Asialoglycoprotein receptor deficiency in mice lacking the minor ...

TIiE JOURNAL OF BIOICGICAL CHEMISTRY Vol. 269, No. 45, Issue of November 11, pp. 27803-27806, 1994 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

Communication

structure and themolecular assembly of this heterooligomeric (approxireceptor. ASGPR is abundantly expressed in the liver mately 500,000 receptorshepatocyte) and is thought to function physiologically in the removal and degradation of desialylated circulating proteins (reviewed in Ashwell and Harford (Received for publication, August 29, 1994, and in revised form, (1982) and Geffen and Spiess (1992)). Normally, the penultimategalactose residues of many oligoSeptember 15, 1994) saccharide side chainson glycosylated proteins are maskedby Shun Ishibashi$§, Robert E. Hammerll, and terminal sialic acid residues. When these galactose residues Joachim Herz$ll are exposed by the actionof sialidases the thus tagged proteins From the Departments of $.Molecular Genetics and become substrates for ASGPR. High affinity binding requires Wiochemistry and the1Woward Hughes Medical the receptorto be assembled as a heterooligomer consisting of Institute, Uniuersity of Texas Southwestern Medical the two highly homologous subunits termed hepatic lectin (HL) Centeer, Dallas, Texas 75235 1and 2. Studies addressing the roles of the individual subunits The asialoglycoprotein receptoris an abundant het- in intracellular transport andcell surface expression ofASGPR erooligomeric endocytic receptor that is predominantly have yielded different results dependingon whether polarized expressed on the sinusoidal surface of the hepatocytes. or unpolarized cell lines were used in the transfection experiProposedphysiologicalandpathophysiologicalfuncments (Geffen et al., 1989; Shia and Lodish, 1989; Braiterman (HL)include the tions ascribed to this hepatic lectin et al., 1989; Graeve et al., 1990). removal of desialylated serum glycoproteins and apop- A number of diverse physiological roles have been proposed totic cells, clearance of chylomicron remnants, and a for ASGPR over the years. The terminal sialic acid residues role as a homing receptor for lymphatic and metastatic that coat many secreted and cellular proteins are thought to act cells. The assembly of two homologous subunits, H L - 1 as effective biological masks (Ashwell and Morell, 1974; and HL-2, is required to form functional, high affinity receptors on the cell surface. However, the importance Schauer, 1985) and have beenproposed to suppress antigenicof the individual subunits for receptor transport to the ity and to protect tumorcells from recognition by the immune cell surface has been controversial.To explore the s i g system. Loss of cell surface sialylation has beenfound to corcell metastasistotheliver (Schleppernificance oftheminorHL-2subunitforreceptor ex- relatewithtumor Schafer et al., 1981; Yeatman et al., 1989). The clearance of pression and function in uiuo, we have disrupted the HL-2 gene in mice. Homozygous HL-2-deficient animals apoptotic cells in the liver hasalso been attributed toASGPR are superficially normal. However, HL-1 expression in (Dini et al., 1992). the liver is greatly reduced, indicating that HL-2 may Important regulatory roles have been ascribed to the oligopromote HL-1 stability. Although these mice are comsaccharides on glycoprotein hormones, which are thought to pletely unable to clear asialoorosomucoid, a high affin- contribute to the serum lifetime of the polypeptide (reviewedin ity ligand for asialoglycoprotein receptor, they do not Drickamer (1991)). An example for a carbohydrate-specific reaccumulate desialylated glycoproteins or lipoproteins ceptor that has been shownt o have such a regulatory function in their circulation. is the SO,-GalNAc-specific receptor, which is present on the Kupffer cellsand the endothelial cells of the liver. This receptor regulates the serum lifetime of the pituitary glycohormone The discovery of the asialoglycoprotein receptor (ASGPR)’ lutropin (Fiete et al., 1991). In analogy, ASGPR might be the resulted from the initial observation by Ashwell and Morell counterpart for other glycohormones that could either be syn(19’741, who found that serum glycoproteins containing termi- thesized with their galactose residues exposed or that could be nal galactose residues were rapidlyremoved from the circula- desialylated at inflammatory sites (Cross and Wright,1991) or tion by a hepatocyte-specific receptor-mediated mechanism. in the circulation. Further studiesby Drickamer, Spiess, McPhaul, and colleagues A possible role for ASGPR in the clearance of lipoproteins (Drickamer et al.,1984; Spiess et al., 1985; Spiess andLodish, from the bloodstream has been proposed by Windler etal. 1985; McPhaul and Berg, 1986, 1987) revealed the primary (1991). This hypothesis was supported by the findingthat apolipoprotein E, a constituent of the carriers of dietary choles* This work was supported by grants from the National Institutes of terol, the chylomicron remnants, can become desialylated in Health (HL20948),the Keck Foundation, andthe Perot Family Foun- plasma (Zannis et al., 1984). dation and by the Howard Hughes Medical Institute. The costsof pubIn the current study we have addressed several of these lication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” unresolved questions by disrupting the gene for the minor (HL-2) subunit of ASGPR in mice and analysisof the resulting in accordance with 18 U.S.C. Section 1734 solelyto indicate this fact. 5 Supported by a postdoctoral fellowship fromthe Sasakawa Health phenotype. Science Foundation,Tokyo. Present address: 3rd Dept.of Internal Medicine, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, BunkyoMATERIALS AND METHODS ku, Tokyo 113, Japan. C57BW6 mice werepurchased from Jackson Laboratories;AB1 cells 11 Supported by the SyntexScholarProgram;Lucille P. Markey were a generous gift from Dr. Allan Bradley, Baylor College, Houston; Scholar. [14Clsialic acidand [3Hlsodium borohydrideand the ECL detection sysThe abbreviations used are: ASGPR,asialoglycoproteinreceptor; tem were from AmershamCorp.; a-2,6-sialyltransferase wasfrom HL, hepatic lectin; ASOR, asialoorosomucoid;MHL,murinehepatic lectin; LDL, low densitylipoprotein;ECL,enhancedchemiluminesGenzyme Corp. (Cambridge, MA); orosomucoid (a,-acidic glycoprotein) l-~2-deoxy-2-fluoro-~-~-arabinofuranosyl)-5-iodouracil; cence; FIAU, andneuraminidasetype XA attached toagarosebeadswerefrom FPLC, fast protein liquid chromatography; ES, embryonic stem. Sigma; G418 (Geneticin)was obtainedfromLifeTechnologies, Inc.,

Asialoglycoprotein Receptor Deficiency in Mice Lacking the Minor Receptor Subunit*

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and FIAU ~1-~2-deoxy-2-fluoro-~-~-arabinofuranosyl~-5-iodouracil~ was 2+34567 8 9 I*o Tar@lng Vector from Oclassen Pharmaceuticals, San Rafael, CA. E E X X S XE Culture of Embryonic StemCells and Generation of Knockout MiceAB1 embryonicstem cells werecultured a s describedpreviously 2 34567 8 9 1 Wildtype Allak (Ishibashi et al., 1993). After electroporation of the linearized targeting X ES E E X X S vector into ES cells G418@/FIAU@ clones were analyzed by polymerase 8 9 chain reaction (Soriano et al., 1991) using primers (indicated by the arDisruptad Allda rows above the disrupted allele Fig. in 1) located withinthe neo cassette E E X X S (5'-GATTGGGAAGACAATAGCAGGCATGC-3') andintron 1 (5'-CTkb 5.6 DisruptadAll* GAGCGTATAGGAAGCTGTGGCTGT-3'). Chimeric mice weregenerE E ated by injecting targeted ES cell clones into C57BU6 blastocysts following standardprocedures(Bradley,1987).Threeindependently 6 k b Wildtype A l k k E E targeted stem cell clones were injected into blastocysts and yielded a total of 14 malemice with coatcolor chimerism rangingfrom 30 to 100%. Of these 14 chimeras 3 gave offspring that carried the disrupted MHL-2 allele. Immunoblot a n d Northern Analysis-Liver membrane proteins (50 pgflane) were prepared a s described (Kowal et al., 1990) and separated on 10% SDS-polyacrylamide gels under non-reducing conditions.Proteins were transferred to nitrocellulose, and immunoblot analysis was performedusing specific rabbit polyclonal antipeptide antibodies (5 pg/ml) generated against carboxyl-terminal sequences of MHL-1 (CETKLDKAN) and MHL-2 (CEKRRNITH) and an ECL detection system (Amersham). Gels were calibrated using a RainbowTMhigh molecular weight marker (Amersham Corp.). For Northern analysis 20ofpg total MUTliver RNMane werehybridizedwith :'?P-labeled randomhexamerL J primed probes generated from the respective mouse cDNA sequences FIG.1. Targeted disruption of the MHL-2 gene. A, a targeting (Sanford andDoyle, 1990). Hybridization and washings were performed vector of the replacement type was constructed by inserting thepol2neo under stringent conditions. cassette (Soriano etal., 1991) intoa BamHI site in exon 2. The long arm Radiolabeling of Plasma Proteins withExposed Terminal Galactose of the targetingvector containing exons3-9 was cloned from a genomic Residues-Labeling of mouse plasma proteins using the sialyltransfer-A library of SacI-restricted mouse DNA(C57BU6 strain). The short arm ase reaction was performed essentially as described (Weinstein et al., is a 0.8-kilobase (kb) BamHI fragment containing partsof exon 2 and 1982). Briefly, 20 pl of mouse plasma were diluted with 180 p1 of water. intron 1. This fragment was part of another A clone that was isolated 2 of CMP['4ClNeuAc, and 0.8 from a commercial B6/CBA library (Stratagene). The 2.5 pl of 0.5 h~ sodium cacodylate (pH 6), pl MHL-2 sequences milliunit of a-2,6-sialyltransferase were added, and the mixture was between the BamHI site in exon 2 and the Sac1 site in intron 2 are incubated for 2 ha t 37 "C. 25 pl of the samples were then subjected to deleted. Two copies of the herpes simplex thymidine kinase(5°K)gene two-dimensional gel electrophoresis (O'Farrell, 1975). flank the short arm a t t h e5' end. Transcriptional orientationof the neo Homologous Plasma Zhrnouer Experiments-ASOR was prepared by incubating and herpes simplex thymidine kinase genes are indicated. 100 mg of orosomucoid a t 37 "C in 10 ml of 0.1 M sodium acetate buffer recombination events can be detected by polymerase chain reaction containing 2 mM CaCI,, pH 5, together with 1 unit of neuraminidase (position of diagnostic primers indicated by the arrows above the disof enzyme unit was rupted allele)or by digesting genomic DNA with EcoRI and subsequent type XA attached to agarose beads. After 4 h another of EcoRI fragments added, and the incubation was continued overnight. Both orosomucoid hybridization witha cDNA probe. The expected size and ASOR were labeled withlZsI using theIODO-GEN procedure. Spe- of the digested alleles is shownbelow the disruptedallele. B , genotypes cific activities of '2'II-orosomucoidand lZ51-ASORwere 56 and c35 p d n g , of offspring from matings of MHL-2'" mice. Tail DNA was digested with 200 p1 of saline contain- EcoRI and analyzed by Southern blotting a s described above. The porespectively. 20 pg of the iodinated proteins in of wild type ( W T )alleles are ing 2mg/ml bovine serum albumin were injected intravenously into thesitions of migration of mutant (MUT) and indicated. jugular vein of anesthetized male mice that were wild type, heterozygous, or homozygous for the MHL-2 gene disruption. Blood was collected at the indicated intervals from the retroorbital plexus, and tri- lB)with a normal Mendelian distribution (--1:2:1, data not chloroaceticacid-precipitableradioactivity of 20 pl of plasmawas shown). determined as described (Herz and Gerard, 1993). The disruption of the MHL-2 gene resulted in the complete Analysis of Mouse Plasma Lipoprotein Profiles-Plasma lipoprotein absence of the encoded protein in -1- animals. Thiswas asceral., 1993). analysiswasdone a s describedpreviously(Ishibashiet tained by immunoblotting of liver membrane proteins (Fig. 2 A ) Briefly, mice were bled from the retroorbital plexus, and the blood was collected into EDTA-containing microcuvettes (Sarstedt). Plasmafrom of +I+(lanes 1 and 4 ) , +I- (lune Z ) , and 4- (lane 3 ) animals three individual animals of the samegenotype waspooled, and 0.6 ml of with an MHL-2-specific anti-peptide antibody. No MHL-2 proof -1- mice (lane 3 ) .Expression the plasma was loaded on a Superose 6 (Sigma) FPLC column. The tein wasdetectable in the livers cholesterolcontent of eachfractionwasdeterminedenzymatically of the HL-1 subunit had previously been reported to be inde(Sigma cholesterol assay kit). pendent of the coexpression of HL-2 in culturedcells. However,

A.

+*Wnnnn

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.

I

in MHL-2-deficient mice expression of endogenous MHL-1 protein was significantly reduced (Fig. 2 B , lanes 3 and 5).HetThe murineHL-2 (MHL-2) gene was cloned by hybridization erozygous animals also expressedreduced levels of MHL-1 proscreening of a mouse genomic library using a mouse cDNA tein (lune 2). To control for equal loading, filters were stained probe (Sanford and Doyle, 1990). A gene replacement vector with Ponceau S after transferof the proteins, and anequivalent was constructed in which the pol2neo expression cassette in- blot was processed in parallel using ananti-LDL receptor anterrupts exon 2 of the MHL-2 gene (Fig. lA). Following elec- tibody (not shown). The decrease in MHL-1 protein expression troporation of the linearized targeting vector into AJ31, ES cells is apparently due toreduced stability in theabsence of MHL-2 and positive/negative selection chimeric mice were generated polypeptide. Inagreementwiththisinterpretation, MHL-1 from threeindependentlytargetedstem cell clones using mRNAlevels werenot affected by the disruptionof MHL-2 (Fig. standard procedures. Only one targeted stem cell line contrib- 2C, lune 2 ) . Although MHL-2-deficient (-/-) mice still express some reuted to thegerm line of three chimeric foundermice. Chimeras were bred with wild type female C57BIJ6 animals. Heterozy- sidual MHL-1 protein, they are unable toclear a desialylated gous offspring (F, generation) were crossed with each other and 12sI-labeledglycoprotein, '2sI-ASOR,from their circulation (Fig. gave rise t o F, offspring that were wild type (+/+), heterozygous 3A, closed symbols). Heterozygous mice cleared '''I-ASOR with (+I-),or homozygous (-/-I for the disruptedMHL-2 allele (Fig. intermediate efficiency. Non-desialylated '251-labeledorosomuRESULTS

Mice Asialoglycoprotein Knockout Receptor

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FIG. 2.Western and Northern blot analysis ofASGPR subunits in mouse livers. A, Western blot analysis of MHL-2 in the livers of female mice wild type (+/+, lanes 1 and 4 ), heterozygous (+/-, lane 2 ) ,or homozygous (-I-, lane 3 ) for the MHL-2 gene disruption. Bandsa t -38 and 83 kDa represent monomeric and dimeric formsof ASGPR, respectively. B , Western blot analysis of MHL-1 in the liversof female mice wild type (+/+, lanes 1 and 4 ), heterozygous (+/-, lane Z ) , or homozygous (-/-, lanes 3 and 5 ) for the MHL-2 gene disruption. The arrow indicates the MHL-1 monomer migratinga t 35 kDa. C, Northern blot analysis of MHL-1 (lanes1 and 2 ) and MHL-2 (lanes 3 and 4 ) in wild type (+/+, lanes I and 3 ) and MHL-2-deficient mice (lanes 2 and 4 ) .Arrows indicate single mRNAspeciesfor MHL-1 4 1 . 3 kilobases) andMHL-2 (-1.4 kilobases).

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FIG.4. FPLC profiles of mouse plasma lipoproteins. A, blood was collected from the retroorbital plexusof MHL-2-deficient mice that either expressed (open circles) or lacked (closed circles) functionalLDL receptors. The latter animals were generated by cross-breeding MHL2-deficient mice with LDL receptor (LDLRhdeficient animals (Ishibashi et al., 1993). 600 pl of pooled plasma from three animalsof the indicated genotype were subjected to gel filtration on a Superose 6 FPLC column, and the cholesterol contentof the individual fractions was determined enzymatically (Ishibashi et al., 1993). The elution positions of chylomicron remnants (CR), LDL, and high density lipoproteins (HDL)are indicated. B, FPLC profile of control animals expressing normallevels of functional ASGPR.

resolution limits of this gel system no major reproducible differences could be detected between the ['4C]sialic acid-labeled protein patterns of animals of either genotype (Fig. 3B). Similar experiments (notshown) in which we used other reagents specific for terminal galactose residues on glycoproteins (galactose oxidase/[3H]sodium borohydride labeling followed by oneand two-dimensional gel electrophoresis or galactose-specific +I+ -1lectin detection on one-dimensionalprotein blots) also failed to FIG.3. A, disappearance of 1251-labeledglycoproteins from plasma. detect any significant accumulation of asialoglycoproteins in Asialoorosomucoid was prepared from orosomucoid (Sigma) by desial- the plasma of MHL-2 knockout mice as compared with wild ylation with immobilized neuraminidase type XA (Sigma). Both glycotype controls. proteins were labeled with lZ5Iusing IODO-GEN (Pierce). Individual To test whether ASGPR is involved in chylomicron remnant clearance kinetics for two animals/group of the indicated genotype injected with 20 pg of either 12511-orosomucoid (open circles)or '2sI-ASOR clearance by the liver we analyzed the plasmalipoprotein pro(closed circles) are shown. Values shown are trichloroacetic acid-insol- file of MHL-2-deficient mice (Fig. 4 A , open circles) and of MHLuble radioactivity remaining in plasma a t t h eindicated time points and 2-deficient mice that also lack LDL receptors (Fig. 4 A , closed are calculated a s percent of radioactivity present in plasma a t 1 min after injection of the label. B , two-dimensional analysis of [14Clsialic circles). Plasma lipoprotein profiles ofASGPR-deficient mice in acid-labeled plasma glycoproteins from wild type (+/+) and MHL-2 de- the absence or presence of functional LDL receptors are indisficient (-/-) mice. u-2,6-Sialyltransferase reactions on mouse plasma tinguishable from those of control animals expressingwild type proteins were performeda s described (Weinstein et al., 1982). Proteins levels of functional ASGPR (Fig. 4B). Neither in the presence were separated by isoelectric focusing (first dimension, 4 parts of ampholine, pH 5-7, mixed with 1 part, pH 3-10; effective pH range, - nor absence of LDL receptors did ASGPR-defective mice accu4-6.5) and 10% SDS-polyacrylamidegel electrophoresis (second dimen- mulate any chylomicron remnants in their plasma. sion), treated withEN3HANCE, and exposed to x-ray film for 3 days. DISCUSSION

We have generated mice lacking functional asialoglycoprocoid (Fig. 3 A , open symbols) was not clearedby animals of any genotype (+I+, +I-, or -I-). tein receptors by destroying the gene for the minor MHL-2 It has previously been suggested that ASGPR has a central receptor subunit by homologous recombination in embryonic regulatory role in the turnover of desialylated plasma glyco- stem cells. Homozygous receptor-deficient mice are viable and display no obvious phenotype under laboratory housingcondiproteins. Such proteins would be expected to accumulate in MHL-2-deficient mice that lack functional, high affinity ASG- tions. In particular, their fertility is not impaired and the aniPRs. We attempted toidentify such plasma proteins containing mals appear to have a normal lifespan (exceeding16 months a t exposed terminal galactose residues inwild type andknockout the time of writing). Lack of expression of the MHL-2 subunit mice by labeling them with['4C]sialic acid and separating the leads to a pronounced reduction of expression of the major proteins by two-dimensional gel electrophoresis.Within the MHL-1 subunit in vivo. This suggests that in polarized liver

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cells MHL-2 promotes post-translational stabilityof the major MHL-1 subunit of ASGPR, probably by oligomerization in an early compartment of the secretory pathway. In an analogous situation, the minor subunit appears to be unstable and is rapidly degraded in the endoplasmic reticulum in the absence of coexpression of the major subunit (Wikstrom and Lodish, 1992; Yuk and Lodish, 1993). Expression of MHL-1 mRNA is not affected by the disruption of the MHL-2 gene. Inagreementwith previous studiesintransfected cells (McPhaul and Berg, 1987; Shia andLodish, 1989; Geffen et al., 1989; Braiterman et al., 1989; Graeve et al., 1990) we find that the minor receptor subunit is absolutely required for normal receptor function. ASOR requires the heterooligomeric (probably HL-1JHL-2,) form of ASGPR to bind with high affinity to the receptor. The remaining low level of MHL-1 protein that can be detected by Western blot analysis in theknockout mice is apparently unable to induce a measurable clearance of 1251labeled asialoorosomucoid. Whether a fraction of the expressed MHL-1 protein escapes intracellular degradation and appears on the cell surface has not been explored. These data further suggest that ASGPR is non-redundant and that the homologous galactose-specific lectin expressed on macrophages (Ozaki et al., 1992) does not contributesignificantly to theclearance of systemically circulating desialylated glycoproteins. A role of ASGPR in the normal turnoverof plasma proteins and lymphocytes (Samlowski et al., 1984) has been frequently discussed but has never been proven experimentally. If this were indeed the case we would expect knockout animals to accumulate substantial amountsof desialylated glycoproteins and lymphocytes in their circulation. Using three independent assay procedures we failed to detect such increased plasma protein levels. While these results do not rule out a role of ASGPR in the regulationof the plasma levels of minor serum glycoproteins or hormones(Drickamer, 19911, theyargue against a general function of ASGPR in thehomeostasis of the major glycosylated plasma components. Furthermore, in multiple experiments notshown here by fluorescence-activatedcell sorting analysis we were also unable to detect a n increase in the number of circulating lymphocytes with elevated cell surface galactose levels. Windler and colleagues (1991) proposed a possible function of ASGPR in hepaticlipoprotein metabolism. We have tested this hypothesis by crossing the ASGPR-deficient mice with animals lacking the receptor for LDL (Ishibashi et al., 1993). The LDL receptor is the major hepatic lipoproteinreceptor and efflciently removes LDL as well as chylomicron remnants from the circulation. Any effect of ASGPR on lipoprotein metabolism might therefore be effectively masked inmice expressing functional LDL receptors. On the other hand,if ASGPR would play any significant physiological role in the hepatic uptake of the remnants in mice, this should result in the accumulation of measurable amountsof these lipoproteins in the plasma of mice deficient for both LDL receptor and ASGPR. However, the plasma lipoprotein profile of ASGPFVLDL receptor double deficient mice did not differ from that of the mice lacking solely the LDL receptor (Ishibashi e t al., 1993). In contrast, apoEdeficient mice or LDL receptor-deficient animals in which the LDL receptor-related protein has been transiently inactivated

Mice accumulate large amounts of remnants (Plump et al., 1992; Zhang e t al., 1992; Willnow et al., 1994). These results argue against a major role of ASGPR in lipoprotein metabolism. In summary, using genetically receptor-defective mice we failed to detect physiological evidence for several of the postulated functions that had previously been ascribed to ASGPR. Although this major endocytic receptor has been extensively characterized on the cellular level, its physiological relevance is still largely unknown. The availability of viable ASGPR-defective mice now forms a basis on which the physiology of this receptor can be explored. Acknowledgments-We thank Lucy Lundquist, Wen-Ling Niu, John Dawson, ScottClark, and the staff of the Parkland pathology laboratory for expert technical assistance and Miguel Seabra, James Bryant, and Guy James for helpful advice. We are indebted to Mike Brown, Joe Goldstein, Mike McPhaul, and Mark Lehrman for reading the manuscript and for critical comments during this study. REFERENCES Ashwell, G., and Harford, J. (1982)Annu. Reu. Biochem. 61,531-554 Ashwell, G., and Morell, A. G. (1974) Adv. Enzymol. Relat. Areas Mol. Biol. 41, 99-128 Bradley, A. (1987) in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach (Robertson, E.J., ed) pp. 113-151, IRL Press, Oxford Braiterman, L. T., Chance, S. C., Porter, W. R., Lee, Y. C., Townsend, R. R., and Hubbard, A. L. (1989) J. Biol. Chem. 264, 1682-1688 Cross, A. S., and Wright, D. G. (1991) J. Clin. Invest. 88, 2067-2076 Dini, L., Autuori, F., Lentini, A,, Oliverio, S., and Piacentini, M. (1992) FEBS Lett. 296, 174-178 Drickamer, K. (1991) Cell 67, 1029-1032 Drickamer, K, Mamon, J. E , Binns, G., and h u n g , J. 0.(1984)J. Biol. Chem. 269, 770-778 Fiete, D., Srivastava, V., Hindsgaul, O., and Baenziger, J. U.(1991) Cell 67, 11031110 Geffen, I., and Spiess, M. (1992) Int. Reu. Cytol. 137B, 181-219 Geffen, I., Wessels, H. P., Roth, J., Shia, M. A., and Spiesa, M. (1989) EMBO J. 8, 2855-2861 Graeve, L., Patzak, A,, Drickamer, K., and Rodriguez-Boulan, E. (1990) J. Biol. Chem. 266, 1216-1224 Herz, J., and Gerard, R. D.(1993) Proc. Natl. Acad. Sci. U.S. A. 90,2812-2816 Ishibashi, S., Brown, M. S., Goldstein, J. L., Gerard, R. D., Hammer, R. E., and Herz, J. (1993) J. Clin. Invest. 92, 883-893 Kowal, R. C., Herz, J., Weisgraber, K. H., Mahley, R. W., Brown, M. S., and Goldstein, J. L. (1990) J. Biol. Chem. 266,10771-10779 McPhaul, M., and Berg, P. (1986) Proc. Natl. Acad. Sci. U.S. A. 83,8863-8867 McPhaul, M., and Berg, P. (1987) Mol. Cell. Biol. 7, 1841-1847 O'Farrell, P. H. (1975) J. Biol. Chem. 260,4007-4021 Ozaki, K , Itoh, N., and Kawasaki, T. (1992) J. Bid. Chem. 267,922%9235 Plump, A. S., Smith, J. D., Hayek, T., Aalto-SetiilP, K., Walsh, A,, Verstuyft, J. G., Rubin, E. M., and Breslow, J. L. (1992) Cell 71, 343-353 Samlowski, W. E., Spangrude, G. J., and Daynes, R.A. (1984) Cell. Immunol. 88, 309322 Sanford, J. P., and Doyle, D. (1990) Biochim. Biophys. Acta 1087,259-261 Schauer, R. (1985) ?Fends Biochem. Sci. 10,357-360 Schlepper-SchPfer,J., Friedrich, E., and Kolh, H. (1981) Eur: J. Cell Biol. 26, 95-102 Shia, M. A,, and Lodish, H. F. (1989) Proc. Natl. Acad. Sci. U. S. A. 66,115%1162 Soriano, P., Montgomery, C., Geske, R., and Bradley, A. (1991) Cell 64,693-702 S p e , M., and Lodish, H. F. (1985) Proc. Natl. Acad. Sci. U. S. A. 82,6465-6469 Spless, M., Schwartz,A. L., and Lodish, H. F. (1985)J. Biol. Chem. 260, 1979-1982 Weinstein, J., de Souza-e-Silva, U., and Paulson, J. C. (1982) J. Biol. Chem. 267, 13835-13844 Wikstrom, L., and Lodish, H. F. (1992) J. Biol. Chem. 267, 5 4 Willnow, T. E., Sheng, Z., Ishibashi, S., and Herz, J. (1994) Science 264,1471-1474 Windler. E., Greeve. J.. Levkau, B., Kolb-Bachofen, V., Daerr, W., and Greten, H. (199i) Bcochem. J . 276, 79-87 Yeatman, T. J.,Bland, K. I., Copeland, E. M., and Kimura, A. K. (1989)J. Surg. Res. 46,567-571 Yuk, M. H., and Lodish, H. F. (1993) J. Cell Biol. 123, 1735-1749 Zannis. V.I., McPherson, J., Goldberger,G.,Karathanasis, S. K , and Breslow, J. L. (1984) J. Bid. Chem.' 259, 5495-5499 Zhang, S. H., Reddick, R. L., Piedrahita, J. A,, and Maeda, N. (1992) Science 268, 468-471