Allosteric modulation of ligand binding to low density lipoprotein receptor-related protein by the receptor-associated protein requires critical lysine residues within its carboxyl-terminal domain.
The low density lipoprotein receptor-related protein (LRP) is a large endocytic receptor that recognizes more than 30 different ligands and plays important roles in protease and lipoprotein catabolism. Ligand binding to newly synthesized LRP is modulated by the receptor-associated protein (RAP), an endoplasmic reticulum-resident protein that functions as a molecular chaperone and prevents ligands from associating with LRP via an allosteric-type mechanism. RAP is a multidomain protein that contains two independent LRP binding sites, one located at the amino-terminal portion of the molecule and the other at the carboxyl-terminal portion of the molecule. The objective of the present investigation was to gain insight into how these two regions of RAP interact with LRP and function to modulate its ligand binding properties. These objectives were accomplished by random mutagenesis of RAP, which identified two critical lysine residues, Lys-256 and Lys-270, within the carboxyl-terminal domain that are necessary for binding of this region of RAP to LRP and to heparin. RAP molecules in which either of these two lysine residues was mutated still bound LRP but with reduced affinity. Furthermore, the mutant RAPs were significantly impaired in their ability to inhibit alpha(2)M* binding to LRP via allosteric mechanisms. In contrast, the mutant RAP molecules were still effective at inhibiting uPA.PAI-1 binding to LRP. These results confirm that both LRP binding sites within RAP cooperate to inhibit ligand binding via an allosteric mechanism.
Migliorini
Mary M
MM
Department of Vascular Biology, Jerome H. Holland Laboratory for the Biomedical Sciences, American Red Cross, Rockville, Maryland 20855, USA.
Behre
Evan H
EH
Brew
Shelesa
S
Ingham
Kenneth C
KC
Strickland
Dudley K
DK
D000495:Allosteric Site; D001667:Binding, Competitive; D002151:Calorimetry; D002847:Chromatography, Agarose; D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006493:Heparin / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D026563:LDL-Receptor Related Protein-Associated Protein / Q000737:chemistry* / Q000378:metabolism*; D026502:LDL-Receptor Related Proteins / Q000378:metabolism*; D008024:Ligands; D008239:Lysine / Q000737:chemistry; D008954:Models, Biological; D016296:Mutagenesis; D009154:Mutation; D017354:Point Mutation; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary
D026563:LDL-Receptor Related Protein-Associated Protein; D026502:LDL-Receptor Related Proteins; D008024:Ligands; D006493:Heparin; D008239:Lysine