Binding Of Receptor-associated Protein Research Articles

Members of the low‐density lipoprotein receptor‐related proteins provide a differential molecular signature between parental and CD133(+) DAOY medulloblastoma cells

Members of the low-density lipoprotein receptor-related protein (LRP) family are involved in metabolic stress and resistance phenotypes of cancer cells. New breakthroughs in brain cancer therapy have exploited that molecular signature and proved that efficient delivery of therapeutic agents involve LRP-mediated mechanisms. We performed gene expression profiling of CD133, a cell surface cancer stem cell marker, and of LRP in response to in vitro nutrient deprivation. We found that CD133 was selectively induced in serum-starved DAOY medulloblastoma cells but not in U87MG glioblastoma cells. Such CD133 induction was correlated to increases in LRP-1 and LRP-1b gene and protein expression. When a specific CD133(+) DAOY cell population was sorted from parental DAOY, we found increases in LRP-5 and LRP-8. Uptake of alpha(2)-macroglobulin, a specific LRP-1/1b ligand, was increased in serum-starved parental DAOY cells but not in CD133(+) DAOY cells, and receptor-associated protein (RAP), which binds to all cell surface LRPs, was able to compete for that uptake. Conversely, RAP binding was increased in serum-starved parental DAOY but alpha(2)-macroglobulin was unable to compete for such uptake. Strategies aiming at targeting cancer stem cell metabolic adaptative responses, such as that through LRP differential expression within the brain tissue microenvironmental niche, can now be envisioned.

Dissection of RAP-LRP interactions: Binding of RAP and RAP fragments to complement-like repeats 7 and 8 from ligand binding cluster II of LRP

The receptor associated protein (RAP) is a three domain 38 kDa ER-resident chaperone that helps folding of LRP and other LDL receptor family members and prevents premature binding of protein ligands. It competes strongly with all known LRP ligands. To further understanding of the specificity of RAP-LRP interactions, the binding of RAP and RAP fragments to two domains (CR7–CR8) from one of the main ligand-binding regions of LRP has been examined by 2D HSQC NMR spectroscopy and isothermal titration calorimetry. We found that RAP contains two binding sites for CR7–CR8, with the higher affinity site ( K d ∼ 1 μM) located in the C-terminal two-thirds and the weaker site ( K d ∼ 5 μM) in the N-terminal third of RAP. Residues from both CR7 and CR8 are involved in binding at each RAP site. The presence of more than one binding site on RAP for CR domains from LRP, together with the previous demonstration by others that RAP can bind to CR5–CR6 with comparably low affinities suggest an explanation for the dual roles of RAP as a folding chaperone and a tight competitive inhibitor of ligand binding.

Interaction of Lipoprotein Lipase and Receptor-associated Protein

Receptor-associated protein (RAP) is a recognized chaperone/escort protein for members of the low density lipoprotein receptor family. In this report, we show that RAP binds to lipoprotein lipase (LPL) and may play a role in the maturation of LPL. Binding of highly purified RAP to LPL was demonstrated in vitro by solid phase assays, surface plasmon resonance, and rate zonal centrifugation. The dissociation constant for this interaction measured by the first two techniques ranged between 2.4 and 13 nM, values similar to those reported for the binding of RAP to LRP or gp330. The specificity of the interaction was demonstrated by competition with a panel of LPL monoclonal antibodies. Rate zonal centrifugation demonstrated the presence of a stable complex with an apparent Mr consistent with the formation of a complex between monomeric LPL and RAP. RAP x LPL complexes were co-immunoprecipitated in adipocyte lysates or from solutions of purified LPL and RAP. The interaction was also demonstrated in whole cells by cross-linking experiments. RAP-deficient adipocytes secreted LPL with a specific activity 2.5-fold lower than the lipase secreted by control cells. Heparin addition to cultured RAP-deficient adipocytes failed to stimulate LPL secretion in the medium, suggesting defective binding of the lipase to the plasma membrane. These studies demonstrate that RAP binds to LPL with high affinity both in purified systems and cell extracts and that RAP-deficient adipocytes secrete poorly assembled LPL. A function of RAP may be to prevent premature interaction of LPL with binding partners in the secretory pathway, namely LRP and heparan sulfate proteoglycan.

Mapping of the receptor-associated protein (RAP) binding proteins on living fibroblast cells using an atomic force microscope

The distribution of the receptor-associated protein (RAP) binding protein and the adhesion forces between RAP and its binding protein on living fibroblast cells were examined using an atomic force microscope (AFM). The distribution of RAP binding protein was obtained on 256 (16×16) locations in 2×2 μm sections over the surface of living cells. The adhesion forces between RAP and the binding protein were measured with an AFM tip functionalized with RAP. In the presence of RAP in the scanning solution, the number of force curves with large adhesion force decreased. These results indicate that the adhesive forces observed here represent specific binding between RAP and the binding protein. This method will be a useful application of AFM to examine receptors on cell surfaces in high resolution.

Dominant thermodynamic role of the third independent receptor binding site in the receptor-associated protein RAP.

The 39 kDa receptor-associated protein (RAP) is a three-domain escort protein in the secretory pathway for several members of the low-density lipoprotein receptor (LDLR) family of endocytic receptors, including the LDLR-related protein (LRP). The minimal functional unit of LRP required for efficient binding to RAP is composed of complement-type repeat (CR)-domain pairs, located in clusters on the extracellular part of LRP. Here we investigate the binding of full-length RAP and isolated RAP domains 1-3 to an ubiquitin-fused CR-domain pair consisting of the fifth and sixth CR domains of LRP (U-CR56). As shown by isothermal titration calorimetric analysis of simple RAP domains as well as adjoined RAP domains, all three RAP domains bind to this CR-domain pair in a noncooperative way. The binding of U-CR56 to RAP domains 1 and 2 is (at room temperature) enthalpically driven with an entropy penalty (K(D) = 2.77 x 10(-6) M and 1.85 x 10(-5) M, respectively), whereas RAP domain 3 binds with a substantially lower enthalpy, but is favored due to a positive entropic contribution (K(D) = 1.71 x 10(-7) M). The heat capacity change for complex formation between RAP domain 1 and the CR-domain pair is -1.65 kJ K(-1) mol(-1). There is an indication of a conformational change in RAP domain 3 upon binding in the surface plasmon resonance analysis of the interaction. The different mechanisms of binding to RAP domains 1 and 3 are further substantiated by the different effects on binding of mutations of the Asp and Trp residues in the LRP CR5 or CR6 domains, which are important for the recognition of several ligands.

Binding of the low density lipoprotein receptor-associated protein (RAP) to thyroglobulin (Tg): putative role of RAP in the Tg secretory pathway.

The 39-44 kDa protein known as the receptor-associated protein binds to members of the low density lipoprotein receptor family and is found within cells that express these receptors. The receptor-associated protein has been shown to prevent premature binding of ligands to the receptors in the endoplasmic reticulum and to promote proper folding and transport of the receptors in the secretory pathway. In the thyroid, megalin (a low-density lipoprotein receptor family member) serves as an endocytic receptor for thyroglobulin. Here we present evidence that the receptor-associated protein can bind to thyroglobulin, which suggests a novel function of the receptor-associated protein, namely binding of certain megalin ligands possibly during the biosynthetic pathway. In solid-phase assays thyroglobulin was shown to bind to the receptor-associated protein with moderately high affinity (mean between K(d) and K(i) = 39.8 nM), in a calcium-dependent and saturable manner. The receptor-associated protein also bound to a native carboxyl-terminal 230-kDa thyroglobulin polypeptide, which markedly reduced binding of intact thyroglobulin to the receptor associated protein, indicating that the receptor-associated protein binding sites of thyroglobulin are located in the carboxyl-terminal portion of the molecule. In addition to thyroglobulin, the receptor-associated protein specifically bound to another megalin ligand, namely lipoprotein lipase. Because lipoprotein lipase markedly reduced receptor-associated protein binding to thyroglobulin, we concluded that the receptor-associated protein uses the same binding site/s to bind to thyroglobulin and lipoprotein lipase. Evidence of thyroglobulin binding to the receptor-associated protein was also obtained in vivo and in cultured thyroid cells. Thus, anti-receptor-associated protein antibodies precipitated intact thyroglobulin from extracts prepared from rat thyroids and cultured thyroid cells (FRTL-5 cells). Chase experiments after inhibition of protein synthesis in FRTL-5 cells showed that thyroglobulin interacts with the receptor-associated protein shortly after the beginning of thyroglobulin biosynthesis.

High Affinity Binding of Receptor-associated Protein to Heparin and Low Density Lipoprotein Receptor-related Protein Requires Similar Basic Amino Acid Sequence Motifs

The 39-kDa receptor-associated protein (RAP) is a specialized chaperone for members of the low density lipoprotein receptor gene family, which also binds heparin. Previous studies have identified a triplicate repeat sequence within RAP that appears to exhibit differential functions. Here we generated a series of truncated and site-directed RAP mutants in order to define the sites within RAP that are important for interacting with heparin and low density lipoprotein receptor-related protein (LRP). We found that high affinity binding of RAP to heparin is mediated by the carboxyl-terminal repeat of RAP, whereas both the carboxyl-terminal repeat and a combination of amino and central repeats exhibit high affinity binding to LRP. Several motifs were found to mediate the binding of RAP to heparin, and each contained a cluster of basic amino acids; among them, an intact R(282)VSR(285)SR(287)EK(289) motif is required for high affinity binding of RAP to heparin, whereas two other motifs, R(203)LR(205)R(206) and R(314)ISR(317)AR(319), also contribute to this interaction. We also found that intact motifs of both R(203)LR(205)R(206) and R(282)VSR(285)SR(287)EK(289) are required for high affinity binding of RAP to LRP, with the third motif, R(314)ISR(317)AR(319), contributing little to RAP-LRP interaction. We conclude that electrostatic interactions likely contribute significantly in the binding of RAP to both heparin and LRP and that high affinity interaction with both heparin and LRP appears to require mostly overlapping sequence motifs within RAP.

Analysis of a two-domain binding site for the urokinase-type plasminogen activator-plasminogen activator inhibitor-1 complex in low-density-lipoprotein-receptor-related protein.

The low-density-lipoprotein-receptor (LDLR)-related protein (LRP) is composed of several classes of domains, including complement-type repeats (CR), which occur in clusters that contain binding sites for a multitude of different ligands. Each approximately 40-residue CR domain contains three conserved disulphide linkages and an octahedral Ca(2+) cage. LRP is a scavenging receptor for ligands from extracellular fluids, e.g. alpha(2)-macroglobulin (alpha(2)M)-proteinase complexes, lipoprotein-containing particles and serine proteinase-inhibitor complexes, like the complex between urokinase-type plasminogen activator (uPA) and the plasminogen activator inhibitor-1 (PAI-1). In the present study we analysed the interaction of the uPA-PAI-1 complex with an ensemble of fragments representing a complete overlapping set of two-domain fragments accounting for the ligand-binding cluster II (CR3-CR10) of LRP. By ligand blotting, solid-state competition analysis and surface-plasmon-resonance analysis, we demonstrate binding to multiple CR domains, but show a preferential interaction between the uPA-PAI-1 complex and a two-domain fragment comprising CR domains 5 and 6 of LRP. We demonstrate that surface-exposed aspartic acid and tryptophan residues at identical positions in the two homologous domains, CR5 and CR6 (Asp(958,CR5), Asp(999,CR6), Trp(953,CR5) and Trp(994,CR6)), are critical for the binding of the complex as well as for the binding of the receptor-associated protein (RAP) - the folding chaperone/escort protein required for transport of LRP to the cell surface. Accordingly, the present work provides (1) an identification of a preferred binding site within LRP CR cluster II; (2) evidence that the uPA-PAI-1 binding site involves residues from two adjacent protein domains; and (3) direct evidence identifying specific residues as important for the binding of uPA-PAI-1 as well as for the binding of RAP.

Analysis of a two-domain binding site for the urokinase-type plasminogen activator–plasminogen activator inhibitor-1 complex in low-density-lipoprotein-receptor-related protein

The low-density-lipoprotein-receptor (LDLR)-related protein (LRP) is composed of several classes of domains, including complement-type repeats (CR), which occur in clusters that contain binding sites for a multitude of different ligands. Each ≈ 40-residue CR domain contains three conserved disulphide linkages and an octahedral Ca2+ cage. LRP is a scavenging receptor for ligands from extracellular fluids, e.g. α2-macroglobulin (α2M)–proteinase complexes, lipoprotein-containing particles and serine proteinase–inhibitor complexes, like the complex between urokinase-type plasminogen activator (uPA) and the plasminogen activator inhibitor-1 (PAI-1). In the present study we analysed the interaction of the uPA–PAI-1 complex with an ensemble of fragments representing a complete overlapping set of two-domain fragments accounting for the ligand-binding cluster II (CR3–CR10) of LRP. By ligand blotting, solid-state competition analysis and surface-plasmon-resonance analysis, we demonstrate binding to multiple CR domains, but show a preferential interaction between the uPA–PAI-1 complex and a two-domain fragment comprising CR domains 5 and 6 of LRP. We demonstrate that surface-exposed aspartic acid and tryptophan residues at identical positions in the two homologous domains, CR5 and CR6 (Asp958,CR5, Asp999,CR6, Trp953,CR5 and Trp994,CR6), are critical for the binding of the complex as well as for the binding of the receptor-associated protein (RAP)–the folding chaperone/escort protein required for transport of LRP to the cell surface. Accordingly, the present work provides (1) an identification of a preferred binding site within LRP CR cluster II; (2) evidence that the uPA–PAI-1 binding site involves residues from two adjacent protein domains; and (3) direct evidence identifying specific residues as important for the binding of uPA–PAI-1 as well as for the binding of RAP.

An Extrahepatic Receptor-associated Protein-sensitive Mechanism Is Involved in the Metabolism of Triglyceride-rich Lipoproteins

We have used adenovirus-mediated gene transfer in mice to investigate low density lipoprotein receptor (LDLR) and LDLR-related protein (LRP)-independent mechanisms that control the metabolism of chylomicron and very low density lipoprotein (VLDL) remnants in vivo. Overexpression of receptor-associated protein (RAP) in mice that lack both LRP and LDLR (MX1cre(+)LRP(flox/flox)LDLR(-/-)) in their livers elicited a marked hypertriglyceridemia in addition to the pre-existing hypercholesterolemia in these animals, resulting in a shift in the distribution of plasma lipids from LDL-sized lipoproteins to large VLDL-sized particles. This dramatic increase in plasma lipids was not due to a RAP-mediated inhibition of a unknown hepatic high affinity binding site involved in lipoprotein metabolism, because no RAP binding could be detected in livers of MX1cre(+)LRP(flox/flox)LDLR(-/-) mice using both membrane binding studies and ligand blotting experiments. Remarkably, RAP overexpression also resulted in a 7-fold increase (from 13.6 to 95.6 ng/ml) of circulating, but largely inactive, lipoprotein lipase (LPL). In contrast, plasma hepatic lipase levels and activity were unaffected. In vitro studies showed that RAP binds to LPL with high affinity (K(d) = 5 nM) but does not affect its catalytic activity, in vitro or in vivo. Our findings suggest that an extrahepatic RAP-sensitive process that is independent of the LDLR or LRP is involved in metabolism of triglyceride-rich lipoproteins. There, RAP may affect the functional maturation of LPL, thus causing the accumulation of triglyceride-rich lipoproteins in the circulation.

The Second and Fourth Cluster of Class A Cysteine-rich Repeats of the Low Density Lipoprotein Receptor-related Protein Share Ligand-binding Properties

The low density lipoprotein receptor-related protein (LRP) is a multifunctional endocytic cell-surface receptor that binds and internalizes a diverse array of ligands. The receptor contains four putative ligand-binding domains, generally referred to as clusters I, II, III, and IV. In this study, soluble recombinant receptor fragments, representing each of the four individual clusters, were used to map the binding sites of a set of structurally and functionally distinct ligands. Using surface plasmon resonance, we studied the binding of these fragments to methylamine-activated alpha(2)-macroglobulin, pro-urokinase-type plasminogen activator, tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor-1, t-PA.plasminogen activator inhibitor-1 complexes, lipoprotein lipase, apolipoprotein E, tissue factor pathway inhibitor, lactoferrin, the light chain of blood coagulation factor VIII, and the intracellular chaperone receptor-associated protein (RAP). No binding of the cluster I fragment to any of the tested ligands was observed. The cluster III fragment only bound to the anti-LRP monoclonal antibody alpha(2)MRalpha3 and weakly to RAP. Except for t-PA, we found that each of the ligands tested binds both to cluster II and to cluster IV. The affinity rate constants of ligand binding to clusters II and IV and to LRP were measured, showing that clusters II and IV display only minor differences in ligand-binding kinetics. Furthermore, we demonstrate that the subdomains C3-C7 of cluster II are essential for binding of ligands and that this segment partially overlaps with a RAP-binding site on cluster II. Finally, we show that one RAP molecule can bind to different clusters simultaneously, supporting a model in which RAP binding to LRP induces a conformational change in the receptor that is incompatible with ligand binding.

Functional domains of the very low density lipoprotein receptor: molecular analysis of ligand binding and acid-dependent ligand dissociation mechanisms.

The very low density lipoprotein (VLDL) receptor is closely related in structure to the low density lipoprotein receptor. The ectodomain of these endocytic receptors is composed of modules which include clusters of cysteine-rich class A repeats, epidermal growth factor (EGF)-like repeats, tyrosine-tryptophan-threonine-aspartic acid (YWTD) repeats and an O-linked sugar domain. To identify important functional regions within the ectodomain of the VLDL receptor, we produced a mutant receptor in which the EGF, YWTD and O-linked sugar domains were deleted. Cells transfected with the mutant receptor were able to bind and internalize (125)I-labeled receptor associated protein (RAP). In contrast to the wild-type receptor, however, RAP did not dissociate from the mutant receptor and consequently was not degraded. Immunofluoresence data indicated that once bound to the mutant receptor, fluorescent-labeled RAP co-localized with markers of the endosomal pathway, whereas, in cells expressing the wild-type receptor, RAP fluorescence co-localized with lysosomal markers. Thus this deleted region is responsible for ligand uncoupling within the endosomes. To identify regions responsible for ligand recognition, soluble receptor fragments containing the eight cysteine-rich class A repeats were produced. (125)I-RAP and (125)I-labeled urokinase-type plasminogen activator:plasminogen activator inhibitor type I (uPA:PAI-1) complexes bound to the soluble fragment with K(D, app) values of 0.3 and 14 nM, respectively. Deletion analysis demonstrate that high affinity RAP binding requires the first four cysteine-rich class A repeats (L1-4) in the VLDL receptor while the second repeat (L2) appears responsible for binding uPA:PAI-1 complexes. Together, these results confirm that ligand uncoupling occurs via an allosteric-type mechanism in which pH induced changes in the EGF and/or YWTD repeats alter the ligand binding properties at the amino-terminal portion of the molecule.

Ligand Binding Properties of the Very Low Density Lipoprotein Receptor: ABSENCE OF THE THIRD COMPLEMENT-TYPE REPEAT ENCODED BY EXON 4 IS ASSOCIATED WITH REDUCED BINDING OF Mr 40,000 RECEPTOR-ASSOCIATED PROTEIN

The very low density lipoprotein receptor (VLDLR) binds, among other ligands, the Mr 40,000 receptor-associated protein (RAP) and a variety of serine proteinase-serpin complexes, including complexes of the proteinase urokinase-type plasminogen activator (uPA) with the serpins plasminogen activator inhibitor-1 (PAI-1) and protease nexin-1 (PN-1). We have analyzed the binding of RAP, uPA.PAI-1, and uPA.PN-1 to two naturally occurring VLDLR variants, VLDLR-I, containing all eight complement-type repeats, and VLDLR-III, lacking the third complement-type repeat, encoded by exon 4. VLDLR-III displayed approximately 4-fold lower binding of RAP than VLDLR-I and approximately 10-fold lower binding of the most C-terminal one of the three domains of RAP. In contrast, the binding of uPA.PAI-1 and uPA.PN-1 to the two VLDLR variants was indistinguishable. Surprisingly, uPA.PN-1, but not uPA.PAI-1, competed RAP binding to both VLDLR variants. These observations show that the third complement-type repeat plays a crucial role in maintaining the contact sites needed for optimal recognition of RAP, but does not affect the proteinase-serpin complex contact sites, and that two ligands can show full cross-competition without sharing the same contacts with the receptor. These results elucidate the mechanisms of molecular recognition of ligands by receptors of the low density lipoprotein receptor family.

Sortilin/Neurotensin Receptor-3 Binds and Mediates Degradation of Lipoprotein Lipase

Lipoprotein lipase and the receptor-associated protein (RAP) bind to overlapping sites on the low density lipoprotein receptor-related protein/alpha2-macroglobulin receptor (LRP). We have investigated if lipoprotein lipase interacts with the RAP binding but structurally distinct receptor sortilin/neurotensin receptor-3. We show, by chemical cross-linking and surface plasmon resonance analysis, that soluble sortilin binds lipoprotein lipase with an affinity similar to that of LRP. The binding was inhibited by heparin and RAP and by the newly discovered sortilin ligand neurotensin. In 35S-labeled 3T3-L1 adipocytes treated with the cross-linker dithiobis(succinimidyl propionate), lipoprotein lipase-containing complexes were isolated by anti-sortilin antibodies. To elucidate function in cells, sortilin-negative Chinese hamster ovary cells were transfected with full-length sortilin and shown to express about 8% of the receptors on the cell surface. These cells degraded 125I-labeled lipoprotein lipase much faster than the wild-type cells. The degradation was inhibited by unlabeled lipoprotein lipase, indicating a saturable pathway, and by RAP and heparin. Moreover, inhibition by the weak base chloroquine suggested that degradation occurs in an acidic vesicle compartment. The results demonstrate that sortilin is a multifunctional receptor that binds lipoprotein lipase and, when expressed on the cell surface, mediates its endocytosis and degradation.

Propeptide cleavage conditions sortilin/neurotensin receptor-3 for ligand binding.

We recently reported the isolation and sequencing of sortilin, a new putative sorting receptor that binds receptor-associated protein (RAP). The luminal N-terminus of sortilin comprises a consensus sequence for cleavage by furin, R41WRR44, which precedes a truncation originally found in sortilin isolated from human brain. We now show that the truncation results from cellular processing. Sortilin is synthesized as a proform which, in late Golgi compartments, is converted to the mature receptor by furin-mediated cleavage of a 44 residue N-terminal propeptide. We further demonstrate that the propeptide exhibits pH-dependent high affinity binding to fully processed sortilin, that the binding is competed for by RAP and the newly discovered sortilin ligand neurotensin, and that prevention of propeptide cleavage essentially prevents binding of RAP and neurotensin. The findings evidence that the propeptide sterically hinders ligands from gaining access to overlapping binding sites in prosortilin, and that cleavage and release of the propeptide preconditions sortilin for full functional activity. Although proteolytic processing is involved in the maturation of several receptors, the described exposure of previously concealed ligand-binding sites after furin-mediated cleavage of propeptide represents a novel mechanism in receptor activation.

Characterization of an Epithelial ∼460-kDa Protein That Facilitates Endocytosis of Intrinsic Factor-Vitamin B12 and Binds Receptor-associated Protein

By using receptor-associated protein (RAP) as an affinity target, an intrinsic factor-vitamin B12 (IF-B12)-binding renal epithelial protein of approximately 460 kDa was copurified together with the transcobalamin-B12-binding 600-kDa receptor, megalin. IF-B12 affinity chromatography of renal cortex membrane from rabbit and man yielded the same approximately 460-kDa protein. Binding studies including surface plasmon resonance analyses of the protein demonstrated a calcium-dependent and high affinity binding of IF-B12 to a site distinct from the RAP binding site. The high affinity binding of IF-B12 was dependent on complex formation with vitamin B12. Light and electron microscope autoradiography of rat renal cortex cryosections incubated directly with IF-57Co-B12 and rat proximal tubules microinjected in vivo with the radioligand demonstrated binding of the ligand to endocytic invaginations of proximal tubule membranes followed by endocytosis and targeting of vitamin B12 to lysosomes. Polyclonal antibodies recognizing the approximately 460-kDa receptor inhibited the uptake. Immunohistochemistry of kidney and intestine showed colocalization of the IF-B12 receptor and megalin in both tissues. In conclusion, we have identified the epithelial IF-B12-binding receptor as a approximately 460-kDa RAP-binding protein facilitating endocytosis.

The solution structure of the N-terminal domain of alpha2-macroglobulin receptor-associated protein.

The three-dimensional structure of the N-terminal domain (residues 18-112) of alpha2-macroglobulin receptor-associated protein (RAP) has been determined by NMR spectroscopy. The structure consists of three helices composed of residues 23-34, 39-65, and 73-88. The three helices are arranged in an up-down-up antiparallel topology. The C-terminal 20 residues were shown not to be in a well defined conformation. A structural model for the binding of RAP to the family of low-density lipoprotein receptors is proposed. It defines a role in binding for both the unordered C terminus and the structural scaffold of the core structure. Pathogenic epitopes for the rat disease Heymann nephritis, an experimental model of human membranous glomerulonephritis, have been identified in RAP and in the large endocytic receptor gp330/megalin. Here we provide the three-dimensional structure of the pathogenic epitope in RAP. The amino acid residues known to form the epitope are in a helix-loop-helix conformation, and from the structure it is possible to rationalize the published results obtained from studies of fragments of the N-terminal domain.

Molecular Analysis of Ligand Binding to the Second Cluster of Complement-type Repeats of the Low Density Lipoprotein Receptor-related Protein: EVIDENCE FOR AN ALLOSTERIC COMPONENT IN RECEPTOR-ASSOCIATED PROTEIN-MEDIATED INHIBITION OF LIGAND BINDING

The low density lipoprotein receptor-related protein (LRP), a member of the low density lipoprotein receptor gene family, mediates the cellular uptake of a diversity of ligands. A folding chaperone, the 39-kDa receptor-associated protein (RAP) that resides in the early compartments of the secretory pathway inhibits the binding of all ligands to the receptor and may serve to prevent premature binding of ligands to the receptor during the trafficking to the cell surface. To elucidate the molecular interactions that underlie the interplay between the receptor, RAP, and the ligands, we have analyzed and delineated the binding sites of plasminogen activator inhibitor-1 (PAI-1), tissue-type plasminogen activator (t-PA).PAI-1 complexes, RAP, and the anti-LRP Fab fragment Fab A8. To that end, we have generated a series of soluble recombinant fragments spanning the second cluster of complement-type repeats (C3-C10) and the amino-terminal flanking epidermal growth factor repeat (E4) of LRP (E4-C10; amino acids 787-1165). All fragments were expressed by stably transfected baby hamster kidney cells and purified by affinity chromatography. A detailed study of ligand binding to the fragments using surface plasmon resonance revealed the presence of three distinct, Ca2+-dependent ligand binding sites in the cluster II domain (Cl-II) of LRP. t-PA.PAI-1 complexes as well as PAI-1 bind to a domain located in the amino-terminal portion of Cl-II, spanning repeats E4-C3-C7. Adjacent to this site and partially overlapping is a high affinity RAP-binding site located on repeats C5-C7. Fab A8, a pseudo-ligand of the receptor, binds to a third Ca2+-dependent binding site on repeats C8-C10 at the carboxyl-terminal end of Cl-II. Next, we studied the RAP-mediated inhibition of ligand binding to LRP and to Cl-II. As expected, we observed a strong inhibition of t-PA.PAI-1 complex and Fab A8 binding to LRP by RAP (IC50 congruent with 0.3 nM), whereas in the reverse experiment, competition of t-PA. PAI-1 complexes and Fab A8 for RAP binding to LRP could only be shown at high concentrations of competitors (>/=1 microM). Interestingly, even though the equilibrium dissociation constants for the binding of RAP to LRP and to Cl-II are similar, the binding of the ligands to Cl-II is only prevented by RAP at concentrations that are at least 2 orders of magnitude higher than those required for inhibition of ligand binding to LRP. Our results favor models that propose RAP-induced allosteric inhibition of ligand binding to LRP that may require LRP moieties that are located outside Cl-II of the receptor.

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.

The receptor-associated protein (RAP) binds calmodulin and is phosphorylated by calmodulin-dependent kinase II.

The receptor-associated protein, RAP, is an intracellular protein that may function as a chaperone for the LDL-receptor family receptors. Here we report calmodulin as the first identified RAP binding protein outside of the LDL-receptor family members. We demonstrate that RAP binds calmodulin in a Ca2+- and pH-dependent manner characteristic of calmodulin-dependent enzymes, and present evidence that RAP is a substrate for calmodulin-dependent enzymes. Thus, CaM-kinase II and calcineurin readily phosphorylate and dephosphorylate, respectively, serine residues in RAP, and in the individual RAP domains D2 (amino acids 113-218) and D3 (amino acids 219-323) which both contain sites for CaM-kinase II-mediated phosphorylation and for calmodulin binding. In addition, we provide evidence that RAP is phosphorylated by other kinases such as casein kinase II. Studies of 32[ortho]P-labelled cell cultures demonstrate that RAP is phosphorylated in vivo. Our results suggest that RAP may have hitherto unknown functions implicating phosphorylation and calmodulin-mediated modulation.