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. 1989 Dec;86(23):9318–9322. doi: 10.1073/pnas.86.23.9318

Lipoprotein binding and endosomal itinerary of the low density lipoprotein receptor-related protein in rat liver.

H Lund 1, K Takahashi 1, R L Hamilton 1, R J Havel 1
PMCID: PMC298486  PMID: 2594771

Abstract

The high affinity of 45Ca binding to the low density lipoprotein receptor (LDL-R) and the LDL-R-related protein (LRP) was utilized to study the subcellular distribution of these two proteins in rat liver. Like the LDL-R, LRP was manyfold enriched in rat liver endosomal membranes with a relative distribution in early and late endosomal compartments consistent with recycling between endosomes and the cell surface. The high concentration of LRP in hepatic endosomal membranes greatly facilitated demonstration of Ca-dependent binding of apolipoprotein E- and B-containing lipoproteins in ligand blots. LRP was severalfold more abundant than the LDL-R in hepatic parenchymal cells, showed extensive degradation in hepatic endosomes, and was found in high concentrations in the Golgi apparatus and endoplasmic reticulum. These data suggest a high rate of synthesis of LRP that appeared to be unaffected by treatment of rats with estradiol. The repeating cysteine-rich A-motif found in the ligand-binding domain of LRP appeared to be responsible for Ca binding by LRP, LDL-R, and complement factor C9 and accounted for immunological cross-reactivity among these proteins. Weaker ligand-blotting properties and an extraordinary susceptibility to proteolysis most likely contribute to the difficulty of detecting LRP in conventional assays for lipoprotein receptors. Our data suggest an extensive proteolytic processing of this protein and are consistent with a functional role of LRP in lipoprotein metabolism.

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Selected References

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

  1. 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]
  2. Belcher J. D., Hamilton R. L., Brady S. E., Hornick C. A., Jaeckle S., Schneider W. J., Havel R. J. Isolation and characterization of three endosomal fractions from the liver of estradiol-treated rats. Proc Natl Acad Sci U S A. 1987 Oct;84(19):6785–6789. doi: 10.1073/pnas.84.19.6785. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bensadoun A., Kompiang I. P. Role of lipoprotein lipase in plasma triglyceride removal. Fed Proc. 1979 Nov;38(12):2622–2626. [PubMed] [Google Scholar]
  4. Berry M. N., Friend D. S. High-yield preparation of isolated rat liver parenchymal cells: a biochemical and fine structural study. J Cell Biol. 1969 Dec;43(3):506–520. doi: 10.1083/jcb.43.3.506. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  6. Coudrier E., Reggio H., Louvard D. Characterization of an integral membrane glycoprotein associated with the microfilaments of pig intestinal microvilli. EMBO J. 1983;2(3):469–475. doi: 10.1002/j.1460-2075.1983.tb01446.x. [DOI] [PMC free article] [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. 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. Dobbs L. G., Gonzalez R., Williams M. C. An improved method for isolating type II cells in high yield and purity. Am Rev Respir Dis. 1986 Jul;134(1):141–145. doi: 10.1164/arrd.1986.134.1.141. [DOI] [PubMed] [Google Scholar]
  10. Glossmann H., Neville D. M., Jr Glycoproteins of cell surfaces. A comparative study of three different cell surfaces of the rat. J Biol Chem. 1971 Oct 25;246(20):6339–6346. [PubMed] [Google Scholar]
  11. HAVEL R. J., EDER H. A., BRAGDON J. H. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J Clin Invest. 1955 Sep;34(9):1345–1353. doi: 10.1172/JCI103182. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hamilton R. L., Fielding P. E. Nascent very low density lipoproteins from rat hepatocytic Golgi fractions are enriched in phosphatidylethanolamine. Biochem Biophys Res Commun. 1989 Apr 14;160(1):162–173. doi: 10.1016/0006-291x(89)91635-5. [DOI] [PubMed] [Google Scholar]
  13. Havel R. J., Chao Y., Windler E. E., Kotite L., Guo L. S. Isoprotein specificity in the hepatic uptake of apolipoprotein E and the pathogenesis of familial dysbetalipoproteinemia. Proc Natl Acad Sci U S A. 1980 Jul;77(7):4349–4353. doi: 10.1073/pnas.77.7.4349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Herz J., Hamann U., Rogne S., Myklebost O., Gausepohl H., Stanley K. K. Surface location and high affinity for calcium of a 500-kd liver membrane protein closely related to the LDL-receptor suggest a physiological role as lipoprotein receptor. EMBO J. 1988 Dec 20;7(13):4119–4127. doi: 10.1002/j.1460-2075.1988.tb03306.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hubbard A. L., Wall D. A., Ma A. Isolation of rat hepatocyte plasma membranes. I. Presence of the three major domains. J Cell Biol. 1983 Jan;96(1):217–229. doi: 10.1083/jcb.96.1.217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Imaizumi K., Fainaru M., Havel R. J. Composition of proteins of mesenteric lymph chylomicrons in the rat and alterations produced upon exposure of chylomicrons to blood serum and serum proteins. J Lipid Res. 1978 Aug;19(6):712–722. [PubMed] [Google Scholar]
  17. Jaeckle S., Brady S. E., Havel R. J. Membrane binding sites for plasma lipoproteins on endosomes from rat liver. Proc Natl Acad Sci U S A. 1989 Mar;86(6):1880–1884. doi: 10.1073/pnas.86.6.1880. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kita T., Goldstein J. L., Brown M. S., Watanabe Y., Hornick C. A., Havel R. J. Hepatic uptake of chylomicron remnants in WHHL rabbits: a mechanism genetically distinct from the low density lipoprotein receptor. Proc Natl Acad Sci U S A. 1982 Jun;79(11):3623–3627. doi: 10.1073/pnas.79.11.3623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kovanen P. T., Brown M. S., Goldstein J. L. Increased binding of low density lipoprotein to liver membranes from rats treated with 17 alpha-ethinyl estradiol. J Biol Chem. 1979 Nov 25;254(22):11367–11373. [PubMed] [Google Scholar]
  20. Kowal R. C., Herz J., Goldstein J. L., Esser V., Brown M. S. Low density lipoprotein receptor-related protein mediates uptake of cholesteryl esters derived from apoprotein E-enriched lipoproteins. Proc Natl Acad Sci U S A. 1989 Aug;86(15):5810–5814. doi: 10.1073/pnas.86.15.5810. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Maruyama K., Mikawa T., Ebashi S. Detection of calcium binding proteins by 45Ca autoradiography on nitrocellulose membrane after sodium dodecyl sulfate gel electrophoresis. J Biochem. 1984 Feb;95(2):511–519. doi: 10.1093/oxfordjournals.jbchem.a134633. [DOI] [PubMed] [Google Scholar]
  22. McFARLANE A. S. Efficient trace-labelling of proteins with iodine. Nature. 1958 Jul 5;182(4627):53–53. doi: 10.1038/182053a0. [DOI] [PubMed] [Google Scholar]
  23. Ohsumi Y., Ishikawa T., Kato K. A rapid and simplified method for the preparation of lysosomal membranes from rat liver. J Biochem. 1983 Feb;93(2):547–556. [PubMed] [Google Scholar]
  24. Olmsted J. B. Affinity purification of antibodies from diazotized paper blots of heterogeneous protein samples. J Biol Chem. 1981 Dec 10;256(23):11955–11957. [PubMed] [Google Scholar]
  25. Rajavashisth T. B., Dawson P. A., Williams D. L., Shackleford J. E., Lebherz H., Lusis A. J. Structure, evolution, and regulation of chicken apolipoprotein A-I. J Biol Chem. 1987 May 25;262(15):7058–7065. [PubMed] [Google Scholar]
  26. Stanley K. K., Kocher H. P., Luzio J. P., Jackson P., Tschopp J. The sequence and topology of human complement component C9. EMBO J. 1985 Feb;4(2):375–382. doi: 10.1002/j.1460-2075.1985.tb03639.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Tschopp J., Masson D., Stanley K. K. Structural/functional similarity between proteins involved in complement- and cytotoxic T-lymphocyte-mediated cytolysis. 1986 Aug 28-Sep 3Nature. 322(6082):831–834. doi: 10.1038/322831a0. [DOI] [PubMed] [Google Scholar]
  28. Windler E. E., Greeve J., Daerr W. H., Greten H. Binding of rat chylomicrons and their remnants to the hepatic low-density-lipoprotein receptor and its role in remnant removal. Biochem J. 1988 Jun 1;252(2):553–561. doi: 10.1042/bj2520553. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Windler E., Chao Y., Havel R. J. Determinants of hepatic uptake of triglyceride-rich lipoproteins and their remnants in the rat. J Biol Chem. 1980 Jun 10;255(11):5475–5480. [PubMed] [Google Scholar]
  30. Wright J. R., Borchelt J. D., Hawgood S. Lung surfactant apoprotein SP-A (26-36 kDa) binds with high affinity to isolated alveolar type II cells. Proc Natl Acad Sci U S A. 1989 Jul;86(14):5410–5414. doi: 10.1073/pnas.86.14.5410. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Yamada N., Shames D. M., Stoudemire J. B., Havel R. J. Metabolism of lipoproteins containing apolipoprotein B-100 in blood plasma of rabbits: heterogeneity related to the presence of apolipoprotein E. Proc Natl Acad Sci U S A. 1986 May;83(10):3479–3483. doi: 10.1073/pnas.83.10.3479. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. van Driel I. R., Goldstein J. L., Südhof T. C., Brown M. S. First cysteine-rich repeat in ligand-binding domain of low density lipoprotein receptor binds Ca2+ and monoclonal antibodies, but not lipoproteins. J Biol Chem. 1987 Dec 25;262(36):17443–17449. [PubMed] [Google Scholar]

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