Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1986 May 1;102(5):1567–1575. doi: 10.1083/jcb.102.5.1567

Unusual forms of low density lipoprotein receptors in hamster cell mutants with defects in the receptor structural gene

PMCID: PMC2114229  PMID: 3517003

Abstract

The structure and processing of low density lipoprotein (LDL) receptors in wild-type and LDL receptor-deficient mutant Chinese hamster ovary cells was examined using polyclonal anti-receptor antibodies. As previously reported for human LDL receptors, the LDL receptors in wild- type Chinese hamster ovary cells were synthesized as precursors which were extensively processed by glycosylation to a mature form. In the course of normal receptor turnover, an apparently unglycosylated portion of the cysteine-rich N-terminal LDL binding domain of the receptor is proteolytically removed. The LDL receptor-deficient mutants fall into four complementation groups, ldlA, ldlB, ldlC, and ldlD; results of the analysis of ldlB, ldlC, and ldlD mutants are described in the accompanying paper (Kingsley, D. M., K. F. Kozarsky, M. Segal, and M. Krieger, 1986, J. Cell. Biol, 102:1576-1585). Analysis of ldlA cells has identified three classes of mutant alleles at the ldlA locus: null alleles, alleles that code for normally processed receptors that cannot bind LDL, and alleles that code for abnormally processed receptors. The abnormally processed receptors were continually converted to novel unstable intracellular intermediates. We also identified a compound-heterozygous mutant and a heterozygous revertant which indicate that the ldlA locus is diploid. In conjunction with other genetic and biochemical data, the finding of multiple mutant forms of the LDL receptor in ldlA mutants, some of which appeared together in the same cell, confirm that the ldlA locus is the structural gene for the LDL receptor.

Full Text

The Full Text of this article is available as a PDF (1.8 MB).

Selected References

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

  1. Beisiegel U., Kita T., Anderson R. G., Schneider W. J., Brown M. S., Goldstein J. L. Immunologic cross-reactivity of the low density lipoprotein receptor from bovine adrenal cortex, human fibroblasts, canine liver and adrenal gland, and rat liver. J Biol Chem. 1981 Apr 25;256(8):4071–4078. [PubMed] [Google Scholar]
  2. Beisiegel U., Schneider W. J., Brown M. S., Goldstein J. L. Immunoblot analysis of low density lipoprotein receptors in fibroblasts from subjects with familial hypercholesterolemia. J Biol Chem. 1982 Nov 10;257(21):13150–13156. [PubMed] [Google Scholar]
  3. Beisiegel U., Schneider W. J., Goldstein J. L., Anderson R. G., Brown M. S. Monoclonal antibodies to the low density lipoprotein receptor as probes for study of receptor-mediated endocytosis and the genetics of familial hypercholesterolemia. J Biol Chem. 1981 Nov 25;256(22):11923–11931. [PubMed] [Google Scholar]
  4. Bode V. C. Ethylnitrosourea mutagenesis and the isolation of mutant alleles for specific genes located in the T region of mouse chromosome 17. Genetics. 1984 Oct;108(2):457–470. doi: 10.1093/genetics/108.2.457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brenner S. The genetics of Caenorhabditis elegans. Genetics. 1974 May;77(1):71–94. doi: 10.1093/genetics/77.1.71. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cummings R. D., Kornfeld S., Schneider W. J., Hobgood K. K., Tolleshaug H., Brown M. S., Goldstein J. L. Biosynthesis of N- and O-linked oligosaccharides of the low density lipoprotein receptor. J Biol Chem. 1983 Dec 25;258(24):15261–15273. [PubMed] [Google Scholar]
  7. Daniel T. O., Schneider W. J., Goldstein J. L., Brown M. S. Visualization of lipoprotein receptors by ligand blotting. J Biol Chem. 1983 Apr 10;258(7):4606–4611. [PubMed] [Google Scholar]
  8. Doyle C., Roth M. G., Sambrook J., Gething M. J. Mutations in the cytoplasmic domain of the influenza virus hemagglutinin affect different stages of intracellular transport. J Cell Biol. 1985 Mar;100(3):704–714. doi: 10.1083/jcb.100.3.704. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dunphy W. G., Fries E., Urbani L. J., Rothman J. E. Early and late functions associated with the Golgi apparatus reside in distinct compartments. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7453–7457. doi: 10.1073/pnas.78.12.7453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Goldberg D. E., Kornfeld S. Evidence for extensive subcellular organization of asparagine-linked oligosaccharide processing and lysosomal enzyme phosphorylation. J Biol Chem. 1983 Mar 10;258(5):3159–3165. [PubMed] [Google Scholar]
  11. Goldstein J. L., Anderson R. G., Brown M. S. Coated pits, coated vesicles, and receptor-mediated endocytosis. Nature. 1979 Jun 21;279(5715):679–685. doi: 10.1038/279679a0. [DOI] [PubMed] [Google Scholar]
  12. Goldstein J. L., Brown M. S., Anderson R. G., Russell D. W., Schneider W. J. Receptor-mediated endocytosis: concepts emerging from the LDL receptor system. Annu Rev Cell Biol. 1985;1:1–39. doi: 10.1146/annurev.cb.01.110185.000245. [DOI] [PubMed] [Google Scholar]
  13. Haguenauer-Tsapis R., Hinnen A. A deletion that includes the signal peptidase cleavage site impairs processing, glycosylation, and secretion of cell surface yeast acid phosphatase. Mol Cell Biol. 1984 Dec;4(12):2668–2675. doi: 10.1128/mcb.4.12.2668. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hancock K., Tsang V. C. India ink staining of proteins on nitrocellulose paper. Anal Biochem. 1983 Aug;133(1):157–162. doi: 10.1016/0003-2697(83)90237-3. [DOI] [PubMed] [Google Scholar]
  15. Hawkes R., Niday E., Gordon J. A dot-immunobinding assay for monoclonal and other antibodies. Anal Biochem. 1982 Jan 1;119(1):142–147. doi: 10.1016/0003-2697(82)90677-7. [DOI] [PubMed] [Google Scholar]
  16. Hercz A., Harpaz N. Characterization of the oligosaccharides of liver Z variant alpha 1-antitrypsin. Can J Biochem. 1980 Aug;58(8):644–648. doi: 10.1139/o80-089. [DOI] [PubMed] [Google Scholar]
  17. Johnson D. A., Elder J. H. Antibody directed to determinants of a Moloney virus derived MCF GP70 recognizes a thymic differentiation antigen. J Exp Med. 1983 Nov 1;158(5):1751–1756. doi: 10.1084/jem.158.5.1751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kingsley D. M., Kozarsky K. F., Segal M., Krieger M. Three types of low density lipoprotein receptor-deficient mutant have pleiotropic defects in the synthesis of N-linked, O-linked, and lipid-linked carbohydrate chains. J Cell Biol. 1986 May;102(5):1576–1585. doi: 10.1083/jcb.102.5.1576. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kingsley D. M., Krieger M. Receptor-mediated endocytosis of low density lipoprotein: somatic cell mutants define multiple genes required for expression of surface-receptor activity. Proc Natl Acad Sci U S A. 1984 Sep;81(17):5454–5458. doi: 10.1073/pnas.81.17.5454. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Krieger M., Brown M. S., Goldstein J. L. Isolation of Chinese hamster cell mutants defective in the receptor-mediated endocytosis of low density lipoprotein. J Mol Biol. 1981 Aug 5;150(2):167–184. doi: 10.1016/0022-2836(81)90447-2. [DOI] [PubMed] [Google Scholar]
  21. Krieger M. Complementation of mutations in the LDL pathway of receptor-mediated endocytosis by cocultivation of LDL receptor-defective hamster cell mutants. Cell. 1983 Jun;33(2):413–422. doi: 10.1016/0092-8674(83)90423-3. [DOI] [PubMed] [Google Scholar]
  22. Krieger M., Martin J., Segal M., Kingsley D. Amphotericin B selection of mutant Chinese hamster cells with defects in the receptor-mediated endocytosis of low density lipoprotein and cholesterol biosynthesis. Proc Natl Acad Sci U S A. 1983 Sep;80(18):5607–5611. doi: 10.1073/pnas.80.18.5607. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Krieger M., McPhaul M. J., Goldstein J. L., Brown M. S. Replacement of neutral lipids of low density lipoprotein with esters of long chain unsaturated fatty acids. J Biol Chem. 1979 May 25;254(10):3845–3853. [PubMed] [Google Scholar]
  24. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [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. Laskey R. A. The use of intensifying screens or organic scintillators for visualizing radioactive molecules resolved by gel electrophoresis. Methods Enzymol. 1980;65(1):363–371. doi: 10.1016/s0076-6879(80)65047-2. [DOI] [PubMed] [Google Scholar]
  27. Lehrman M. A., Goldstein J. L., Brown M. S., Russell D. W., Schneider W. J. Internalization-defective LDL receptors produced by genes with nonsense and frameshift mutations that truncate the cytoplasmic domain. Cell. 1985 Jul;41(3):735–743. doi: 10.1016/s0092-8674(85)80054-4. [DOI] [PubMed] [Google Scholar]
  28. Novick P., Ferro S., Schekman R. Order of events in the yeast secretory pathway. Cell. 1981 Aug;25(2):461–469. doi: 10.1016/0092-8674(81)90064-7. [DOI] [PubMed] [Google Scholar]
  29. Robbins A. R., Oliver C., Bateman J. L., Krag S. S., Galloway C. J., Mellman I. A single mutation in Chinese hamster ovary cells impairs both Golgi and endosomal functions. J Cell Biol. 1984 Oct;99(4 Pt 1):1296–1308. doi: 10.1083/jcb.99.4.1296. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Rose J. K., Bergmann J. E. Altered cytoplasmic domains affect intracellular transport of the vesicular stomatitis virus glycoprotein. Cell. 1983 Sep;34(2):513–524. doi: 10.1016/0092-8674(83)90384-7. [DOI] [PubMed] [Google Scholar]
  31. Roth J., Berger E. G. Immunocytochemical localization of galactosyltransferase in HeLa cells: codistribution with thiamine pyrophosphatase in trans-Golgi cisternae. J Cell Biol. 1982 Apr;93(1):223–229. doi: 10.1083/jcb.93.1.223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Russell D. W., Schneider W. J., Yamamoto T., Luskey K. L., Brown M. S., Goldstein J. L. Domain map of the LDL receptor: sequence homology with the epidermal growth factor precursor. Cell. 1984 Jun;37(2):577–585. doi: 10.1016/0092-8674(84)90388-x. [DOI] [PubMed] [Google Scholar]
  33. Schauer I., Emr S., Gross C., Schekman R. Invertase signal and mature sequence substitutions that delay intercompartmental transport of active enzyme. J Cell Biol. 1985 May;100(5):1664–1675. doi: 10.1083/jcb.100.5.1664. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Schneider W. J., Beisiegel U., Goldstein J. L., Brown M. S. Purification of the low density lipoprotein receptor, an acidic glycoprotein of 164,000 molecular weight. J Biol Chem. 1982 Mar 10;257(5):2664–2673. [PubMed] [Google Scholar]
  35. Schneider W. J., Brown M. S., Goldstein J. L. Kinetic defects in the processing of the low density lipoprotein receptor in fibroblasts from WHHL rabbits and a family with familial hypercholesterolemia. Mol Biol Med. 1983 Oct;1(3):353–367. [PubMed] [Google Scholar]
  36. Schneider W. J., Goldstein J. L., Brown M. S. Partial purification and characterization of the low density lipoprotein receptor from bovine adrenal cortex. J Biol Chem. 1980 Dec 10;255(23):11442–11447. [PubMed] [Google Scholar]
  37. Sege R. D., Kozarsky K., Nelson D. L., Krieger M. Expression and regulation of human low-density lipoprotein receptors in Chinese hamster ovary cells. Nature. 1984 Feb 23;307(5953):742–745. doi: 10.1038/307742a0. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. Tolleshaug H., Hobgood K. K., Brown M. S., Goldstein J. L. The LDL receptor locus in familial hypercholesterolemia: multiple mutations disrupt transport and processing of a membrane receptor. Cell. 1983 Mar;32(3):941–951. doi: 10.1016/0092-8674(83)90079-x. [DOI] [PubMed] [Google Scholar]
  40. Yamamoto T., Davis C. G., Brown M. S., Schneider W. J., Casey M. L., Goldstein J. L., Russell D. W. The human LDL receptor: a cysteine-rich protein with multiple Alu sequences in its mRNA. Cell. 1984 Nov;39(1):27–38. doi: 10.1016/0092-8674(84)90188-0. [DOI] [PubMed] [Google Scholar]

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

RESOURCES