Identification of the second cluster of ligand-binding repeats in megalin as a site for receptor-ligand interactions.
Megalin is a large cell surface receptor that mediates the binding and internalization of a number of structurally and functionally distinct ligands from the lipoprotein and protease:protease inhibitor families. To begin to address how megalin is able to bind ligands with unique structurally properties, we have mapped a binding site for apolipoprotein E (apoE)-beta very low density lipoprotein (beta VLDL), lipoprotein lipase, aprotinin, lactoferrin, and the receptor-associated protein (RAP) within the primary sequence of the receptor. RAP is known to inhibit the binding of all ligands to megalin. We identified a ligand-binding site on megalin by raising mAb against purified megalin, selected for a mAb whose binding to megalin is inhibited by RAP, and mapped the epitope for this mAb. mAb AC10 inhibited the binding of apoE-beta VLDL, lipoprotein lipase, aprotinin, and lactoferrin to megalin in a concentration-dependent manner. When cDNA fragments encoding the four cysteine-rich ligand-binding repeats in megalin were expressed in a baculovirus system and immunoblotted with AC10, it recognized only the second cluster of ligand-binding repeats. The location of the epitope recognized by mAb AC10 within this domain was pinpointed to amino acids 1111-1210. From these studies we conclude that the binding of apoE-beta VLDL, lactoferrin, aprotinin, lipoprotein lipase, and RAP to megalin is either competitively or sterically inhibited by mAb AC10 suggesting that these ligands bind to the same or closely overlapping sites within the second cluster of ligand-binding repeats.
Orlando
R A
RA
Department of Pathology, University of California, San Diego, La Jolla 92093-0651, USA.
Exner
M
M
Czekay
R P
RP
Yamazaki
H
H
Saito
A
A
Ullrich
R
R
Kerjaschki
D
D
Farquhar
M G
MG
D000818:Animals; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000378:metabolism; D003545:Cysteine; D006023:Glycoproteins / Q000378:metabolism; D026541:Heymann Nephritis Antigenic Complex; D015151:Immunoblotting; D007668:Kidney / Q000276:immunology / Q000378:metabolism*; D007700:Kinetics; D026563:LDL-Receptor Related Protein-Associated Protein; D008024:Ligands; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D008871:Microvilli / Q000378:metabolism*; D008961:Models, Structural; D011487:Protein Conformation; D051381:Rats; D011973:Receptors, LDL / Q000737:chemistry / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D006023:Glycoproteins; D026541:Heymann Nephritis Antigenic Complex; D026563:LDL-Receptor Related Protein-Associated Protein; D008024:Ligands; D008562:Membrane Glycoproteins; D011973:Receptors, LDL; D003545:Cysteine