Identification of a cell line that expresses a cell surface and a soluble form of the gp330/receptor-associated protein (RAP) Heymann nephritis antigenic complex.
gp330 is a large glycoprotein located in clathrin-coated pits at the surface of the glomerular and proximal tubule epithelia in the rat kidney. It was originally identified as the target of autoimmune antibodies in Heymann nephritis (HN) and has since been shown to be a member of the low density lipoprotein receptor gene family and to form a stable association with receptor-associated protein (RAP), which together constitute the HN antigen complex (HNAC). Progress in defining the normal functions of gp330 as well as the molecular mechanisms of HN has been hampered by the lack of an available kidney cell line that expresses this protein. We here report the identification of a rat yolk sac carcinoma cell line (L2) that synthesizes HNAC and expresses it in coated pits at the cell surface. gp330 and RAP from L2 cells are immunologically identical to their kidney counterparts, and peptide maps of gp330 yielded identical peptide fragments. Characterization of the cell line revealed that there are 3.3 x 10(4) gp330 molecules per L2 cell and that the cells produce a soluble form of gp330 that is released into the medium. Heparin ligand blot analysis demonstrated that RAP but not gp330 binds heparin. By heparin affinity chromatography, gp330 and RAP copurify, indicating that the glycosaminoglycan binding site within RAP is accessible when the subunit is complexed with gp330. These results indicate that the L2 cell line provides a valid and useful model for studies on the function of HNAC and the pathogenesis of HN.
Orlando
R A
RA
Division of Cellular and Molecular Medicine, University of California, San Diego, La Jolla 92093.
Farquhar
M G
MG
D000818:Animals; D001324:Autoantigens / Q000302:isolation & purification / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism*; D010450:Endopeptidases; D005982:Glutathione Transferase / Q000378:metabolism; D006493:Heparin / Q000378:metabolism; D026541:Heymann Nephritis Antigenic Complex; D007668:Kidney / Q000276:immunology; D008562:Membrane Glycoproteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008871:Microvilli / Q000276:immunology; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification; D051381:Rats; D011973:Receptors, LDL / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D001324:Autoantigens; D026541:Heymann Nephritis Antigenic Complex; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; D011973:Receptors, LDL; D011993:Recombinant Fusion Proteins; D006493:Heparin; D005982:Glutathione Transferase; D010450:Endopeptidases