Structural Organization of the Receptor Associated Protein

Lazic, Ana; Dolmer, Klavs; Strickland, Dudley K.; Gettins, Peter G.W.

doi:10.1021/bi035779e

Cited by 14 publications

(20 citation statements)

References 16 publications

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“…These observations are all consistent with previously reported studies (7,9). To test the hypothesis that low pH-induced bundle unfolding drives dissociation of RAP-receptor complexes, we sought to construct variants of the RAP-D3 helical bundle that were thermostable, retained near-native affinity for LA repeat pairs, and exhibited increased resistance to unfolding induced by low pH.…”

Section: Resultssupporting

confidence: 84%

Unfolding of the RAP-D3 Helical Bundle Facilitates Dissociation of RAP−Receptor Complexes

2008

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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.

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Section: Resultssupporting

confidence: 84%

Unfolding of the RAP-D3 Helical Bundle Facilitates Dissociation of RAP−Receptor Complexes

2008

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“…We asked whether turnover of matrix fibronectin in FN-null MF depends on LRP by examining whether the LRP inhibitor, receptor-associated protein (RAP), could inhibit loss or degradation of matrix fibronectin. RAP binds to LRP and competes for binding with LRP ligands, thus preventing their internalization and degradation (Herz et al, 1991;Godyna et al, 1995a;Lazic et al, 2003). As shown in Figure 6A, cells incubated with RAP degraded similar levels of fibronectin as control cells.…”

Section: Fibronectin Degradation In Fn-null Mf Is Not Lipoprotein Recmentioning

confidence: 84%

Fibronectin Matrix Turnover Occurs through a Caveolin-1–dependent Process

Sottile

Chandler

2005

MBoC

123

143

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Extracellular matrix remodeling occurs during development, tissue repair, and in a number of pathologies, including fibrotic disorders, hypertension, and atherosclerosis. Extracellular matrix remodeling involves the complex interplay between extracellular matrix synthesis, deposition, and degradation. Factors that control these processes are likely to play key roles in regulating physiological and pathological extracellular matrix remodeling. Our data show that fibronectin polymerization into the extracellular matrix regulates the deposition and stability of other extracellular matrix proteins, including collagen I and thrombospondin-1 (Sottile and Hocking, 2002. Mol. Biol. Cell 13, 3546). In the absence of continual fibronectin polymerization, there is a loss of fibronectin matrix fibrils, and increased levels of fibronectin degradation. Fibronectin degradation occurs intracellularly after endocytosis and can be inhibited by chloroquine, an inhibitor of lysosomal degradation, and by caveolae-disrupting agents. Down-regulation of caveolin-1 by RNAi inhibits loss of fibronectin matrix fibrils, fibronectin internalization, and fibronectin degradation; these processes can be restored by reexpression of caveolin-1. These data show that fibronectin matrix turnover occurs through a caveolin-1-dependent process. Caveolin-1 regulation of fibronectin matrix turnover is a novel mechanism regulating extracellular matrix remodeling. INTRODUCTIONExtracellular matrix (ECM) remodeling is a dynamic, cellmediated process that occurs during development, tissue repair, and in a variety of pathological events including atherosclerosis, hypertension, and ischemic injury (Clark, 1996;Prescott et al., 1999;Streuli, 1999;Newby and Zaltsman, 2000;Intengan and Schiffrin, 2001). Furthermore, abnormal matrix remodeling is associated with fibrosis, arthritis, reduced angiogenesis, and developmental abnormalities (Liu et al., 1995;Vu et al., 1998;Holmbeck et al., 1999). During tissue repair, the precise deposition of ECM molecules, including collagen I and fibronectin, is required to preserve the structural and functional integrity of tissues (Clark, 1996). Excessive or inappropriate deposition of ECM molecules, as occurs during fibrosis, disrupts normal tissue architecture, leading to impaired organ function (Mutsaers et al., 1997;Zeisberg et al., 2000). The mechanisms that control ECM organization and homeostasis are incompletely understood. We have recently shown that fibronectin matrix polymerization is essential for the organization, as well as the maintenance of ECM architecture . Our data show that the cell-dependent process of polymerizing fibronectin into the ECM is required for the deposition and maintenance of fibrillar fibronectin, collagen-I, and thrombosponin-1 . These data are consistent with other studies showing that collagen I and collagen III deposition into the ECM are regulated by fibronectin (McDonald et al., 1982;Velling et al., 2002). Fibronectin has also been implicated in regulating the deposition of tenascin C (Chu...

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“…Indeed, deficiency in either LDLR or LRP in mice prolonged the half-life of FVIII about 1.5-fold, whereas the combined deficiency resulted in ϳ4.8-fold prolongation (2). Furthermore, inhibition of both receptors by their highly specific ligand ␣-2-macroglobulin receptor-associated protein (RAP) (3,4) increased its half-life in mice (5,6). In humans, polymorphism in either LDLR or LRP is associated with elevated levels of FVIII (7)(8)(9)(10).…”

Section: Factor VIII (Fviii)mentioning

confidence: 99%

Mapping the Binding Region on the Low Density Lipoprotein Receptor for Blood Coagulation Factor VIII

Kurasawa

Shestopal

Karnaukhova

et al. 2013

Journal of Biological Chemistry

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Background: Low density lipoprotein receptor (LDLR) mediates clearance of blood coagulation factor VIII (FVIII). Results:The region of complement-type repeats 2-5 in LDLR was identified as the binding site for FVIII and for ␣-2-macroglobulin receptor-associated protein (RAP). Conclusion: Binding sites of LDLR for FVIII, and also for RAP, were characterized. Significance: This provides new data on LDLR structure and function and on FVIII catabolism.

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Structural Organization of the Receptor Associated Protein

Cited by 14 publications

References 16 publications

Unfolding of the RAP-D3 Helical Bundle Facilitates Dissociation of RAP−Receptor Complexes

Unfolding of the RAP-D3 Helical Bundle Facilitates Dissociation of RAP−Receptor Complexes

Fibronectin Matrix Turnover Occurs through a Caveolin-1–dependent Process

Mapping the Binding Region on the Low Density Lipoprotein Receptor for Blood Coagulation Factor VIII

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