title: Unfolding of the RAP-D3 helical bundle facilitates dissociation of RAP-receptor complexes.
abstract: The receptor-associated protein (RAP) functions as an escort protein for receptors of the low-density lipoprotein receptor (LDLR) family by preventing premature intracellular binding of ligands and assisting with delivery of mature receptors to the cell surface. The modulation of affinity by pH is believed to play an important role in the escort function of RAP, because RAP binds tightly to proteins of the LDLR family at near-neutral pH early in the secretory pathway where its high affinity precludes premature binding of ligands but then dissociates from bound receptors at the lower pH of the Golgi compartment. The third domain of RAP (RAP-D3), which forms a three-helix bundle, is sufficient to reconstitute the escort activity. Here, we test the hypothesis that low-pH induced unfolding of the RAP-D3 helical bundle facilitates dissociation of RAP-receptor complexes. First, variants of RAP-D3 resistant to low pH-induced unfolding were constructed by replacing interior histidine residues with phenylalanines. In contrast to native RAP-D3, which exhibits an unfolding pKa of 6.3 and a Tm of 42 degrees C, the most hyperstable variant of RAP-D3, in which four histidine residues are replaced with phenylalanine, has an unfolding pKa of 4.8, and a Tm of 58 degrees C. The phenylalanine substitutions in RAP-D3 confer increased stability to pH-induced dissociation of complexes formed between RAP-D3 and a two-repeat fragment of the LDLR (LA3-4). When introduced into full-length RAP, the four mutations that confer hyperstability on RAP-D3 interfere with transport of endogenous LRP-1 to the cell surface in a dominant negative fashion under conditions where expression of normal RAP has no effect on LRP-1 transport. Our studies support a model in which low pH-dependent unfolding of RAP-D3 facilitates dissociation of RAP from the LA repeats of LDLR family proteins in the mildly acidic pH of the Golgi.
authors.affiliation: Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Kristine
authors.initials: K
authors.lastname: Estrada
authors.forename: Carl
authors.initials: C
authors.lastname: Fisher
authors.forename: Stephen C
authors.initials: SC
authors.lastname: Blacklow
mesh_terms: D002942:Circular Dichroism; D006863:Hydrogen-Ion Concentration; D026563:LDL-Receptor Related Protein-Associated Protein / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D011489:Protein Denaturation
chemical_list: D026563:LDL-Receptor Related Protein-Associated Protein