Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1991 Mar 1;112(5):853–861. doi: 10.1083/jcb.112.5.853

Mutagenesis of the human transferrin receptor: two cytoplasmic phenylalanines are required for efficient internalization and a second- site mutation is capable of reverting an internalization-defective phenotype

PMCID: PMC2288869  PMID: 1900298

Abstract

Site-specific mutagenesis has been used to define the sequences required for efficient internalization of the human transferrin receptor. It has previously been shown that the sole cytoplasmic tyrosine, at position 20, is required for efficient internalization. When two other cytoplasmic aromatic residues, the phenylalanines at positions 13 and 23, are substituted with alanines internalization is also reduced. The phenylalanine 23 mutation decreases the internalization rate constant approximately threefold, and mutation of phenylalanine 13 decreases it by approximately twofold. The mutation at position 23 has as serious an effect on internalization as substitution with a nonaromatic amino acid for the single tyrosine. These results demonstrate the importance of several aromatic amino acids in maintaining efficient internalization of the transferrin receptor. Substitution of a tyrosine at a second site, for a serine at position 34, within the cytoplasmic domain of a transferrin receptor with a nonaromatic amino acid at position 20, results in a complete reversion of the internalization-defective phenotype. This reversion is completely dependent upon a tyrosine, as phenylalanine substituted at position 34 does not revert the internalization-defective phenotype. This result demonstrates that a tyrosine placed outside of its native context can still function in the internalization of the transferrin receptor, suggesting a flexibility in surrounding sequences required for efficient internalization.

Full Text

The Full Text of this article is available as a PDF (1,021.8 KB).

Selected References

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

  1. Ajioka R. S., Kaplan J. Intracellular pools of transferrin receptors result from constitutive internalization of unoccupied receptors. Proc Natl Acad Sci U S A. 1986 Sep;83(17):6445–6449. doi: 10.1073/pnas.83.17.6445. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Alvarez E., Gironès N., Davis R. J. A point mutation in the cytoplasmic domain of the transferrin receptor inhibits endocytosis. Biochem J. 1990 Apr 1;267(1):31–35. doi: 10.1042/bj2670031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Breitfeld P. P., Casanova J. E., McKinnon W. C., Mostov K. E. Deletions in the cytoplasmic domain of the polymeric immunoglobulin receptor differentially affect endocytotic rate and postendocytotic traffic. J Biol Chem. 1990 Aug 15;265(23):13750–13757. [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  6. Chou P. Y., Fasman G. D. Prediction of the secondary structure of proteins from their amino acid sequence. Adv Enzymol Relat Areas Mol Biol. 1978;47:45–148. doi: 10.1002/9780470122921.ch2. [DOI] [PubMed] [Google Scholar]
  7. Davis C. G., Lehrman M. A., Russell D. W., Anderson R. G., Brown M. S., Goldstein J. L. The J.D. mutation in familial hypercholesterolemia: amino acid substitution in cytoplasmic domain impedes internalization of LDL receptors. Cell. 1986 Apr 11;45(1):15–24. doi: 10.1016/0092-8674(86)90533-7. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Dickson R. B., Willingham M. C., Pastan I. alpha 2-macroglobulin adsorbed to colloidal gold: a new probe in the study of receptor-mediated endocytosis. J Cell Biol. 1981 Apr;89(1):29–34. doi: 10.1083/jcb.89.1.29. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Garnier J., Osguthorpe D. J., Robson B. Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. J Mol Biol. 1978 Mar 25;120(1):97–120. doi: 10.1016/0022-2836(78)90297-8. [DOI] [PubMed] [Google Scholar]
  11. Gibrat J. F., Garnier J., Robson B. Further developments of protein secondary structure prediction using information theory. New parameters and consideration of residue pairs. J Mol Biol. 1987 Dec 5;198(3):425–443. doi: 10.1016/0022-2836(87)90292-0. [DOI] [PubMed] [Google Scholar]
  12. Glickman J. N., Conibear E., Pearse B. M. Specificity of binding of clathrin adaptors to signals on the mannose-6-phosphate/insulin-like growth factor II receptor. EMBO J. 1989 Apr;8(4):1041–1047. doi: 10.1002/j.1460-2075.1989.tb03471.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Hopkins C. R. The appearance and internalization of transferrin receptors at the margins of spreading human tumor cells. Cell. 1985 Jan;40(1):199–208. doi: 10.1016/0092-8674(85)90323-x. [DOI] [PubMed] [Google Scholar]
  15. Hopkins C. R., Trowbridge I. S. Internalization and processing of transferrin and the transferrin receptor in human carcinoma A431 cells. J Cell Biol. 1983 Aug;97(2):508–521. doi: 10.1083/jcb.97.2.508. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Jameson B. A., Wolf H. The antigenic index: a novel algorithm for predicting antigenic determinants. Comput Appl Biosci. 1988 Mar;4(1):181–186. doi: 10.1093/bioinformatics/4.1.181. [DOI] [PubMed] [Google Scholar]
  17. Jing S. Q., Spencer T., Miller K., Hopkins C., Trowbridge I. S. Role of the human transferrin receptor cytoplasmic domain in endocytosis: localization of a specific signal sequence for internalization. J Cell Biol. 1990 Feb;110(2):283–294. doi: 10.1083/jcb.110.2.283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. 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]
  22. Maxfield F. R., Scheraga H. A. Status of empirical methods for the prediction of protein backbone topography. Biochemistry. 1976 Nov 16;15(23):5138–5153. doi: 10.1021/bi00668a030. [DOI] [PubMed] [Google Scholar]
  23. McClelland A., Kühn L. C., Ruddle F. H. The human transferrin receptor gene: genomic organization, and the complete primary structure of the receptor deduced from a cDNA sequence. Cell. 1984 Dec;39(2 Pt 1):267–274. doi: 10.1016/0092-8674(84)90004-7. [DOI] [PubMed] [Google Scholar]
  24. McGraw T. E., Dunn K. W., Maxfield F. R. Phorbol ester treatment increases the exocytic rate of the transferrin receptor recycling pathway independent of serine-24 phosphorylation. J Cell Biol. 1988 Apr;106(4):1061–1066. doi: 10.1083/jcb.106.4.1061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. McGraw T. E., Greenfield L., Maxfield F. R. Functional expression of the human transferrin receptor cDNA in Chinese hamster ovary cells deficient in endogenous transferrin receptor. J Cell Biol. 1987 Jul;105(1):207–214. doi: 10.1083/jcb.105.1.207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. McGraw T. E., Maxfield F. R. Human transferrin receptor internalization is partially dependent upon an aromatic amino acid on the cytoplasmic domain. Cell Regul. 1990 Mar;1(4):369–377. doi: 10.1091/mbc.1.4.369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Rothenberger S., Iacopetta B. J., Kühn L. C. Endocytosis of the transferrin receptor requires the cytoplasmic domain but not its phosphorylation site. Cell. 1987 May 8;49(3):423–431. doi: 10.1016/0092-8674(87)90295-9. [DOI] [PubMed] [Google Scholar]
  29. Sandvig K., Olsnes S., Petersen O. W., Van Deurs B. Control of coated-pit function by cytoplasmic pH. Methods Cell Biol. 1989;32:365–382. doi: 10.1016/s0091-679x(08)61181-4. [DOI] [PubMed] [Google Scholar]
  30. Verrey F., Gilbert T., Mellow T., Proulx G., Drickamer K. Endocytosis via coated pits mediated by glycoprotein receptor in which the cytoplasmic tail is replaced by unrelated sequences. Cell Regul. 1990 May;1(6):471–486. doi: 10.1091/mbc.1.6.471. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Wiley H. S., Cunningham D. D. The endocytotic rate constant. A cellular parameter for quantitating receptor-mediated endocytosis. J Biol Chem. 1982 Apr 25;257(8):4222–4229. [PubMed] [Google Scholar]
  32. Willingham M. C., Pastan I. H., Sahagian G. G., Jourdian G. W., Neufeld E. F. Morphologic study of the internalization of a lysosomal enzyme by the mannose 6-phosphate receptor in cultured Chinese hamster ovary cells. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6967–6971. doi: 10.1073/pnas.78.11.6967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. von Heijne G. Signal sequences. The limits of variation. J Mol Biol. 1985 Jul 5;184(1):99–105. doi: 10.1016/0022-2836(85)90046-4. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES