., White M. F. Tyrosine phosphorylation of the insulin receptor is not required for receptor internalization: studies in 2,4-dinitrophenol-treated cells. Proc Natl Acad Sci U S A. 1989 May;86(9):3209–3213. doi: 10.1073/pnas.86.9.3209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Backer J. M., Myers M. G., Jr, Shoelson S. E., Chin D. J., Sun X. J., Miralpeix M., Hu P., Margolis B., Skolnik E. Y., Schlessinger J. Phosphatidylinositol 3'-kinase is activated by association with IRS-1 during insulin stimulation. EMBO J. 1992 Sep;11(9):3469–3479. doi: 10.1002/j.1460-2075.1992.tb05426.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Backer J. M., Shoelson S. E., Haring E., White M. F. Insulin receptors internalize by a rapid, saturable pathway requiring receptor autophosphorylation and an intact juxtamembrane region. J Cell Biol. 1991 Dec;115(6):1535–1545. doi: 10.1083/jcb.115.6.1535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Backer J. M., Shoelson S. E., Weiss M. A., Hua Q. X., Cheatham R. B., Haring E., Cahill D. C., White M. F. The insulin receptor juxtamembrane region contains two independent tyrosine/beta-turn internalization signals. J Cell Biol. 1992 Aug;118(4):831–839. doi: 10.1083/jcb.118.4.831. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bansal A., Gierasch L. M. The NPXY internalization signal of the LDL receptor adopts a reverse-turn conformation. Cell. 1991 Dec 20;67(6):1195–1201. doi: 10.1016/0092-8674(91)90295-a. [DOI] [PubMed] [Google Scholar]
  9. Barazzone P., Carpentier J. L., Gorden P., Van Obberghen E., Orci L. Polar redistribution of 125I-labelled insulin on the plasma membrane of cultured human lymphocytes. Nature. 1980 Jul 24;286(5771):401–403. doi: 10.1038/286401a0. [DOI] [PubMed] [Google Scholar]
  10. Beltzer J. P., Spiess M. In vitro binding of the asialoglycoprotein receptor to the beta adaptin of plasma membrane coated vesicles. EMBO J. 1991 Dec;10(12):3735–3742. doi: 10.1002/j.1460-2075.1991.tb04942.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Berhanu P., Ibrahim-Schneck R. H., Anderson C., Wood W. M. The NPEY sequence is not necessary for endocytosis and processing of insulin-receptor complexes. Mol Endocrinol. 1991 Dec;5(12):1827–1835. doi: 10.1210/mend-5-12-1827. [DOI] [PubMed] [Google Scholar]
  12. Bourguignon L. Y., Singer S. J. Transmembrane interactions and the mechanism of capping of surface receptors by their specific ligands. Proc Natl Acad Sci U S A. 1977 Nov;74(11):5031–5035. doi: 10.1073/pnas.74.11.5031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Canfield W. M., Johnson K. F., Ye R. D., Gregory W., Kornfeld S. Localization of the signal for rapid internalization of the bovine cation-independent mannose 6-phosphate/insulin-like growth factor-II receptor to amino acids 24-29 of the cytoplasmic tail. J Biol Chem. 1991 Mar 25;266(9):5682–5688. [PubMed] [Google Scholar]
  15. Carpentier J. L., Dayer J. M., Lang U., Silverman R., Orci L., Gorden P. Down-regulation and recycling of insulin receptors. Effect of monensin on IM-9 lymphocytes and U-937 monocyte-like cells. J Biol Chem. 1984 Nov 25;259(22):14190–14195. [PubMed] [Google Scholar]
  16. Carpentier J. L., Fehlmann M., Van Obberghen E., Gorden P., Orci L. Redistribution of 125I-insulin on the surface of rat hepatocytes as a function of dissociation time. Diabetes. 1985 Oct;34(10):1002–1007. doi: 10.2337/diab.34.10.1002. [DOI] [PubMed] [Google Scholar]
  17. Carpentier J. L., Gazzano H., Van Obberghen E., Fehlmann M., Freychet P., Orci L. Internalization and recycling of 125I-photoreactive insulin-receptor complexes in hepatocytes in primary culture. Mol Cell Endocrinol. 1986 Oct;47(3):243–255. doi: 10.1016/0303-7207(86)90118-8. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Carpentier J. L., Gorden P., Anderson R. G., Goldstein J. L., Brown M. S., Cohen S., Orci L. Co-localization of 125I-epidermal growth factor and ferritin-low density lipoprotein in coated pits: a quantitative electron microscopic study in normal and mutant human fibroblasts. J Cell Biol. 1982 Oct;95(1):73–77. doi: 10.1083/jcb.95.1.73. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Carpentier J. L., Paccaud J. P., Gorden P., Rutter W. J., Orci L. Insulin-induced surface redistribution regulates internalization of the insulin receptor and requires its autophosphorylation. Proc Natl Acad Sci U S A. 1992 Jan 1;89(1):162–166. doi: 10.1073/pnas.89.1.162. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Carpentier J. L. The cell biology of the insulin receptor. Diabetologia. 1989 Sep;32(9):627–635. doi: 10.1007/BF00274248. [DOI] [PubMed] [Google Scholar]
  22. Carpentier J. L., Van Obberghen E., Gorden P., Orci L. Surface redistribution of 125I-insulin in cultured human lymphocytes. J Cell Biol. 1981 Oct;91(1):17–25. doi: 10.1083/jcb.91.1.17. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Chen W. J., Goldstein J. L., Brown M. S. NPXY, a sequence often found in cytoplasmic tails, is required for coated pit-mediated internalization of the low density lipoprotein receptor. J Biol Chem. 1990 Feb 25;265(6):3116–3123. [PubMed] [Google Scholar]
  24. Chou C. K., Dull T. J., Russell D. S., Gherzi R., Lebwohl D., Ullrich A., Rosen O. M. Human insulin receptors mutated at the ATP-binding site lack protein tyrosine kinase activity and fail to mediate postreceptor effects of insulin. J Biol Chem. 1987 Feb 5;262(4):1842–1847. [PubMed] [Google Scholar]
  25. Collawn J. F., Stangel M., Kuhn L. A., Esekogwu V., Jing S. Q., Trowbridge I. S., Tainer J. A. Transferrin receptor internalization sequence YXRF implicates a tight turn as the structural recognition motif for endocytosis. Cell. 1990 Nov 30;63(5):1061–1072. doi: 10.1016/0092-8674(90)90509-d. [DOI] [PubMed] [Google Scholar]
  26. Davis C. G., van Driel I. R., Russell D. W., Brown M. S., Goldstein J. L. The low density lipoprotein receptor. Identification of amino acids in cytoplasmic domain required for rapid endocytosis. J Biol Chem. 1987 Mar 25;262(9):4075–4082. [PubMed] [Google Scholar]
  27. Eberle W., Sander C., Klaus W., Schmidt B., von Figura K., Peters C. The essential tyrosine of the internalization signal in lysosomal acid phosphatase is part of a beta turn. Cell. 1991 Dec 20;67(6):1203–1209. doi: 10.1016/0092-8674(91)90296-b. [DOI] [PubMed] [Google Scholar]
  28. Fan J. Y., Carpentier J. L., Gorden P., Van Obberghen E., Blackett N. M., Grunfeld C., Orci L. Receptor-mediated endocytosis of insulin: role of microvilli, coated pits, and coated vesicles. Proc Natl Acad Sci U S A. 1982 Dec;79(24):7788–7791. doi: 10.1073/pnas.79.24.7788. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Fuhrer C., Geffen I., Spiess M. Endocytosis of the ASGP receptor H1 is reduced by mutation of tyrosine-5 but still occurs via coated pits. J Cell Biol. 1991 Aug;114(3):423–431. doi: 10.1083/jcb.114.3.423. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Gironès N., Alverez E., Seth A., Lin I. M., Latour D. A., Davis R. J. Mutational analysis of the cytoplasmic tail of the human transferrin receptor. Identification of a sub-domain that is required for rapid endocytosis. J Biol Chem. 1991 Oct 5;266(28):19006–19012. [PubMed] [Google Scholar]
  31. Hari J., Roth R. A. Defective internalization of insulin and its receptor in cells expressing mutated insulin receptors lacking kinase activity. J Biol Chem. 1987 Nov 15;262(32):15341–15344. [PubMed] [Google Scholar]
  32. Herrera R., Petruzzelli L. M., Rosen O. M. Antibodies to deduced sequences of the insulin receptor distinguish conserved and nonconserved regions in the IGF-I receptor. J Biol Chem. 1986 Feb 25;261(6):2489–2491. [PubMed] [Google Scholar]
  33. Heuser J. Effects of cytoplasmic acidification on clathrin lattice morphology. J Cell Biol. 1989 Feb;108(2):401–411. doi: 10.1083/jcb.108.2.401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Jadot M., Canfield W. M., Gregory W., Kornfeld S. Characterization of the signal for rapid internalization of the bovine mannose 6-phosphate/insulin-like growth factor-II receptor. J Biol Chem. 1992 Jun 5;267(16):11069–11077. [PubMed] [Google Scholar]
  35. Kahn C. R., White M. F. The insulin receptor and the molecular mechanism of insulin action. J Clin Invest. 1988 Oct;82(4):1151–1156. doi: 10.1172/JCI113711. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Ktistakis N. T., Thomas D., Roth M. G. Characteristics of the tyrosine recognition signal for internalization of transmembrane surface glycoproteins. J Cell Biol. 1990 Oct;111(4):1393–1407. doi: 10.1083/jcb.111.4.1393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Lazarovits J., Roth M. A single amino acid change in the cytoplasmic domain allows the influenza virus hemagglutinin to be endocytosed through coated pits. Cell. 1988 Jun 3;53(5):743–752. doi: 10.1016/0092-8674(88)90092-x. [DOI] [PubMed] [Google Scholar]
  38. Lehmann L. E., Eberle W., Krull S., Prill V., Schmidt B., Sander C., von Figura K., Peters C. The internalization signal in the cytoplasmic tail of lysosomal acid phosphatase consists of the hexapeptide PGYRHV. EMBO J. 1992 Dec;11(12):4391–4399. doi: 10.1002/j.1460-2075.1992.tb05539.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Lin H. C., Moore M. S., Sanan D. A., Anderson R. G. Reconstitution of clathrin-coated pit budding from plasma membranes. J Cell Biol. 1991 Sep;114(5):881–891. doi: 10.1083/jcb.114.5.881. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Lobel P., Fujimoto K., Ye R. D., Griffiths G., Kornfeld S. Mutations in the cytoplasmic domain of the 275 kd mannose 6-phosphate receptor differentially alter lysosomal enzyme sorting and endocytosis. Cell. 1989 Jun 2;57(5):787–796. doi: 10.1016/0092-8674(89)90793-9. [DOI] [PubMed] [Google Scholar]
  41. Majercik M. H., Bourguignon L. Y. Insulin receptor capping and its correlation with calmodulin-dependent myosin light chain kinase. J Cell Physiol. 1985 Sep;124(3):403–410. doi: 10.1002/jcp.1041240308. [DOI] [PubMed] [Google Scholar]
  42. McClain D. A., Maegawa H., Lee J., Dull T. J., Ulrich A., Olefsky J. M. A mutant insulin receptor with defective tyrosine kinase displays no biologic activity and does not undergo endocytosis. J Biol Chem. 1987 Oct 25;262(30):14663–14671. [PubMed] [Google Scholar]
  43. Moore M. S., Mahaffey D. T., Brodsky F. M., Anderson R. G. Assembly of clathrin-coated pits onto purified plasma membranes. Science. 1987 May 1;236(4801):558–563. doi: 10.1126/science.2883727. [DOI] [PubMed] [Google Scholar]
  44. Myers M. G., Backer J. M., Siddle K., White M. F. The insulin receptor functions normally in Chinese hamster ovary cells after truncation of the C terminus. J Biol Chem. 1991 Jun 5;266(16):10616–10623. [PubMed] [Google Scholar]
  45. Ohta Y., Stossel T. P., Hartwig J. H. Ligand-sensitive binding of actin-binding protein to immunoglobulin G Fc receptor I (Fc gamma RI). Cell. 1991 Oct 18;67(2):275–282. doi: 10.1016/0092-8674(91)90179-3. [DOI] [PubMed] [Google Scholar]
  46. Paccaud J. P., Siddle K., Carpentier J. L. Internalization of the human insulin receptor. The insulin-independent pathway. J Biol Chem. 1992 Jun 25;267(18):13101–13106. [PubMed] [Google Scholar]
  47. Pearse B. M. Receptors compete for adaptors found in plasma membrane coated pits. EMBO J. 1988 Nov;7(11):3331–3336. doi: 10.1002/j.1460-2075.1988.tb03204.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Pearse B. M., Robinson M. S. Clathrin, adaptors, and sorting. Annu Rev Cell Biol. 1990;6:151–171. doi: 10.1146/annurev.cb.06.110190.001055. [DOI] [PubMed] [Google Scholar]
  49. Perlman R., Bottaro D. P., White M. F., Kahn C. R. Conformational changes in the alpha- and beta-subunits of the insulin receptor identified by anti-peptide antibodies. J Biol Chem. 1989 May 25;264(15):8946–8950. [PubMed] [Google Scholar]
  50. Rajagopalan M., Neidigh J. L., McClain D. A. Amino acid sequences Gly-Pro-Leu-Tyr and Asn-Pro-Glu-Tyr in the submembranous domain of the insulin receptor are required for normal endocytosis. J Biol Chem. 1991 Dec 5;266(34):23068–23073. [PubMed] [Google Scholar]
  51. Reddy S. S., Lauris V., Kahn C. R. Insulin receptor function in fibroblasts from patients with leprechaunism. Differential alterations in binding, autophosphorylation, kinase activity, and receptor-mediated internalization. J Clin Invest. 1988 Oct;82(4):1359–1365. doi: 10.1172/JCI113739. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Reynet C., Caron M., Magré J., Cherqui G., Clauser E., Picard J., Capeau J. Mutation of tyrosine residues 1162 and 1163 of the insulin receptor affects hormone and receptor internalization. Mol Endocrinol. 1990 Feb;4(2):304–311. doi: 10.1210/mend-4-2-304. [DOI] [PubMed] [Google Scholar]
  53. Russell D. S., Gherzi R., Johnson E. L., Chou C. K., Rosen O. M. The protein-tyrosine kinase activity of the insulin receptor is necessary for insulin-mediated receptor down-regulation. J Biol Chem. 1987 Aug 25;262(24):11833–11840. [PubMed] [Google Scholar]
  54. Schlessinger J., Van Obberghen E., Kahn C. R. Insulin and antibodies against insulin receptor cap on the membrane of cultured human lymphocytes. Nature. 1980 Aug 14;286(5774):729–731. doi: 10.1038/286729a0. [DOI] [PubMed] [Google Scholar]
  55. Smith R. M., Seely B. L., Shah N., Olefsky J. M., Jarett L. Tyrosine kinase-defective insulin receptors undergo insulin-induced microaggregation but do not concentrate in coated pits. J Biol Chem. 1991 Sep 15;266(26):17522–17530. [PubMed] [Google Scholar]
  56. Sun X. J., Rothenberg P., Kahn C. R., Backer J. M., Araki E., Wilden P. A., Cahill D. A., Goldstein B. J., White M. F. Structure of the insulin receptor substrate IRS-1 defines a unique signal transduction protein. Nature. 1991 Jul 4;352(6330):73–77. doi: 10.1038/352073a0. [DOI] [PubMed] [Google Scholar]
  57. Thies R. S., Ullrich A., McClain D. A. Augmented mitogenesis and impaired metabolic signaling mediated by a truncated insulin receptor. J Biol Chem. 1989 Aug 5;264(22):12820–12825. [PubMed] [Google Scholar]
  58. Thies R. S., Webster N. J., McClain D. A. A domain of the insulin receptor required for endocytosis in rat fibroblasts. J Biol Chem. 1990 Jun 15;265(17):10132–10137. [PubMed] [Google Scholar]
  59. Trischitta V., Wong K. Y., Brunetti A., Scalisi R., Vigneri R., Goldfine I. D. Endocytosis, recycling, and degradation of the insulin receptor. Studies with monoclonal antireceptor antibodies that do not activate receptor kinase. J Biol Chem. 1989 Mar 25;264(9):5041–5046. [PubMed] [Google Scholar]
  60. 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]
  61. White M. F., Livingston J. N., Backer J. M., Lauris V., Dull T. J., Ullrich A., Kahn C. R. Mutation of the insulin receptor at tyrosine 960 inhibits signal transmission but does not affect its tyrosine kinase activity. Cell. 1988 Aug 26;54(5):641–649. doi: 10.1016/s0092-8674(88)80008-4. [DOI] [PubMed] [Google Scholar]
  62. White M. F., Maron R., Kahn C. R. Insulin rapidly stimulates tyrosine phosphorylation of a Mr-185,000 protein in intact cells. Nature. 1985 Nov 14;318(6042):183–186. doi: 10.1038/318183a0. [DOI] [PubMed] [Google Scholar]
  63. White M. F., Stegmann E. W., Dull T. J., Ullrich A., Kahn C. R. Characterization of an endogenous substrate of the insulin receptor in cultured cells. J Biol Chem. 1987 Jul 15;262(20):9769–9777. [PubMed] [Google Scholar]
  64. Wilden P. A., Backer J. M., Kahn C. R., Cahill D. A., Schroeder G. J., White M. F. The insulin receptor with phenylalanine replacing tyrosine-1146 provides evidence for separate signals regulating cellular metabolism and growth. Proc Natl Acad Sci U S A. 1990 May;87(9):3358–3362. doi: 10.1073/pnas.87.9.3358. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. 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]

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