An Apo A-I Protein A Hybrid Used for the Study of HDL Receptor Binding and Endocytosis

  • H. M. Bond
  • L. Monaco
  • R. Cortese
  • K. E. Howell
Conference paper
Part of the Recent Developments in Lipid and Lipoprotein Research book series (LIPID)

Abstract

HDL (high-density lipoprotein) interacts with cells in reverse cholesterol transport and gains cholesterol. The molecular mechanism and the subcellular localization of this acquisition are not understood. A major problem has been the difficulty in determining whether HDL follows a receptor-mediated pathway, and whether receptor-mediated endocytosis is a prerequisite for reverse cholesterol transport [1–4]. Common steps have been defined for receptor mediated endocytosis of most ligands. They bind to cell surface receptors at 4 °C and at 37 °C, they are rapidly internalized into an early endosomal compartment. In most cases the acidic pH of the endosomal lumen allows the receptor-ligand complex to dissociate. Receptors and ligands may either be degraded in the lysosomes after 15–30 min or recycled, reappearing on the cell surface with a half-time of 5–8 min. In some systems it is possible experimentally to manipulate the proportion of molecules degraded or recycled [5, 6]. With the fundamental properties of receptor-mediated endocytosis in mind, we examined the HDL pathway using as the ligands HDL and an apo A-I containing hybrid protein constructed using recombinant DNA techniques. The apo A-I hybrid permits the analysis of the specific role of this apolipoprotein in HDL function.

Keywords

Cholesterol Urea Europium Oligomer Trypsin 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Schmitz G, Robenek H, Lohamann U, Assmann G (1985) Interaction of high density lipoproteins with cholesteryl ester-laden macrophages biochemical and morphological characterization of cell surface receptor binding, endocytosis and resecretion of high density lipoproteins by macrophages. EMBO J 4: 613–622.PubMedGoogle Scholar
  2. 2.
    Oram JF, Johnson CJ, Brown TA (1987) Interaction of high density lipoprotein with its receptor on cultured fibroblasts and macrophages. J Biol Chem 262: 2405–2410.PubMedGoogle Scholar
  3. 3.
    Pittman RC, Knecht TP, Rosenbaum MS, Taylor CA (1987) A nonendocytotic mechanism for the selective uptake of high density lipoprotein-associated cholesterol esters. J Biol Chem 262: 2443–2450.PubMedGoogle Scholar
  4. 4.
    Karlin JB, Johnson WJ, Benedict CR, Chacko GK, Phillips MC, Rothblat GH (1987) Cholesterol flux between cells and high density lipoprotein. Lack of relationship to specific binding of the lipoprotein to the cell surface. J Biol Chem 262: 12557–12564.Google Scholar
  5. 5.
    Gruenberg J, Howell KE (1987) Fusion in the endocytic pathway reconstituted in a cell-free system using immuno-isolated fractions. In: Cell-free analysis of membrane traffic, DJ Morré, KE Howell and GMW Cook eds. AR Liss, New York pp 77–90Google Scholar
  6. 6.
    Mellman I, Plutner H, Ukkonen P (1984) Internalization and degradation of macrophage Fc receptors bound to monovalent antireceptor antibody possible role of a prelysosomal compartment. J Cell Biol 98: 1163–1169.PubMedCrossRefGoogle Scholar
  7. 7.
    Monaco L, Bond HM, Howell KE, Cortese R (1987) A recombinant apo A-I-protein A hybrid reproduces the binding parameters of HDL to its receptor. EMBO J 6: 3253–3260.PubMedGoogle Scholar
  8. 8.
    Bond HM, Monaco L, Cortese R, Howell KE (1989) Receptor mediated endocytosis of high density lipoproteins studied in relation to cholesterol efflux. (manuscript submitted).Google Scholar
  9. 9.
    Lewis V, Green SA, Marsh M, Vihko P, Helinus A, Mellman I (1985) Glycoproteins of the lysosomal membrane. J Cell Biol 100: 1839–1847.PubMedCrossRefGoogle Scholar
  10. 10.
    Soini E, Hemmila I (1979) Fluoroimmunoassays present status and key problems. Clin Chem 25/3: 353–361.Google Scholar
  11. 11.
    Soini E, Kojola H (1983) Time resolved fluorometer for lanthinide chelates — A new generation of nonisotopic immunoassays. Clin Chem 29/1: 65–68.Google Scholar
  12. 12.
    Hemmilä I, Dakubu S, Mukkala VM, Siitari H, Lövgren T (1984) Europium as a label in time resolved immunofluorometric assays. Anal Biochem 137: 335–343.PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 1989

Authors and Affiliations

  • H. M. Bond
  • L. Monaco
  • R. Cortese
  • K. E. Howell

There are no affiliations available

Personalised recommendations