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. 1984 Mar;3(3):531–537. doi: 10.1002/j.1460-2075.1984.tb01842.x

Biosynthesis of the epidermal growth factor receptor in A431 cells.

E L Mayes, M D Waterfield
PMCID: PMC557382  PMID: 6325174

Abstract

A monoclonal antibody R1 against the human epidermal growth factor receptor has been used to study biosynthesis in the carcinoma cell line A431. Two glycoproteins of apparent mol. wts. 95 000 and 160 000 were immunoprecipitated from cells labelled for short times with [35S]methionine or [3H]mannose. Pulse-chase studies show the 160 000 mol. wt. glycoprotein to be a precursor of the 175 000 mol. wt. receptor, but do not establish a precursor role for the 95 000 mol. wt. glycoprotein. Limited proteolysis, peptide mapping, endoglycosidase digestion and the use of monensin and tunicamycin show that the 95 000 mol. wt. glycoprotein is structurally related to the 160 000 mol. wt. glycoprotein and that both glycoproteins have approximately 22 000 - 28 000 mol. wt. of oligosaccharide side chains. Monensin blocks conversion of the 160 000 to the 175 000 mol. wt. mature receptor, a process which involves complexing several of its N-linked oligosaccharide chains. Pulse-chase studies showed that an immunoprecipitable polypeptide of 115 000 mol. wt., or 95 000 mol. wt., in the presence of monensin, was secreted into the medium at late chase times. The possible mechanisms for the origins of all the receptor-related polypeptides are discussed.

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Selected References

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

  1. Adamson E. D., Rees A. R. Epidermal growth factor receptors. Mol Cell Biochem. 1981 Feb 11;34(3):129–152. doi: 10.1007/BF02359619. [DOI] [PubMed] [Google Scholar]
  2. Anderson R. G., Brown M. S., Beisiegel U., Goldstein J. L. Surface distribution and recycling of the low density lipoprotein receptor as visualized with antireceptor antibodies. J Cell Biol. 1982 Jun;93(3):523–531. doi: 10.1083/jcb.93.3.523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Antoniades H. N., Williams L. T. Human platelet-derived growth factor: structure and function. Fed Proc. 1983 Jun;42(9):2630–2634. [PubMed] [Google Scholar]
  4. Baker J. B., Simmer R. L., Glenn K. C., Cunningham D. D. Thrombin and epidermal growth factor become linked to cell surface receptors during mitogenic stimulation. Nature. 1979 Apr 19;278(5706):743–745. doi: 10.1038/278743a0. [DOI] [PubMed] [Google Scholar]
  5. Bonner W. M., Laskey R. A. A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem. 1974 Jul 1;46(1):83–88. doi: 10.1111/j.1432-1033.1974.tb03599.x. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Brown M. S., Kovanen P. T., Goldstein J. L. Receptor-mediated uptake of lipoprotein-cholesterol and its utilization for steroid synthesis in the adrenal cortex. Recent Prog Horm Res. 1979;35:215–257. doi: 10.1016/b978-0-12-571135-7.50009-6. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Cassel D., Glaser L. Proteolytic cleavage of epidermal growth factor receptor. A Ca2+-dependent, sulfhydryl-sensitive proteolytic system in A431 cells. J Biol Chem. 1982 Aug 25;257(16):9845–9848. [PubMed] [Google Scholar]
  10. Cheng H. L., Blattner F. R., Fitzmaurice L., Mushinski J. F., Tucker P. W. Structure of genes for membrane and secreted murine IgD heavy chains. Nature. 1982 Apr 1;296(5856):410–415. doi: 10.1038/296410a0. [DOI] [PubMed] [Google Scholar]
  11. Cohen S., Fava R. A., Sawyer S. T. Purification and characterization of epidermal growth factor receptor/protein kinase from normal mouse liver. Proc Natl Acad Sci U S A. 1982 Oct;79(20):6237–6241. doi: 10.1073/pnas.79.20.6237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Cohen S., Ushiro H., Stoscheck C., Chinkers M. A native 170,000 epidermal growth factor receptor-kinase complex from shed plasma membrane vesicles. J Biol Chem. 1982 Feb 10;257(3):1523–1531. [PubMed] [Google Scholar]
  13. Comens P. G., Simmer R. L., Baker J. B. Direct linkage of 125I-EGF to cell surface receptors. A useful artifact of chloramine-T treatment. J Biol Chem. 1982 Jan 10;257(1):42–45. [PubMed] [Google Scholar]
  14. Czech M. P., Oppenheimer C. L., Massagué J. Interrelationships among receptor structures for insulin and peptide growth factors. Fed Proc. 1983 Jun;42(9):2598–2601. [PubMed] [Google Scholar]
  15. Czech M. P. Structural and functional homologies in the receptors for insulin and the insulin-like growth factors. Cell. 1982 Nov;31(1):8–10. doi: 10.1016/0092-8674(82)90399-3. [DOI] [PubMed] [Google Scholar]
  16. Davies R. L., Grosse V. A., Kucherlapati R., Bothwell M. Genetic analysis of epidermal growth factor action: assignment of human epidermal growth factor receptor gene to chromosome 7. Proc Natl Acad Sci U S A. 1980 Jul;77(7):4188–4192. doi: 10.1073/pnas.77.7.4188. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Deutsch P. J., Wan C. F., Rosen O. M., Rubin C. S. Latent insulin receptors and possible receptor precursors in 3T3-L1 adipocytes. Proc Natl Acad Sci U S A. 1983 Jan;80(1):133–136. doi: 10.1073/pnas.80.1.133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Early P., Rogers J., Davis M., Calame K., Bond M., Wall R., Hood L. Two mRNAs can be produced from a single immunoglobulin mu gene by alternative RNA processing pathways. Cell. 1980 Jun;20(2):313–319. doi: 10.1016/0092-8674(80)90617-0. [DOI] [PubMed] [Google Scholar]
  19. Fabricant R. N., De Larco J. E., Todaro G. J. Nerve growth factor receptors on human melanoma cells in culture. Proc Natl Acad Sci U S A. 1977 Feb;74(2):565–569. doi: 10.1073/pnas.74.2.565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Goldstein J. L., Brown M. S. The low-density lipoprotein pathway and its relation to atherosclerosis. Annu Rev Biochem. 1977;46:897–930. doi: 10.1146/annurev.bi.46.070177.004341. [DOI] [PubMed] [Google Scholar]
  21. Hendrix R. W., Casjens S. R. Protein fusion: a novel reaction in bacteriophage lambda head assembly. Proc Natl Acad Sci U S A. 1974 Apr;71(4):1451–1455. doi: 10.1073/pnas.71.4.1451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Jacobs S., Kull F. C., Jr, Cuatrecasas P. Monensin blocks the maturation of receptors for insulin and somatomedin C: identification of receptor precursors. Proc Natl Acad Sci U S A. 1983 Mar;80(5):1228–1231. doi: 10.1073/pnas.80.5.1228. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kasuga M., Hedo J. A., Yamada K. M., Kahn C. R. The structure of insulin receptor and its subunits. Evidence for multiple nonreduced forms and a 210,000 possible proreceptor. J Biol Chem. 1982 Sep 10;257(17):10392–10399. [PubMed] [Google Scholar]
  24. Klenk H. D., Rott R. Cotranslational and posttranslational processing of viral glycoproteins. Curr Top Microbiol Immunol. 1980;90:19–48. doi: 10.1007/978-3-642-67717-5_2. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Linsley P. S., Blifeld C., Wrann M., Fox C. F. Direct linkage of epidermal growth factor to its receptor. Nature. 1979 Apr 19;278(5706):745–748. doi: 10.1038/278745a0. [DOI] [PubMed] [Google Scholar]
  27. Linsley P. S., Fox C. F. Controlled proteolysis of EGF receptors: evidence for transmembrane distribution of the EGF binding and phosphate acceptor sites. J Supramol Struct. 1980;14(4):461–471. doi: 10.1002/jss.400140405. [DOI] [PubMed] [Google Scholar]
  28. Maki R., Roeder W., Traunecker A., Sidman C., Wabl M., Raschke W., Tonegawa S. The role of DNA rearrangement and alternative RNA processing in the expression of immunoglobulin delta genes. Cell. 1981 May;24(2):353–365. doi: 10.1016/0092-8674(81)90325-1. [DOI] [PubMed] [Google Scholar]
  29. Omary M. B., Trowbridge I. S. Biosynthesis of the human transferrin receptor in cultured cells. J Biol Chem. 1981 Dec 25;256(24):12888–12892. [PubMed] [Google Scholar]
  30. Owen M. J., Kissonerghis A. M., Lodish H. F. Biosynthesis of HLA-A and HLA-B antigens in vivo. J Biol Chem. 1980 Oct 25;255(20):9678–9684. [PubMed] [Google Scholar]
  31. Rogers J., Choi E., Souza L., Carter C., Word C., Kuehl M., Eisenberg D., Wall R. Gene segments encoding transmembrane carboxyl termini of immunoglobulin gamma chains. Cell. 1981 Oct;26(1 Pt 1):19–27. doi: 10.1016/0092-8674(81)90029-5. [DOI] [PubMed] [Google Scholar]
  32. Schneider C., Sutherland R., Newman R., Greaves M. Structural features of the cell surface receptor for transferrin that is recognized by the monoclonal antibody OKT9. J Biol Chem. 1982 Jul 25;257(14):8516–8522. [PubMed] [Google Scholar]
  33. Struck D. K., Lennarz W. J. Evidence for the participation of saccharide-lipids in the synthesis of the oligosaccharide chain of ovalbumin. J Biol Chem. 1977 Feb 10;252(3):1007–1013. [PubMed] [Google Scholar]
  34. Tartakoff A. M. Perturbation of vesicular traffic with the carboxylic ionophore monensin. Cell. 1983 Apr;32(4):1026–1028. doi: 10.1016/0092-8674(83)90286-6. [DOI] [PubMed] [Google Scholar]
  35. Tolleshaug H., Goldstein J. L., Schneider W. J., Brown M. S. Posttranslational processing of the LDL receptor and its genetic disruption in familial hypercholesterolemia. Cell. 1982 Oct;30(3):715–724. doi: 10.1016/0092-8674(82)90276-8. [DOI] [PubMed] [Google Scholar]
  36. Tyler B. M., Cowman A. F., Gerondakis S. D., Adams J. M., Bernard O. mRNA for surface immunoglobulin gamma chains encodes a highly conserved transmembrane sequence and a 28-residue intracellular domain. Proc Natl Acad Sci U S A. 1982 Mar;79(6):2008–2012. doi: 10.1073/pnas.79.6.2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Waterfield M. D., Mayes E. L., Stroobant P., Bennet P. L., Young S., Goodfellow P. N., Banting G. S., Ozanne B. A monoclonal antibody to the human epidermal growth factor receptor. J Cell Biochem. 1982;20(2):149–161. doi: 10.1002/jcb.240200207. [DOI] [PubMed] [Google Scholar]
  38. Word C. J., Mushinski J. F., Tucker P. W. The murine immunoglobulin alpha gene expresses multiple transcripts from a unique membrane exon. EMBO J. 1983;2(6):887–898. doi: 10.1002/j.1460-2075.1983.tb01518.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Wrann M. M., Fox C. F. Identification of epidermal growth factor receptors in a hyperproducing human epidermoid carcinoma cell line. J Biol Chem. 1979 Sep 10;254(17):8083–8086. [PubMed] [Google Scholar]
  40. Yeaton R. W., Lipari M. T., Fox C. F. Calcium-mediated degradation of epidermal growth factor receptor in dislodged A431 cells and membrane preparations. J Biol Chem. 1983 Aug 10;258(15):9254–9261. [PubMed] [Google Scholar]

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