title: Receptor-associated protein (RAP) has two high-affinity binding sites for the low-density lipoprotein receptor-related protein (LRP): consequences for the chaperone functions of RAP.
abstract: RAP (receptor-associated protein) is a three domain 38 kDa ER (endoplasmic reticulum)-resident protein that is a chaperone for the LRP (low-density lipoprotein receptor-related protein). Whereas RAP is known to compete for binding of all known LRP ligands, neither the location, the number of binding sites on LRP, nor the domains of RAP involved in binding is known with certainty. We have systematically examined the binding of each of the three RAP domains (D1, D2 and D3) to tandem and triple CRs (complement-like repeats) that span the principal ligand-binding region, cluster II, of LRP. We found that D3 binds with low nanomolar affinity to all (CR)2 species examined. Addition of a third CR domain increases the affinity for D3 slightly. A pH change from 7.4 to 5.5 gave only a 6-fold increase in Kd for D3 at 37 degrees C, whereas temperature change from 22 degrees C to 37 degrees C has a similar small effect on affinity, raising questions about the recently proposed D3-destabilization mechanism of RAP release from LRP. Surprisingly, and in contrast to literature suggestions, D1 and D2 also bind to most (CR)2 and (CR)3 constructs with nanomolar affinity. Although this suggested that there might be three high-affinity binding sites in RAP for LRP, studies with intact RAP showed that only two binding sites are available in the intact chaperone. These findings suggest a new model for RAP to function as a folding chaperone and also for the involvement of YWTD domains in RAP release from LRP in the Golgi.
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, 900 S. Ashland, M/C 669, Chicago, IL 60607, USA.
authors.forename: Jan K
authors.initials: JK
authors.lastname: Jensen
authors.forename: Klavs
authors.initials: K
authors.lastname: Dolmer
authors.forename: Christine
authors.initials: C
authors.lastname: Schar
authors.forename: Peter G W
authors.initials: PG
authors.lastname: Gettins
mesh_terms: D001665:Binding Sites; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D026563:LDL-Receptor Related Protein-Associated Protein / Q000737:chemistry* / Q000378:metabolism; D026503:Low Density Lipoprotein Receptor-Related Protein-1 / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D013696:Temperature
chemical_list: D026563:LDL-Receptor Related Protein-Associated Protein; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D018832:Molecular Chaperones