Abstract
We have recently described a PAI-1-independent pathway of tissue-type plasminogen activator (t-PA) uptake and degradation on the rat MH1C1 hepatoma cell line. Further studies have implicated the low-density-lipoprotein-receptor-related protein (LRP) as the mediator of plasminogen-activator inhibitor type-1-independent t-PA endocytosis. The LRP is a multi-functional receptor which is shared by a variety of ligands, including alpha 2-macroglobulin, apoprotein E-enriched beta-very-low-density lipoprotein, t-PA and Pseudomonas exotoxin A. In each case, binding of ligand to this receptor can be inhibited by addition of the 39 kDa LRP-receptor-associated protein. This protein, which co-purifies with the LRP receptor, is the focus of our present study. 125I-labelled 39 kDa protein binds specifically and with high affinity to a single kinetic binding species on the rat MH1C1 cell surface. Scatchard analysis reveals an equilibrium dissociation constant (Kd) of 3.3 +/- 0.9 (S.D.) nM, with 380,000 +/- 190,000 (S.D.) binding sites per cell. Cross-linking studies indicate that the specific interaction between MH1C1 cells and the 39 kDa protein is mediated by an association with the LRP receptor. The 39 kDa protein strongly inhibits binding of 125I-t-PA, with an apparent Ki value of 0.5 nM. In addition, both unlabelled t-PA and 125I-labelled 39 kDa protein can be co-bound and cross-linked to the same cell-associated LRP receptor. Endocytosis of cell-surface-associated 39 kDa protein was shown to be rapid, with internalized ligand subsequently degraded and released to the extracellular milieu. The rate of uptake and degradation of 125I-labelled 39 kDa protein at 37 degrees C was determined to be 52 fmol/min per 10(6) cells, and supports a model for active recycling of the LRP receptor.
Full text
PDF








Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Ashcom J. D., Tiller S. E., Dickerson K., Cravens J. L., Argraves W. S., Strickland D. K. The human alpha 2-macroglobulin receptor: identification of a 420-kD cell surface glycoprotein specific for the activated conformation of alpha 2-macroglobulin. J Cell Biol. 1990 Apr;110(4):1041–1048. doi: 10.1083/jcb.110.4.1041. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Beisiegel U., Weber W., Ihrke G., Herz J., Stanley K. K. The LDL-receptor-related protein, LRP, is an apolipoprotein E-binding protein. Nature. 1989 Sep 14;341(6238):162–164. doi: 10.1038/341162a0. [DOI] [PubMed] [Google Scholar]
- Bu G., Morton P. A., Schwartz A. L. Identification and partial characterization by chemical cross-linking of a binding protein for tissue-type plasminogen activator (t-PA) on rat hepatoma cells. A plasminogen activator inhibitor type 1-independent t-PA receptor. J Biol Chem. 1992 Aug 5;267(22):15595–15602. [PubMed] [Google Scholar]
- Bu G., Williams S., Strickland D. K., Schwartz A. L. Low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor is an hepatic receptor for tissue-type plasminogen activator. Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7427–7431. doi: 10.1073/pnas.89.16.7427. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ciechanover A., Schwartz A. L., Dautry-Varsat A., Lodish H. F. Kinetics of internalization and recycling of transferrin and the transferrin receptor in a human hepatoma cell line. Effect of lysosomotropic agents. J Biol Chem. 1983 Aug 25;258(16):9681–9689. [PubMed] [Google Scholar]
- Dunn W. A., Connolly T. P., Hubbard A. L. Receptor-mediated endocytosis of epidermal growth factor by rat hepatocytes: receptor pathway. J Cell Biol. 1986 Jan;102(1):24–36. doi: 10.1083/jcb.102.1.24. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Furukawa T., Ozawa M., Huang R. P., Muramatsu T. A heparin binding protein whose expression increases during differentiation of embryonal carcinoma cells to parietal endoderm cells: cDNA cloning and sequence analysis. J Biochem. 1990 Aug;108(2):297–302. doi: 10.1093/oxfordjournals.jbchem.a123197. [DOI] [PubMed] [Google Scholar]
- 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]
- Herz J., Goldstein J. L., Strickland D. K., Ho Y. K., Brown M. S. 39-kDa protein modulates binding of ligands to low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor. J Biol Chem. 1991 Nov 5;266(31):21232–21238. [PubMed] [Google Scholar]
- 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]
- Jensen P. H., Moestrup S. K., Gliemann J. Purification of the human placental alpha 2-macroglobulin receptor. FEBS Lett. 1989 Sep 25;255(2):275–280. doi: 10.1016/0014-5793(89)81105-6. [DOI] [PubMed] [Google Scholar]
- Kounnas M. Z., Morris R. E., Thompson M. R., FitzGerald D. J., Strickland D. K., Saelinger C. B. The alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein binds and internalizes Pseudomonas exotoxin A. J Biol Chem. 1992 Jun 25;267(18):12420–12423. [PubMed] [Google Scholar]
- 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]
- Kristensen T., Moestrup S. K., Gliemann J., Bendtsen L., Sand O., Sottrup-Jensen L. Evidence that the newly cloned low-density-lipoprotein receptor related protein (LRP) is the alpha 2-macroglobulin receptor. FEBS Lett. 1990 Dec 10;276(1-2):151–155. doi: 10.1016/0014-5793(90)80530-v. [DOI] [PubMed] [Google Scholar]
- 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]
- Moestrup S. K., Gliemann J. Analysis of ligand recognition by the purified alpha 2-macroglobulin receptor (low density lipoprotein receptor-related protein). Evidence that high affinity of alpha 2-macroglobulin-proteinase complex is achieved by binding to adjacent receptors. J Biol Chem. 1991 Jul 25;266(21):14011–14017. [PubMed] [Google Scholar]
- Moestrup S. K., Gliemann J. Purification of the rat hepatic alpha 2-macroglobulin receptor as an approximately 440-kDa single chain protein. J Biol Chem. 1989 Sep 15;264(26):15574–15577. [PubMed] [Google Scholar]
- Orth K., Madison E. L., Gething M. J., Sambrook J. F., Herz J. Complexes of tissue-type plasminogen activator and its serpin inhibitor plasminogen-activator inhibitor type 1 are internalized by means of the low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor. Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7422–7426. doi: 10.1073/pnas.89.16.7422. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pietromonaco S., Kerjaschki D., Binder S., Ullrich R., Farquhar M. G. Molecular cloning of a cDNA encoding a major pathogenic domain of the Heymann nephritis antigen gp330. Proc Natl Acad Sci U S A. 1990 Mar;87(5):1811–1815. doi: 10.1073/pnas.87.5.1811. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schwartz A. L., Fridovich S. E., Lodish H. F. Kinetics of internalization and recycling of the asialoglycoprotein receptor in a hepatoma cell line. J Biol Chem. 1982 Apr 25;257(8):4230–4237. [PubMed] [Google Scholar]
- Schwartz A. L., Rup D. Biosynthesis of the human asialoglycoprotein receptor. J Biol Chem. 1983 Sep 25;258(18):11249–11255. [PubMed] [Google Scholar]
- Simmons C. F., Jr, Schwartz A. L. Cellular pathways of galactose-terminal ligand movement in a cloned human hepatoma cell line. Mol Pharmacol. 1984 Nov;26(3):509–519. [PubMed] [Google Scholar]
- Strickland D. K., Ashcom J. D., Williams S., Battey F., Behre E., McTigue K., Battey J. F., Argraves W. S. Primary structure of alpha 2-macroglobulin receptor-associated protein. Human homologue of a Heymann nephritis antigen. J Biol Chem. 1991 Jul 15;266(20):13364–13369. [PubMed] [Google Scholar]
- Strickland D. K., Ashcom J. D., Williams S., Burgess W. H., Migliorini M., Argraves W. S. Sequence identity between the alpha 2-macroglobulin receptor and low density lipoprotein receptor-related protein suggests that this molecule is a multifunctional receptor. J Biol Chem. 1990 Oct 15;265(29):17401–17404. [PubMed] [Google Scholar]
- 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]