Biochimica et Biophysica Acta 1741 (2005) 234 – 239 http://www.elsevier.com/locate/bba Rapid report LRP1B functions as a receptor for Pseudomonas exotoxin

Diana V. Pastrana a, Alison J. Hanson a,1, Jane Knisely b, Guojun Bu b, David J. FitzGerald a,*

aLaboratory of Molecular Biology, CCR, National Cancer Institute, Bethesda, MD 20892-4263, USA bDepartment of Pediatrics, Washington University School of Medicine, St. Louis, MO 63110, USA

Received 23 March 2005; received in revised form 6 June 2005; accepted 15 June 2005 Available online 20 July 2005

Abstract

Pseudomonas aeruginosa is an opportunistic pathogen that produces several virulence factors, among them Pseudomonas Exotoxin A (PE). Previously, low-density receptor-related 1 (LRP1) was shown to be the primary receptor for PE. In this report, we show that a close family member, LRP1B, can also function as a receptor. Published by Elsevier B.V.

Keywords: Toxin; Receptor; LRP1; LRP1B; Pseudomonas; ADP-ribosylation

1. Rapid report domains containing LDL-A ligand binding repeats. Some ligands interact with only one domain, i.e., tPA:PAI-I (tissue The alpha 2-Macroglobulin Receptor or Low Density type plasminogen activator complexed with plasminogen Lipoprotein Receptor-related Protein (LRP1) functions as activator inhibitor type I); while others require multiple the receptor that Pseudomonas Exotoxin A (PE) utilizes to domains, i.e., alpha-2 Macroglobulin [8]. gain access into mammalian cells [1]. LRP1 belongs to the PE was shown to bind predominantly to Domain IV of Low Density Lipoprotein (LDL) receptor family. In LRP1, and until this report, LRP1 was the only known mammals, the family currently consists of twelve members functional receptor for this toxin [1,9,10]. LRP1 is classified (reviewed in [2,3]), which share some common structural as one of the large (about 600 kDa) LDLR family members motifs. All the family members possess complement-type (reviewed in [11]). Family members include LRP1, megalin, cysteine rich repeats also known as LDLR class A repeats LRP6 and LRP1B. LRP1 binds a variety of unrelated (LDL-A), epidermal growth factor (EGF) repeats, YWTD ligands including such diverse molecules as alpha2 macro- repeats and a cytoplasmic tail with NPXY repeat(s). The globulin–protease complexes, , lipases and LDL-A repeats are generally responsible for ligand binding toxins. Despite a lack of high sequence identity among [4], although EGF repeats can also participate. The LDL-A family members, many of the ligands that bind to LRP1 also repeats also help in the coordination of calcium ions, which bind at least one of the other large LDLR receptors. In this is a requisite for interaction with ligands [5]. The LDL study, we report that a minireceptor of LRP1B, expressing receptor has only one LDL-A-containing domain, and only domain IV and termed mLRP1B4, functions as a individual LDL-A repeats in this domain have been receptor for PE. Based on ligand blots and cytotoxicity implicated in ligand binding [6,7]. In contrast, LRP1 has 4 assays, results suggest that LRP1B is fully functional for delivery of PE to the cell cytosol of mammalian cells. We also report that the receptor-associated protein (RAP), which * Corresponding author. Mailing Address: Biotherapy Section, LMB, 37/ functions as a chaperone for LRP1 and blocks the binding of 5124, 37 Convent Dr., MSC 4264, Bethesda, MD 20892-4264, USA. Tel.: +1 301 496 9457; fax: +1 301 402 1969. most LRP1 ligands including PE, failed to interfere with PE E-mail address: [email protected] (D.J. FitzGerald). uptake into cell lines expressing minireceptor versions of 1 Current Address: Vanderbilt School of Medicine, Nashville, TN, USA. either LRP1 or LRP1B.

0925-4439/$ - see front matter. Published by Elsevier B.V. doi:10.1016/j.bbadis.2005.06.007 D.V. Pastrana et al. / Biochimica et Biophysica Acta 1741 (2005) 234–239 235

Fig. 1. Characterization of PE binding to LRP1 and mLRP1B4. Wild type CHO’s (lanes 1, 4, 7 and 10), CHO LRP-null pcDNA3 vector control (lanes 2, 5, 8 and 11), or CHO LRP-null mLRP1B4 cells (lanes 3, 6, 9 and 12) were used to obtain crude membrane preparations (A and B) or enriched plasma membrane preparations (C and D). Membrane preparations were separated on 4–20% SDS-PAGE under non-reducing conditions and transferred to PVDF membranes. For ligand blots (A and C), PE was incubated overnight with membranes at a concentration of 25 Ag/ml. After 3 rinses with binding solution to remove excess PE, bound PE was detected with an anti-PE monoclonal antibody (M40-1) followed by donkey anti-mouse HRP. For the anti-HA blot (B and D), the minireceptor was detected with anti-HA ascites. The arrowhead marks the position of LRP1 while the empty and filled arrows denote unprocessed and processed mLRP1B4, respectively.

The interaction of PE with cellular LRP1 can be membrane isolation procedure of Stolz and Jacobson [14]. recapitulated in ligand blots [1]. PE binds immobilized Briefly, cells were grown as described above, washed with LRP1 transferred to PVDF membranes after separation on coating buffer (135 mM NaCl, 20 mM MES pH 5.5, 1 mM non-reducing SDS-PAGE. Non-binding PE mutants fail to MgCl2 and 0.5 mM CaCl2), coated with 1% cationic bind when assayed in the same manner. To determine colloidal silica (Sigma Aldrich), washed again, then coated whether wild type PE could also bind the LRP1B mini- with 1 mg/ml polyacrylic acid solution (Sigma-Aldrich) receptor containing domain IV [12], we performed similar and washed again. After incubation for 30 min on ice with experiments. Wild type Chinese hamster ovary cells ‘‘domain lysis’’ buffer (2.5 mM Imidazole, 1 mM MgCl2, (CHO), LRP-null CHOs [9] stably transfected with an and 0.5 mM CaCl2 supplemented with Roche’s complete empty vector (pcDNA3), or the mLRP1B4 stably trans- protease Inhibitors without EDTA), cell lysis was assisted fected LRP-null CHO cell line [12] were used to prepare by the forceful application of lysis buffer through a syringe crude membranes. Cells were grown for 72 h, scraped from needle. The solution was pelleted at 900Âg for 1 min. The their plates, washed and resuspended in lysis solution (100 pellet was resuspended by sonication and the membranes mM KCl, 20 mM HEPES pH 7) with complete protease separated through a 1.03, 1.25, 1.37 g/ml Optiprep (Sigma) inhibitors without EDTA (Roche). The material was step density gradient at 60,000Âg for 30 min. The plasma sonicated; nuclei and other insoluble material were briefly membranes, which pellet to the bottom of the tube under pelleted at 2000Âg for 15 min. The supernatant was spun these conditions, were resuspended in 2% SDS and heated at 80,000Âg for 35 min to produce a high speed at 80 -C for 15 min. The silica was removed by membrane fraction. These high-speed pellets were resus- centrifugation at 14,000Âg for 10 min, and the supernatant pended in 2% SDS by light sonication and the amount of assessed for protein content as above. In CHO mLRP1B4 protein estimated by the BCA assay (Pierce). Forty cells, PE bound a band of about 160 kDa, and one of about micrograms of protein were separated on 4–20% poly- 100 kDa (Fig. 1A). The LRP1B minireceptor has an HA- acrylamide gels under non-reducing conditions and trans- Tag at its amino terminus. In an anti-HA immunoblot (anti- ferred to PVDF membranes. The membranes were blocked, HA from Covance), the presence of this sequence was used and incubated overnight with 25 Ag/ml of wild type (WT) to confirm that the PE-reactive bands were indeed the PE in binding solution (1Â Tris-buffered saline (TBS), recombinantly-expressed LRP1B minireceptor (Fig. 1B). 0.1% Tween 20 and 2 mM CaCl2 pH 5.5). The anti-PE The mLRP1B4 bands are presumably the mini-receptor monoclonal antibody M40-1 [13] reacted with bound PE, precursor, and its furin processed mature form. Liu et al. and donkey anti-mouse HRP conjugated polyclonal anti- [12] had reported the estimated sizes of these bands as 210 bodies (Jackson Immunoresearch) were used to detect the and 120 kDa. The differences with our observed sizes are complex. In wild type CHO cells, PE bound to a high- likely due to the lack of reducing agent in our gels, since molecular weight protein (>250 kDa) consistent with the the presence of such reagents can lead to an increase in the size of LRP1 (Fig. 1A, lane 1). In LRP1B4 transfectants, apparent migration of LRP molecules ([8,15], and our two reactive bands were noted (Fig. 1A, lane 3) and are unpublished observations). The stronger interaction of PE characterized below. In CHO LRP-null cells, only a with mLRP1B4 compared to wild type LRP1 does not background band, present in all three cell-lines, was noted. necessarily reflect a higher affinity of the toxin for this This background band disappeared when purified plasma minireceptor, but more likely the higher number of receptor membranes were used in the experiment (Fig. 1C). We molecules on the cell-surface. The number of mLRP1B4 enriched for plasma membrane , using a slight receptors is about 80,000/cell [8,12], while WT CHO cells modification of the cationic colloidal silica microbead exhibit approximately 1000 receptors/cell [16]. 236 D.V. Pastrana et al. / Biochimica et Biophysica Acta 1741 (2005) 234–239

Fig. 2. Dose response cytotoxicity assay. Increasing concentrations of WT PE, or a binding mutant, PE K57E, were added to CHO WT, CHO LRP-null pcDNA3 (vector control) or CHO LRP-null mLRP1B4 cells. After an overnight incubation, (3H) leucine was added for 1 h. The level of protein synthesis was determined by comparing toxin-treated cells with media-only controls. Data are expressed as % of control, where error bars represent 1 standard deviation of triplicate samples. The entire experiment was performed three times.

In addition to establishing an interaction of PE with cells [17]. To determine whether and how effectively PE mLRP1B4 from crude membranes, we wanted to obtain could kill CHO mLRP1B4, protein synthesis inhibition evidence that PE would interact with this minireceptor when assays were performed [18]. Cells were plated at 1Â105 it was processed and displayed on the surface of cells. For cells per well in 24-well dishes. The next day, cells were this, we used enriched plasma membrane proteins. Due to incubated for 15 min with either WT PE, or a PE with a the high level of mLRP1B4 expressed on the CHO cells, K57E mutation, which renders the toxin 50- to 100-fold less different amounts of protein per individual lane were run on active due to decreased receptor binding [19]. After an the polyacrylamide gels used for the ligand and the anti-HA overnight incubation with toxin or a media-only control, blots of purified plasma membrane proteins. For CHO WT cells were pulsed with 3 ACi/ml of (3H) leucine for 1 h. cells, we used 15 Ag of protein, for CHO pcDNA3 LRP-null Wells were washed with Phosphate Buffered saline, and the cells 18 Ag, and for CHO mLRP1B4 1.8 Ag. Enrichment of proteins precipitated with Trichloroacetic acid (TCA). plasma membrane proteins was achieved. Loading only 1.8 Incorporated tritium was measured as counts per minute Ag of CHO mLRP1B4 purified plasma membrane proteins per well of cells in a liquid scintillation counter. Wild type resulted in an anti-HA signal (Fig. 1D) comparable to or PE inhibited protein synthesis in both CHO WT and CHO greater than the signal obtained by loading 40 Ag of crude mLRP1B4 cells with an IC50 of about 10 ng/ml (Fig. 2). In membrane proteins (Fig. 1B); and the ratio of furin contrast, protein synthesis in CHO pcDNA3 cells (LRP- processed to unprocessed minireceptor was higher in the null) was only marginally reduced at 100 ng/ml. This result purified preparation. PE was able to bind to LRP1 and confirmed that this minireceptor can function as a surface mLRP1B4 from purified plasma membranes (Fig. 1C) and it receptor for PE, transporting the toxin into the cell and appeared to preferentially bind to the processed form of eventually to the cytosol. mLRP1B4. These results suggested that PE might, at least in The mutant PE K57E did not significantly inhibit the absence of LRP1, be able to utilize domain IV of protein synthesis in any of the cell lines. This mutant PE mLRP1B as a receptor to gain access to the cell cytosol. has decreased affinity for domain IV of LRP1, and given In order to be considered a fully functional receptor, the results shown in Fig. 2, it also has decreased affinity LRP1B should bind and allow PE to reach the cytosol, for mLRP1B4. In addition, a ligand blot of the purified where it can exert its toxic effect on cells. PE inhibits plasma membrane protein preparations with the PE K57E mammalian protein synthesis by transferring ADP-ribose mutant failed to show any specific binding in any of the from NAD to elongation factor 2, effectively killing the cell lines (data not shown).

Fig. 3. Toxicity of PE at 10 and 100 ng/ml in the presence or absence of RAP. In panels A–E, inhibition of protein synthesis was used to determine toxin activity. RAP (20 Ag/ml) or media only were added 90 min prior to the addition of PE in the presence of additional RAP (20 Ag/ml). After an overnight incubation, (3H) Leucine was added for 1 h and protein synthesis was determined by measuring the amount of radioactivity incorporated into TCA-precipitable material. Results are expressed as the average counts per minute per well. In panel F, the result of a cell viability assay is shown. RAP and PE were added as described for the protein synthesis assay, but cells were allowed to incubate 48 h before addition of WST-1 reagent. Absorbance at 450 nm was measured, and the average of wells with medium (without cells) was subtracted from all samples. For both protein synthesis and cell viability assays, bars represent the mean of triplicate wells and error bars represent one standard deviation from the triplicate samples. D.V. Pastrana et al. / Biochimica et Biophysica Acta 1741 (2005) 234–239 237 238 D.V. Pastrana et al. / Biochimica et Biophysica Acta 1741 (2005) 234–239

Receptor-associated protein (RAP) functions as a chap- (reviewed in [21]), however, RAP may not be able to erone that helps to properly fold LDLR family members and prevent ligand binding to mini-receptors. it can inhibit the binding of most LRP ligands (reviewed in Ligands are often shared among the LDL receptor family [20,21]). RAP can bind and is internalized by domains 2, 3 members; some infectious agents take advantage of this and 4, but not domain 1 of LRP1, and it can prevent PE promiscuity and can use one or more of these receptors to toxicity in cells that express full-length LRP1 [1,22]. It has gain entry into cells [23,24]. Pseudomonas aeruginosa has a been shown that PE binds exclusively to domain IV of LRP ubiquitous distribution and with its opportunistic nature, it [8]. It was therefore unexpected to observe that RAP would benefit from being able to access more than one prevented PE toxicity in CHO WT cells, which have the receptor for one of its virulence factors. Members of the full-length LRP1 receptor, but was unable to prevent LDL receptor family have been found to be expressed in toxicity in CHO LRP-null cells that had been transfected tissues infected by Pseudomonas. For example, LRP1 with domain IV of either LRP1B, LRP1, or both. To expression has been identified in human alveolar macro- evaluate RAP inhibition of PE toxicity, protein synthesis phages, in Langerhans’ cells [25] and in rat eye cilliary assays were performed as described above, except that the epithelium [26]. Studies of the distribution of LRP1B are cells were preincubated for 90 min at 37 -C with 20 Ag/ml contradictory; while an initial study reports wide distribu- of purified RAP. PE was then added at either 10 or 100 ng/ tion in many tissues [27]; later reports confine it to the brain ml along with an additional 20 Ag/ml of RAP (final RAP and potentially adrenal gland and testis [12,28]. Until a concentration was ¨1 AM). The cells were incubated for 18 consensus is reached on the distribution, we can only h before the addition of (3H) Leucine. In CHO WT cells, speculate about whether PE utilizes LRP1B in vivo during human RAP was able to either completely or partially opportunistic infection. However, it is intriguing to note that abolish the effects of 10 or 100 ng/ml of PE, respectively LRP1B may be expressed on the surface of airway epithelia (Fig. 3A). In contrast, rat RAP was able to abolish the (the gene is deleted in a high percentage of human tumors effects of PE at both concentrations (Fig. 3B). Although rat originating from this tissue) and therefore a possible route of and human RAP are usually interchangeable for inhibiting toxin entry when Cystic Fibrosis patients are infected with ligand-receptor binding interactions, the mechanism (com- Pseudomonas. In mutant LRP1B knockout mice, there is no petitive or steric) of inhibition is not known. With regard to obvious phenotype [28], presumably because receptors like the hamster-derived CHO cells, presumably rat RAP is more LRP1, or other related receptor(s) can substitute for it. active against the hamster encoded LRP1 than the human Replacement of LRP1 by other members of the family is not homolog because it is phylogenetically more similar. feasible during early development, as an absence of LRP1 is Results derived from inhibition of protein synthesis assays lethal for mice [29]. were confirmed by performing cell viability/cytotoxicity In this report, we show that LRP1B can function, at least assays. Cells were seeded at 4000 per well in 96 well plates, in tissue culture, as a receptor for Pseudomonas exotoxin, after 16 h, PE and RAP were added as described above. and can be internalized and transported through the appro- After 48 h, PE induced-cytotoxicity was measured using priate pathway to inhibit protein synthesis in these cells. WST-1 reagent (Roche). Briefly, 10 Al of WST-1 reagent were added to the cells (100 Al total volume), plates were incubated at 37 -C for 1 h and the absorbance read at 450 References nm. The absorbance from medium only wells was sub- [1] M.Z. Kounnas, R.E. Morris, M.R. Thompson, D.J. FitzGerald, D.K. tracted from all samples. A representative example is Strickland, C.B. Saelinger, The alpha 2-macroglobulin receptor/low provided (Fig. 3F). Results indicate that human RAP also density lipoprotein receptor-related protein binds and internalizes completely abolished the effects of 10 ng/ml of PE but only Pseudomonas exotoxin A, J. Biol. Chem. 267 (1992) 12420–12423. partially reversed the effects of 100 ng/ml PE. [2] A. Nykjaer, T.E. Willnow, The low-density lipoprotein receptor gene Inhibition of PE by RAP was tested in CHO mLRP1B4 family: a cellular Swiss army knife? Trends Cell Biol. 12 (2002) 273–280. cells, and in two additional cell lines: (1) an LRP-null CHO [3] J. Gent, I. Braakman, Low-density lipoprotein receptor structure and cell line stably transfected with domain IV of LRP1 (CHO folding, Cell. Mol. Life Sci. 61 (2004) 2461–2470. mLRP4) [12], and 2) an LRP-null CHO cell line that was [4] C.L. North, S.C. Blacklow, Solution structure of the sixth LDL-A transfected with both mLRP1B4 and mLRP4 (CHO module of the LDL receptor, Biochemistry (Mosc.) 39 (2000) mLRP1B4/mLRP4). Because transfection of the cells was 2564–2571. [5] D. Fass, S. Blacklow, P.S. Kim, J.M. Berger, Molecular basis of performed with the human version of the LRP receptor(s), familial hypercholesterolaemia from structure of LDL receptor only human RAP was used as a potential inhibitor. CHO module, Nature 388 (1997) 691–693. mLRP4, CHO mLRP1B4 and CHO mLRP1B4/mLRP4 [6] V. Esser, L.E. Limbird, M.S. Brown, J.L. Goldstein, D.W. Russell, were sensitive to PE at 10 and 100 ng/ml, and RAP was Mutational analysis of the ligand binding domain of the low density unable to rescue the effects of PE at either of these lipoprotein receptor, J. Biol. Chem. 263 (1988) 13282–13290. [7] D.W. Russell, M.S. Brown, J.L. Goldstein, Different combinations of concentrations (Fig. 3C, D and E). LRP minireceptors have cysteine-rich repeats mediate binding of low density lipoprotein been shown to bind and internalize RAP [8], and RAP has receptor to two different proteins, J. Biol. Chem. 264 (1989) been shown to inhibit ligand binding to full-length receptors 21682–21688. D.V. Pastrana et al. / Biochimica et Biophysica Acta 1741 (2005) 234–239 239

[8] L.M. Obermoeller-McCormick, Y. Li, H. Osaka, D.J. FitzGerald, A.L. alpha and Pseudomonas toxin, Proc. Natl. Acad. Sci. U. S. A. 84 Schwartz, G. Bu, Dissection of receptor folding and ligand-binding (1987) 4538–4542. property with functional minireceptors of LDL receptor-related [19] Y. Jinno, V.K. Chaudhary, T. Kondo, S. Adhya, D.J. FitzGerald, I. protein, J. Cell Sci. 114 (2001) 899–908. Pastan, Mutational analysis of domain I of Pseudomonas exotoxin. [9] D.J. FitzGerald, C.M. Fryling, A. Zdanovsky, C.B. Saelinger, M. Mutations in domain I of Pseudomonas exotoxin which reduce cell Kounnas, J.A. Winkles, D. Strickland, S. Leppla, Pseudomonas binding and animal toxicity, J. Biol. Chem. 263 (1988) 13203–13207. exotoxin-mediated selection yields cells with altered expression of [20] G. Bu, A.L. Schwartz, RAP, a novel type of ER chaperone, Trends low-density lipoprotein receptor-related protein, J. Cell Biol. 129 Cell Biol. 8 (1998) 272–276. (1995) 1533–1541. [21] G. Bu, The roles of receptor-associated protein (RAP) as a molecular [10] T.E. Willnow, J. Herz, Genetic deficiency in low density lipoprotein chaperone for members of the LDL receptor family, Int. Rev. Cytol. receptor-related protein confers cellular resistance to Pseudomonas 209 (2001) 79–116. exotoxin A. Evidence that this protein is required for uptake and [22] D. Mucci, J. Forristal, D. Strickland, R. Morris, D. Fitzgerald, C.B. degradation of multiple ligands, J. Cell Sci. 107 (Pt. 3) (1994) Saelinger, Level of receptor-associated protein moderates cellular 719–726. susceptibility to Pseudomonas exotoxin A, Infect. Immun. 63 (1995) [11] J. Gliemann, Receptors of the low density lipoprotein (LDL) receptor 2912–2918. family in man. Multiple functions of the large family members via [23] P. Bates, J.A. Young, H.E. Varmus, A receptor for subgroup A Rous interaction with complex ligands, Biol. Chem. 379 (1998) 951–964. sarcoma virus is related to the low density lipoprotein receptor, Cell 74 [12] C.X. Liu, Y. Li, L.M. Obermoeller-McCormick, A.L. Schwartz, G. (1993) 1043–1051. Bu, The putative tumor suppressor LRP1B, a novel member of the low [24] F. Hofer, M. Gruenberger, H. Kowalski, H. Machat, M. Huettinger, E. density lipoprotein (LDL) receptor family, exhibits both overlapping Kuechler, D. Blass, Members of the low density lipoprotein receptor and distinct properties with the LDL receptor-related protein, J. Biol. family mediate cell entry of a minor-group common cold virus, Proc. Chem. 276 (2001) 28889–28896. Natl. Acad. Sci. U. S. A. 91 (1994) 1839–1842. [13] M. Ogata, I. Pastan, D. FitzGerald, Analysis of Pseudomonas exotoxin [25] S.K. Moestrup, J. Gliemann, G. Pallesen, Distribution of the alpha 2- activation and conformational changes by using monoclonal anti- macroglobulin receptor/low density lipoprotein receptor-related pro- bodies as probes, Infect. Immun. 59 (1991) 407–414. tein in human tissues, Cell Tissue Res. 269 (1992) 375–382. [14] D.B. Stolz, B.S. Jacobson, Examination of transcellular membrane [26] G. Zheng, D.R. Bachinsky, I. Stamenkovic, D.K. Strickland, D. protein polarity of bovine aortic endothelial cells in vitro using Brown, G. Andres, R.T. McCluskey, Organ distribution in rats of the cationic colloidal silica microbead membrane-isolation proce- two members of the low-density lipoprotein receptor gene family, dure, J. Cell Sci. 103 (Pt. 1) (1992) 39–51. gp330 and LRP/alpa 2MR, and the receptor-associated protein [15] M.R. Thompson, J. Forristal, P. Kauffmann, T. Madden, K. Kozak, (RAP), J. Histochem. Cytochem. 42 (1994) 531–542. R.E. Morris, C.B. Saelinger, Isolation and characterization of [27] C.X. Liu, S. Musco, N.M. Lisitsina, E. Forgacs, J.D. Minna, N.A. Pseudomonas aeruginosa exotoxin A binding glycoprotein from Lisitsyn, LRP-DIT, a putative endocytic receptor gene, is frequently mouse LM cells, J. Biol. Chem. 266 (1991) 2390–2396. inactivated in non-small cell lung cancer cell lines, Cancer Res. 60 [16] M. Gu, V.M. Gordon, D.J. Fitzgerald, S.H. Leppla, Furin regulates (2000) 1961–1967. both the activation of Pseudomonas exotoxin A and the quantity of the [28] P. Marschang, J. Brich, E.J. Weeber, J.D. Sweatt, J.M. Shelton, toxin receptor expressed on target cells, Infect. Immun. 64 (1996) J.A. Richardson, R.E. Hammer, J. Herz, Normal development and 524–527. fertility of knockout mice lacking the tumor suppressor gene [17] B.H. Iglewski, D. Kabat, NAD-dependent inhibition of protein LRP1b suggest functional compensation by LRP1, Mol. Cell. Biol. synthesis by Pseudomonas aeruginosa toxin, Proc. Natl. Acad. Sci. 24 (2004) 3782–3793. U. S. A. 72 (1975) 2284–2288. [29] J. Herz, D.E. Clouthier, R.E. Hammer, LDL receptor-related protein [18] V.K. Chaudhary, D.J. FitzGerald, S. Adhya, I. Pastan, Activity of a internalizes and degrades uPA–PAI-1 complexes and is essential for recombinant fusion protein between transforming growth factor type embryo implantation, Cell 71 (1992) 411–421.