Functional domains of the receptor-associated protein (RAP).
The receptor-associated protein (RAP) specifically associates with gp330 and the low density lipoprotein (LDL) receptor-related protein (LRP), the two newest members of the LDL receptor gene family. Results obtained by ligand blotting, affinity chromatography, and density-gradient sedimentation demonstrate that RAP binds to both receptors with high affinity and that the binding is Ca2+ dependent. RAP also binds heparin and is identical to a mouse heparin binding protein (HBP-44) identified in a teratocarcinoma cell line (F9). While biochemical studies have shown that RAP is present on the cell surface and is an effective inhibitor of ligand binding to gp330 and LRP, immunocytochemical findings indicate that RAP is most abundant in the endoplasmic reticulum lumen and may function in receptor folding and/or trafficking. To facilitate the characterization of RAP's function(s) we have mapped its gp330 and heparin binding sites by performing direct binding studies on fusion proteins representing overlapping domains of RAP. gp330 was found to bind to two separate sites on RAP--i.e., between amino acids 85-148 and 178-248. Binding studies with radiolabeled heparin indicate that the heparin binding site is between amino acids 261 and 323, which is consistent with our previously proposed site (residues 287-306) based on the amphipathic nature of the C terminus of RAP. These data demonstrate that the gp330 and heparin binding sites and the Heymann nephritis pathogenic epitope (amino acids 1-86) demonstrated earlier are represented by distinct domains of the RAP polypeptide.
Orlando
R A
RA
Division of Cellular and Molecular Medicine, University of California at San Diego, La Jolla 92093-0651.
Farquhar
M G
MG
D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000276:immunology; D000939:Epitopes; D005921:Glomerulonephritis / Q000276:immunology; D006023:Glycoproteins / Q000737:chemistry* / Q000276:immunology; D006493:Heparin / Q000378:metabolism; D026541:Heymann Nephritis Antigenic Complex; D066298:In Vitro Techniques; D026563:LDL-Receptor Related Protein-Associated Protein; D008562:Membrane Glycoproteins / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins; D013329:Structure-Activity Relationship
D002352:Carrier Proteins; D000939:Epitopes; D006023:Glycoproteins; D026541:Heymann Nephritis Antigenic Complex; D026563:LDL-Receptor Related Protein-Associated Protein; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D006493:Heparin