Proc. Natl. Acad. Sci. USA Vol. 92, pp. 4537-4541, May 1995 Cell Biology Functional expression of low density receptor-related is controlled by receptor-associated protein in vivo THOMAS E. WILLNOW*, SCOTr A. ARMSTRONG*, ROBERT E. HAMMERt, AND JOACHIM HERZ* Departments of *Molecular Genetics and tBiochemistry and Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX 75235 Communicated by Joseph L. Goldstein, The University of Texas Southwestern Medical Center, Dallas, TX, February 15, 1995 ABSTRACT The 39-kDa receptor-associated protein terminal HNEL tetraamino acid motif has prompted Strick- (RAP) associates with the multifunctional low density li- land et al (32) to propose a possible role of the KDEL receptor poprotein (LDL) receptor-related protein (LRP) and thereby in the process. When RAP is overexpressed the retention prevents the binding of all known ligands, including a2- system becomes saturated and RAP is secreted from the cell, macroglobulin and chylomicron remnants. RAP is predomi- resulting in autocrine/paracrine inhibition of LRP function in nantly localized in the , raising the vitro and in vivo. We have used this effect in a previous study possibility that it functions as a chaperone or escort protein (33) to provide evidence that LRP participates in the clearance in the biosynthesis or intracellular transport of LRP. Here we of remnant by the . have used gene targeting to show that RAP promotes the Based on its biochemical properties RAP has been proposed expression of functional LRP in vivo. The amount of mature, to function as a chaperone during biosynthesis, as an escort processed LRP is reduced in liver and brain of RAP-deficient protein within the secretory pathway, and as a short-acting mice. As a result, hepatic clearance of a2-macroglobulin is modulator of LRP activity (2, 25, 26, 31, 34). In this study we impaired and remnant lipoproteins accumulate in the plasma have used gene targeting to disrupt the RAP gene in mice to ofRAP-deficient mice that also lack functional LDL receptors. determine whether RAP is required for normal LRP expres- These results are consistent with the hypothesis that RAP sion and activity. We found that homozygous RAP-deficient stabilizes LRP within the secretory pathway. They also suggest mice are viable and superficially normal. LRP expression in a further mechanism by which the activity of an endocytic liver and brain is significantly reduced in these animals. This receptor may be modulated in vivo. results in impaired clearance of LRP-specific circulating li- gands by the liver. Our results indicate that RAP controls LRP The low density lipoprotein (LDL) receptor-related protein expression, possibly by stabilizing the receptor during biosyn- (LRP) (1) is a ubiquitously expressed endocytic receptor thesis or transport along the secretory pathway. (reviewed in ref. 2) that binds a diverse group of ligands, including lipoproteins (3-6), lipoprotein (7), proteases EXPERIMENTAL PROCEDURES (8, 9), protease inhibitors and protease:inhibitor complexes Methylamine-activated human a2-macroglobulin was a gift (10-15), bacterial toxins (16, 17), viruses (18), and lactoferrin from Dudley Strickland (American Red Cross, Rockville, (19). Some of these macromolecules compete with each other MD); antibodies directed against synaptophysin were provided for a common site on LRP, while others bind to independent by T. Rosahl and T. C. Siidhof (Howard Hughes Medical sites (20). LRP is an essential gene (21, 22), probably because Institute, University of Texas Southwestern Medical Center, it participates in such a large number of diverse biological Dallas). Bovine asialofetuin was purchased from Sigma. Glu- processes. tathione S-transferase/RAP fusion protein was produced as LRP is synthesized as a 600-kDa precursor (LRP 600) that described (25). Mice used in the experiments were C57BL/6J is cleaved to generate an amino-terminal 515-kDa (LRP 515) x 129SvJ hybrid male mice (age, 12-20 weeks) bred in house fragment and a carboxyl-terminal 85-kDa (LRP 85) fragment. and fed ad libitum throughout the course of the experiment This proteolytic processing takes place in a post-Golgi secre- (Teklad 6% mouse/rat diet 7001; Teklad, Madison, WI). tory compartment (23). LRP 515 harbors all known ligand Animal care and experimental procedures involving animals binding sites and remains noncovalently associated with LRP were conducted in accordance with institutional guidelines. 85, which contains the membrane anchor and the cytoplasmic Protein iodinations and DNA manipulations were performed domain. as described (25). Protein content of membrane preparations The binding of ligands to LRP can be blocked in vitro by a was determined using the Coomassie plus protein assay re- 39-kDa polypeptide designated receptor-associated protein agent (Pierce). (RAP) (24-26) that copurifies with LRP on a2-macroglobulin Disruption of the RAP Gene. A 14-kb fragment of the affinity columns (10, 11). RAP also binds tightly to gp330, an murine RAP gene was isolated from a commercial genomic endocytic receptor that resembles LRP and is abundantly library (Stratagene) by hybridization screening with a rat expressed in the kidney (19, 27, 28), and to the very low density cDNA probe (25). The fragment was subcloned into lipoprotein (VLDL) receptor (29). Both are also pGEM5Zf(+) and the pol2sneobpA expression cassette (35) members of the LDL receptor gene family. RAP also interacts was inserted into an Nco I site located within the leader peptide weakly with the LDL receptor (30). sequence. The neo expression cassette was flanked on the 5' Under normal circumstances RAP resides predominantly in side by a 1-kb Nco I fragment (short arm) and on the 3' end the endoplasmic reticulum (ER) and in the Golgi complex, by an adjacent 10-kb Sca I fragment (long arm). Two copies of while only a small percentage is present on the cell surface or the herpes simplex virus thymidine kinase gene were inserted in (27, 30, 31). The mechanism by which RAP is in tandem at the 5' end of the short homology region. retained in the ER is unknown, but the presence of a carboxyl- Abbreviations: LDL, low density lipoprotein; LRP, LDL receptor- The publication costs of this article were defrayed in part by page charge related protein; RAP, receptor-associated protein; CR, chylomicron payment. This article must therefore be hereby marked "advertisement" in remnant(s); apo, (s); VLDL, very low density lipopro- accordance with 18 U.S.C. §1734 solely to indicate this fact. tein; ER, endoplasmic reticulum. 4537 Downloaded by guest on September 28, 2021 4538 Cell Biology: Willnow et at Proc. Natl. Acad. Sci. USA 92 (1995) of the linearized vector into Electroporation replacement A B LIVER BRAIN murine embryonic stem cells (JH1 and AB1) and derivation of I RAP|+ germ-line chimeras from four independent stem cell clones | RAP .+I|-/-|+/- ,+,/+|--,-l-,-1 12kb- S '6*7 8RAP was performed according to standard protocols (36). Targeted --hko RAP- ", .;.1 stem cell clones were identified PCR under the conditions by 8kb- -wt LRP 515- '." .:.:..'. :' described by Soriano et al (35) using a primer located within the 3' untranslated region of the neo expression cassette LRP 85-*- (5'-GATTGGGAAGACAATAGCAGGCATGC-3') and an- LDLR-- ,l1 -Synapt other primer located outside the targeting construct and 12 3 4 upstream of the short homology segment (5'-TGATTGG- TACCATCTCTGGGCTGG-3'). FIG. 2. Southern and immunoblot analysis of wild-type and RAP- Immunoblot and Blot of Membrane Pro- deficient mice. (A) Twenty micrograms of genomic tail DNA from Ligand Analysis and RAP- teins. Membrane were from mouse tissues wild-type (lane 4), heterozygous (lane 1), homozygous proteins prepared deficient mice 2 and was with Sca I and and 50 of lane was on (lanes 3) digested analyzed by ,ug protein per separated 4-15% Southern the rat RAP cDNA as a under conditions with- blotting using hybridization probe. SDS/polyacrylamide gels nonreducing Diagnostic fragments for wild-type (wt) and disrupted allele (ko) are out boiling (final concentration of SDS, 2.3%) and transferred indicated. (B) Fifty micrograms of membrane proteins prepared from to nitrocellulose paper at 4°C as described (5). Filters were liver (lanes 1-4) or brain (lanes 5-8) of wild-type (lanes 1, 2, 5, and 6) incubated with polyclonal rabbit IgGs (5 ,tg/ml). Bound IgG or RAP-deficient mice (lanes 3, 4, 7, and 8) was separated by SDS gel was detected using 125I-labeled goat anti-rabbit IgG (1 x 106 electrophoresis on 4-15% polyacrylamide gels under nonreducing cpm/ml) (5) or by enhanced chemoluminescence using horse- conditions, and the proteins were transferred to nitrocellulose filters. radish peroxidase-coupled donkey anti-rabbit IgG and the Filters were incubated with polyclonal rabbit IgGs (5 ,g/ml) directed ECL system (Amersham) according to the manufacturer's against the extracellular portion of rat LRP (LRP515), the cytoplasmic recommendations. tail of human LRP (LRP 85), rat RAP, bovine LDL receptor (LDLR), or rat synaptophysin (Synapt.). Bound IgG was detected using the Turnover Experiments. 125I-labeled human methylamine- enhanced chemoluminescence system (ECL, Amersham). activated a2-macroglobulin or bovine asialofetuin was injected into the external jugular vein of anesthetized mice (5 ,ug per Mendelian frequency (-1:2:1). We did not observe any vari- animal). At designated time points blood was collected by ation in biochemical or physiological properties among these retro-orbital bleeding and the amount of radioactivity in the individually derived lines. RAP-deficient mice thrived well, plasma was determined as reported earlier (33). were superficially normal, and generally were indistinguish- Feeding Studies. C57BL/6J x 129SvJ hybrid male mice of able from wild-type litter mates. the indicated genotypes were fed normal mouse chow or, Reduced LRP Expression in RAP-/- Animals. To explore where indicated, animals were switched to a - the effect of the of the RAP on LRP enriched'diet from the standard chow to which 0.2% disruption gene expres- prepared sion we performed an immunoblot analysis of both proteins in cholesterol (wt/wt) and 10% coconut oil (vol/wt) had been and brains of and RAP-/- mice added. Blood was collected by retro-orbital bleeding before wild-type (Fig. 2B). and after the and Both LRP and RAP are normally highly expressed in these feeding experiment 50 ,ul of plasma (pools tissues No RAP could be found Western of three to six animals) was analyzed by FPLC (37). (1, 38,39). by blotting in livers (lanes 3 and 4) or brains (lanes 7 and 8) of mice homozygous for the RAP mutation, while it was readily RESULTS detectable in wild-type tissues (lanes 1, 2, 5, and 6). In the Generation of RAP-Deficient Mice. The RAP gene was absence of RAP, LRP expression was significantly reduced in targeted in murine embryonic stem cells by inserting the these organs. This was shown with independent antibodies pol2neo expression cassette into the (Fig. 1) directed against the LRP 515 or LRP 85 subunits. Semiquan- using a replacement-type targeting vector and a G418/FIAU titative immunoblot analysis with an 125I-labeled secondary double-selection strategy (35). Germ-line chimeras from four antibody and a PhosphorImager revealed that the total amount independently targeted stem cell clones were generated using of LRP 515 and LRP 85 in the liver of the knockout animals established procedures (36). All clones gave rise to wild-type was reduced by -75% when compared to matching wild-type (+/+), heterozygous (+/-), and homozygous (-/-) off- controls. In contrast, the amounts of LDL receptor in the liver spring (Fig. 2A) in the F2 generation with the expected (lanes 1-4) and of synaptophysin (40), an intrinsic protein of ..gt cocccag ccc t gca V P Q P I A 5' _ 3' Targeting vector I -1

Wild type allele -- s N NS S - Exon contaning RAP leader peptde

i Wild type Scal fragment I 8 kb Mutant Scal fragment I I 12kb

FIG. 1. Strategy for the disruption of the RAP locus in the mouse genome. A replacement-type targeting vector was constructed by inserting the pol2neo cassette (NEO) into Nco I (N) and Sca I (S) restriction sites within an exon encoding the leader peptide of the RAP gene. The transcriptional direction of neo is antiparallel to that of the RAP gene. The DNA and sequences of RAP preceding the neo insertion site are indicated. Two copies of the herpes simplex virus thymidine kinase (TK) gene flank the 5' homology fragment. Homologous recombination of the targeting vector with the wild-type RAP allele results in the loss of a Sca I site in the targeted allele. Recombination is detected by hybridizing Sca I-digested genomic DNA with the complete RAP cDNA. An 8-kb Sca I fragment is diagnostic for the wild type, a 12-kb Sca I fragment for the disrupted allele. Arrows indicate the position of oligonucleotides used for detection of recombinant embryonic stem cell clones by PCR. Downloaded by guest on September 28, 2021 Cell Biology: Willnow et at Proc. Natl. Acad. Sci USA 92 (1995) 4539 synaptic vesicles in the brain (lanes 5-8), were unaffected by the disruption of the RAP gene. Thus, the RAP knockout apparently selectively affects LRP and not the closely related LDL receptor or an unrelated vesicle protein. LRP Activity Is Reduced in Livers of RAP-Deficient Mice. CR CR In liver LRP functions as a receptor for activated a2- macroglobulin and for remnant lipoproteins as well as other A ALDLD ligands. In brain, the physiological ligands for LRP are as yet 30 10 20 30 0 10 20 30 unknown. We therefore concentrated on the liver in an 75 attempt to determine whether the reduced expression level of LRP resulted in diminished receptor function in vivo. This was 20 LDL H DL achieved by first monitoring the clearance rate of 125I-labeled HDL methylamine-activated a2-macroglobulin, a specific ligand for 10- LRP, from the plasma of wild-type (Fig. 3A, open circles) and CR RAP-/- mice 1251- (closed circles). Intravenously injected 0 labeled a2-macroglobulin disappeared from the circulation of 0 10 20 30 0 10 20 30 knockout mice at one-fourth the rate of control approximately Fraction Number animals, consistent with the 75% reduction in LRP mass. To exclude a possible general effect on receptor-mediated endo- FIG. 4. FPLC profile of mouse lipoproteins. Wild-type (A), cytosis that might be caused by the disruption of the RAP RAP-/- (B), LDLR-/- (C), and RAP-/-, LDLR-/- mice (D) were gene, we also determined the plasma clearance rate of 1251- fed on normal mouse chow containing 6% fat. Blood was collected in labeled asialofetuin in the two mouse strains. This ligand is the evening, and 50 ,l of plasma was analyzed by FPLC on a Superose specific for the asialoglycoprotein receptor (41), which is 6 column (Sigma) as described (37). The animals were then switched unrelated to LRP. As the 125I-labeled asialofetuin to chow supplemented with 0.2% cholesterol and 10% coconut oil. expected, Four later blood was collected in the and clearance rates of wild-type and RAP-deficient animals were days again evening plasma from each other that the lipoproteins were separated by FPLC. The cholesterol content of each indistinguishable (Fig. 3B), indicating fraction was measured spectrofluorimetrically. Profiles of cholesterol- RAP knockout does not indiscriminately affect intracellular fed animals (three to six male mice per group; age, 12-20 weeks; closed transport processes. circles) are shown in comparison to the profiles of the same group Effect on Cholesterol Metabolism. In the liver, a dual animals on normal chow (6% fat, open circles). Fractions containing receptor system composed of LRP and LDL receptor is VLDL CR, LDL, and HDL are indicated. thought to mediate the apolipoprotein (apo) E-dependent hepatic removal of chylomicron remnants, the carriers of (Fig. 4D) that were kept on standard low fat chow (open dietary cholesterol, from the circulation. Even in the absence circles) or were fed a cholesterol-enriched diet for 4 days of functional LDL receptors, LRP is normally expressed by (closed circles). The profiles ofwild-type (Fig. 4A) or RAP-/- at sufficiently high levels to ensure the quantita- mice (Fig. 4B) were not affected by the increased cholesterol tive removal of these lipoproteins from the bloodstream. content of the diet. In particular, the animals did not accu- Similarly, animals in which LRP has been transiently inacti- mulate large lipoprotein particles in the size range of CR. In vated do not accumulate remnants in their plasma as long as LDLR-/- mice, the LDL, but not the CR, fraction was the LDL receptor is present. However, when the function of increased (Fig. 4C). In contrast, animals lacking RAP and both receptors is disrupted large amounts of remnant lipopro- LDL receptor accumulated CR-sized particles even on a teins accumulate in plasma (33). normal chow diet (Fig. 4D). Moreover, the CR as well as the To test whether the reduced expression of LRP in RAP- LDL fraction in these animals were significantly increased. deficient mice also impairs the removal of chylomicron rem- The amount of cholesterol in LDL-sized particles in the nants (CR) from the circulation, we cross-bred LDL receptor- RAP-/-, LDLR-/- animals was somewhat higher than in deficient (LDLR-/-) (37) and RAP-/- animals to obtain a LDLR-/- mice fed the same diet, whereas the HDL fraction strain of mice lacking both proteins. Fig. 4 shows the plasma was slightly decreased. The relative increase of the CR and the lipoprotein profiles of wild-type (+/+, Fig. 4A), RAP-/- LDL fraction in the RAP-/-, LDLR-/- mice was reflected in (Fig. 4B), LDLR-/- (Fig. 4C), and RAP-/-, LDLR-/- mice an almost 2-fold increase of the total plasma cholesterol concentration in RAP-/-, LDLR-/- mice as compared to 1251251-a-macroglobulinIlt 125-asialofetuin RAP+/+, LDLR-/- mice (Table 1). The moderate increase in total levels is consistent with the notion -*a ^ A B plasma that the accumulating lipoprotein particles consist largely of e 60 Table 1. Total plasma cholesterol and triglyceride concentrations of wild-type, RAP-/-, LDLR-/-, and RAP-/-, LDLR-/- mice RA uK Q- |- P-+/ 20 -t-{N . Cholesterol, , 1 Genotype mg/dl mg/dl n 2 0 10 20 0 4 6 8 10 Wild type 92 + 9 94 ± 10 8 Time (min) RAP-/- 103 ± 3* 115 ± 8* 20 LDLR-/- 273 ± 12 100 + 9 10 FIG. 3. Plasma clearance of 125I-labeled in and ligands wild-type RAP-/-, LDLR-/- 487 ± 31** 279 ± 25** 19 RAP-deficient mice. Wild-type (open circles) or RAP-deficient (closed circles) mice were intravenously injected with 5 tug of 125I- Values were determined spectrometrically (37) from the indicated labeled human a2-macroglobulin or bovine asialofetuin (33). At the number of animals fed a normal chow diet containing 6% fat and indicated time points blood samples (=80 tLI) were collected from the analyzed using the unpaired Student's t test. Neither cholesterol nor retro-orbital plexus and the amount of trichloroacetic acid- triglyceride concentrations of RAP-/- animals were significantly precipitable radioactivity in plasma was determined. Values are ex- different from those of wild-type controls (*, P > 0.12). Cholesterol pressed as percent of radioactivity present in plasma at 1 min after and triglycerides were significantly increased when RAP-/-, injection of the label. Variances shown represent the range of absolute LDLR-/- mice were compared to RAP+/+, LDLR-/- animals (**, values measured (four to five animals per group). P = 0.0001). Downloaded by guest on September 28, 2021 4540 Cell Biology: Willnow et al Proc. NatL Acad. Sci. USA 92 (1995)

De NormalDietoralDeh.iet Cho. Diet concentrations of wild-type and RAP-/- animals. The most likely explanation of our results therefore would be a model in RAP ++ -/- +/+ /+ -/- +/+ which RAP acts either as a folding chaperone or as an escort LDLR +/+ +/+ -/. /-+/ +/+ -/- protein. The escort function may be necessitated by the fact that LRP binds a large number of diverse ligands, several of B-100 which (e.g., a2-macroglobulin, al-antitrypsin, apoE, plasmin- B-48 ogen activator inhibitor) are synthesized in large amounts in the liver simultaneously with LRP. RAP might bind to the nascent receptor, preventing association with coexpressed ligands. In the absence of RAP this ligand binding could otherwise lead to aggregation and subsequent degradation of Al - _ _- LRP within the ER or in (42). The notion that RAP may be important in LRP folding is supported by the finding 1 2 3 4 5 6 7 8 that RAP associates with another LDL receptor gene family in and before is FIG. 5. Immunoblot analysis of mouse . Plasma member, gp330, early biosynthesis folding samples (0.5 uLI, pools of three animals) obtained from wild-type (lanes completed (34). 1 and 5), RAP-/- (lanes 2 and 6), LDLR-/- (lanes 3 and 7), and A similar function to the one proposed here for RAP has RAP-/-, LDLR-/- (lanes 4 and 8) mice before (lanes 1-4) and after been described for the invariant chain, which blocks the (lanes 5-8) feeding of the cholesterol-enriched diet were analyzed by association of peptides with major histocompatibility complex immunoblotting as described in the legend to Fig. 2B. Polyclonal rabbit (MHC) class II molecules during their exit from the ER. IgGs used are directed against mouse apoB (B-100 and B-48), rat apoE Deletion of the gene for invariant chain in mice results in (E), or rat apoAI (AI). intracellular accumulation and poor expression of dysfunc- tional MHC class II antigen on the cell surface (43). In contrast lipolyzed remnant lipoproteins. Plasma triglycerides in the to the case of the invariant chain and MHC class II molecules, cholesterol-fed animals were almost exclusively contained however, RAP does not appear to be required for the func- within the CR fraction, which had a triglyceride/cholesterol tional maturation of LRP molecules that escape to later ratio of -1.0 (data not shown). This ratio is typical of compartments. The few LRP molecules that are transported to remnants. the cell surface in RAP-deficient livers appear to be fully Fig. 5 shows an immunoblot analysis ofwhole plasma probed functional. Further experiments will be necessary to determine with antibodies against apoB100, apoB48, apoE, and apoAI. in which intracellular compartment the postulated LRP deg- ApoB100 is the only lipoprotein in LDL, whereas apoB48 is radation is taking place. present in CR and in a large fraction of hepatic VLDL in mice. The current data do not exclude the possibility that RAP ApoE is present in all lipoprotein fractions and apoAI is found acts as a short-acting regulator of LRP activity. Although RAP mainly in HDL. Animals of all four genotypes were fed either is predominantly localized in the ER, significant amounts of standard chow (lanes 1-4) or the cholesterol-enriched diet the protein were detected in the Golgi apparatus, and a small (lanes 5-8). The plasma apolipoprotein composition of amount was also present in endosomes and on the cell surface RAP-/- animals (lanes 2 and 6) did not differ significantly (30, 31). The latter distribution raises the possibility that RAP from that of wild-type controls (lanes 1 and 5) either on the can be released from an intracellular normal or on the cholesterol-enriched diet. RAP+/+, rapidly storage compart- LDLR-/- mice accumulated and ment, after which it would associate with LRP within a primarily apoB100- apoE- recycling vesicle or on the cell surface. Although at present containing lipoproteins without a detectable increase in such a mode of regulation is speculative, a complete block of apoB48 (lanes 3 and 7). RAP-/-, LDLR-/- animals showed LRP a relative increase in with function has been achieved by overexpression of RAP in slight apoB100 compared RAP+/+, vivo and in vitro (33), demonstrating that, in principle, such a LDLR-/- mice, suggesting that RAP can affect LDL produc- mechanism is feasible. tion or turnover. The most striking change in the RAP-/-, The RAP-deficient mice we have in will LDLR-/- animals was a increase in the amount of described this paper large be a valuable tool for further elucidating the biosynthesis, apoB48 (lanes 4 and 8). The plasma of these animals also intracellular and of LRP and other contained increased levels of apoE as would be expected for transport, regulation an accumulation of remnant members of the LDL receptor gene family. They also have lipoproteins. Analysis of the CR provided us with a phenotypically hypomorphic animal model fraction from these mice revealed only apoB48 and apoE (not with partial LRP deficiency that will allow investigation of the shown), confirming that this fraction consisted of chylomicron functions of LRP in adult mice. This had or VLDL remnants. ApoAI did not differ significantly be- multiple physiological tween animals of the various previously not been possible, because embryos that lack LRP genotypes. die early in utero (21, 22). DISCUSSION We thank David Russell, Mike Brown, and Joe Goldstein for critical reading of the manuscript and for helpful comments, Wen-Ling Niu, The present experiments with gene targeting indicate that Teresa Burnett, Lucy Lundquist, and Scott Clark for expert technical RAP is necessary for the normal activity of LRP in liver and assistance, and Thomas Siidhof, Thomas Rosahl, and Dudley Strick- brain. LRP expression in these organs is substantially reduced land for sharing reagents. This work was supported by grants from the in mice lacking RAP. As a consequence of the diminished National Institutes of Health (HL20948), the Keck Foundation, and activity of LRP in the liver, ligands such as c2-macroglobulin the Perot Family Foundation. T.E.W. was a recipient of a postdoctoral are cleared from plasma at a slower rate. Another set of fellowship from the Deutsche Forschungsgemeinschaft. S.A.A. is ligands, the CR, do not accumulate in of RAP-/- mice, supported by Medical Scientists Training Grant GM08014. J.H. was plasma supported by the Syntex Scholar Program and is a Lucille P. Markey but they do accumulate in LDLR-/-, RAP-/- double knock- Scholar. outs. This finding is consistent with the hypothesis that the LDL receptor and LRP both participate in the removal of CR 1. Herz, J., Hamann, U., Rogne, S., Myklebost, O., Gausepohl, H. and that a significant block in clearance requires a reduction & Stanley, K. K. (1988) EMBO J. 7, 4119-4127. in the activity of both receptors. 2. Krieger, M. & Herz, J. (1994) Annu. Rev. Biochem. 63, 601-637. In a series of experiments not shown here we could not 3. Beisiegel, U., Weber, W., Ihrke, G., Herz, J. & Stanley, K. K. detect reproducible differences in the hepatic LRP mRNA (1989) Nature (London) 341, 162-164. Downloaded by guest on September 28, 2021 Cell Biology: Willnow et all Proc. NatL Acad Sci USA 92 (1995) 4541 4. Kowal, R. C., Herz, J., Goldstein, J. L., Esser, V. & Brown, M. S. 24. Moestrup, S. K. & Gliemann, J. (1991) J. Biol. Chem. 266, (1989) Proc. Natl. Acad. Sci. USA 86, 5810-5814. 14011-14017. 5. Kowal, R. C., Herz, J., Weisgraber, K. H., Mahley, R. W., Brown, 25. Herz, J., Goldstein, J. L., Strickland, D. K., Ho, Y. K. & Brown, M. S. & Goldstein, J. L. (1990) J. Biol. Chem. 265, 10771-10779. M. S. (1991) J. Biol. Chem. 266, 21232-21238. 6. Ji, Z.-S., Brecht, W. J., Miranda, R. D., Hussain, M. M., Inner- 26. Williams, S. E., Ashcom, J. D., Argraves, W. S. & Strickland, arity, T. L. & Mahley, R. W. (1993) J. Biol. Chem. 268, 10160- D. K. (1992) J. Biol. Chem. 267, 9035-9040. 10167. 27. Orlando, R. A., Kerjaschki, D., Kurihura, H., Biemesderfer, D. 7. Beisiegel, U., Weber, W. & Bengtsson-Olivecrona, G. (1991) & Farquhar, M. G. (1992) Proc. Natl. Acad. Sci. USA 89, 6698- Proc. Natl. Acad. Sci. USA 88, 8342-8346. 6702. 8. Bu, G., Williams, S., Strickland, D. K. & Schwartz, A. L. (1992) 28. Kounnas, M. Z., Argraves, W. S. & Strickland, D. K. (1992) J. Proc. Natl. Acad. Sci. USA 89, 7427-7431. Biol. Chem. 21162-21166. 9. Kounnas, M. Z., Henkin, J., Argraves, W. S. & Strickland, D. K. 267, (1993) J. Biol. Chem. 268, 21862-21867. 29. Battey, F. D., Gafvels, M. E., FitzGerald, D. J., Argraves, W. S., 10. Strickland, D. K., Ashcom, J. D., Williams, S., Burgess, W. H., Chappell, D. A., Strauss, J. F. & Strickland, D. K. (1994) J. Biol. Migliorini, M. & Argraves, W. S. (1990) J. Biol. Chem. 265, Chem. 269, 23268-23273. 17401-17404. 30. Mokuno, H., Brady, S., Kotite, L., Herz, J. & Havel, R. J. (1994) 11. Kristensen, T., Moestrup, S. K., Gliemann, J., Bendtsen, L., Sand, J. Biol. Chem. 269, 13238-13243. 0. & Sottrup-Jensen, L. (1990) FEBS Lett. 276, 151-155. 31. Bu, G. J., Maksymovitch, E. A., Geuze, H. & Schwartz, A. L. 12. Orth, K., Madison, E. L., Gething, M.-J., Sambrook, J. F. & Herz, (1994) J. Biol. Chem. 269, 29874-29882. J. (1992) Proc. Natl. Acad. Sci. USA 89, 7422-7426. 32. Strickland, D. K., Ashcom, J. D., Williams, S., Battey, F., Behre, 13. Nykjaer, A., Petersen, C. M., Moller, B., Jensen, P. A., Moestrup, E., McTigue, K., Battey, J. F. & Argraves, W. S. (1991) J. Biol. S. K., Holtet, T. L., Etzerodt, M., Thogersen, H. C., Munch, M., Chem. 266, 13364-13369. Andreasen, P.A. & Gliemann, J. (1992) J. Biol. Chem. 267, 33. Willnow, T. E., Sheng, Z., Ishibashi, S. & Herz, J. (1994) Science 14543-14546. 264; 1471-1474. 14. Warshawsky, I., Broze, G. J., Jr., & Schwartz, A. L. (1994) Proc. 34. Biemesderfer, D., Dekan, G., Aronson, P. S. & Farquhar, M. G. Natl. Acad. Sci. USA 91, 6664-6668. (1993) Am. J. Physiol. 264, F1011-F1020. 15. Conese, M., Olson, D. & Blasi, F. (1994) J. Biol. Chem. 269, 35. Soriano, P., Montgomery, C., Geske, R. & Bradley, A. (1991) Cell 17886-17892. 64, 693-702. 16. Kounnas, M. Z., Morris, R. E., Thompson, M. R., FitzGerald, 36. Bradley, A. (1987) in Teratocarcinomas and Embryonic Stem D. J., Strickland, D. K. & Saelinger, C. B. (1992) J. Biol. Chem. Cells: A Practical Approach, ed. Robertson, E. J. (IRL, Oxford), 267, 12420-12423. pp. 113-151. 17. Cavallaro, U., del Vecchio, A., Lappi, D. A. & Soria, M. R. 37. Ishibashi, S., Brown, M. S., Goldstein, J. L., Gerard, R. D., (1993) J. Biol. Chem. 268, 23186-23190. Hammer, R. E. & Herz, J. (1993) J. Clin. Invest. 92, 883-893. 18. Hofer, F., Gruenberger, M., Kowalski, H., Machat, H., G. Cell Tissue Huettinger, M., Kuechler, E. & Blaas, D. (1994) Proc. Natl. Acad. 38. Moestrup, S. K., Gliemann, J. & Pallesen, (1992) Sci. USA 91, 1839-1842. Res. 269, 375-382. 19. Willnow, T. E., Goldstein, J. L., Orth, K., Brown, M. S. & Herz, 39. Zheng, G., Bachinsky, D. R., Stamenkovic, I., Strickland, D. K., J. (1992) J. Biol. Chem. 267, 26172-26180. Brown, D., Andres, G. & McCluskey, R. T. (1994) J. Histochem. 20. Willnow, T. E., Orth, K. & Herz, J. (1994) J. Biol. Chem. 269, Cytochem. 42, 531-542. 15827-15832. 40. Johnston, P. A., Jahn, R. & Siidhof, T. C. (1989) J. Biol. Chem. 21. Herz, J., Clouthier, D.E. & Hammer, R.E. (1992) Cell 71, 264, 1268-1273. 411-421. 41. Ashwell, G. & Harford, J. (1982) Annu. Rev. Biochem. 51, 22. Herz, J., Clouthier, D. E. & Hammer, R. E. (1993) Cell 73, 428 531-554. (lett.). 42. Klausner, R. D. & Sitia, R. (1990) Cell 62, 611-614. 23. Herz, J., Kowal, R. C., Goldstein, J. L. & Brown, M. S. (1990) 43. Viville, S., Neefjes, J., Lotteau, V., Dierich, A., Lemeur, M., EMBO J. 9, 1769-1776. Ploegh, H., Benoist, C. & Mathis, D. (1993) Cell 72, 635-648. Downloaded by guest on September 28, 2021