The EMBO Journal vol.7 no.13 pp.4119-4127, 1988

Surface location and high affinity for calcium of a 500-kd liver membrane closely related to the LDL-receptor suggest a physiological role as receptor

Joachim Herz, Ute Hamann, Sissel Rogne1, animals or patients with defective LDL receptors (Kita et Ola Myklebost2, Heinrich Gausepohl and Keith al., 1982; Brown and Goldstein, 1983). Furthermore, in K.Stanley transgenic mice over-expressing the LDL-receptor, LDL but not HDLc concentrations are reduced (Hofmann et al., European Molecular Biology Laboratory, Meyerhofstrasse 1, Postfach 1988). In liver, which is the central organ for lipoprotein 10.2209, D-6900 Heidelberg, FRG, 'Institute for Animal Science, The metabolism two low mol. wt apo E-binding have Agricultural University, N-1432 As, Norway, and 2Biochemistry Department, Institute for Cancer Research, Montebello N-0310, Oslo been found by affinity chromatography and by ligand blotting 3, Norway (Mahley, 1988), but these turned out not to be genuine apo E receptors (Beisiegel et al., 1987). A further high mol. wt Communicated by B.Dobberstein lipoprotein receptor has been demonstrated in liver by ligand We describe a cell surface protein that is abundant in blotting (Hoeg et al., 1986). Different receptors for apo E liver and has close structural and biochemical similarities may also be involved in its other postulated functions of to the low density lipoprotein (LDL) receptor. The regenerating damaged neural tissue, modulation of the complete sequence of the protein containing 4544 amino immune response and modulating cell growth and differen- acids is presented. From the sequence a remarkable tiation (for review see Mahley, 1988). Until now, however, resemblance to the LDL-receptor and epidermal growth none of these receptors has been purified or cloned. We factor (EGF) precursor is apparent. Three types of present here the complete sequence of a human cDNA clone repeating sequence motifs entirely account for the encoding a cell surface protein with strong homology to the extracellular domain of the molecule. These are arranged LDL-receptor and the epidermal growth factor (EGF)- in a manner resembling four copies of the ligand binding precursor. and the EGF-precursor homologous region of the LDL- receptor. Following a proline-rich segment of 17 amino acids are found six consecutive repeats with close Results homology to EGF. A single membrane-spanning segment Isolation and characterization of cDNA clones precedes a carboxy-terminal 'tail' of 100 amino acids. Molecular cloning and site-directed mutagenesis of the LDL- This contains two seven-amino acid sequences with receptor and one of its two ligands, E (apo striking homology to the cytoplasmic tail of the LDL- E), have shown that the principal interaction between ligand receptor in the region that contains the signal for and receptor involves a basic region on the ligand (Lalazar clustering into coated pits. The mRNA for this protein et al., 1988) and an acidic region present on each of seven is most abundant in liver, brain and lung. By using an cysteine-rich domains at the amino-terminus of the receptor antibody raised against a 13-amino acid peptide cor- (Yamamoto et al., 1984). Homologous sequences with the responding to the deduced amino acid sequence of the same pattern of cysteine residues (termed class A cysteine- carboxy-terminus of the protein we have demonstrated rich motifs; Stanley et al., 1986) have also been found in its existence on the cell surface and its abundance in liver. the terminal complement components (Stanley et al., 1985; Like the LDL-receptor this protein also strongly binds Haefliger et al., 1987; Rao et al., 1987; DiScipio et al., calcium, a cation absolutely required for binding of 1988) and complement factor I (Catterall et al., 1987). Only B and E to their receptors. We propose in the former group however is the acidic region conserved that this LDL-receptor related protein (LRP) is a and here it has been argued that they serve a similar function recycling lipoprotein receptor with possible growth- as in the LDL-receptor, binding to apo E-like regions of the modulating effects. proteins in order to stabilize the cytolytic conformation of Key words: /atherosclerosis/endocytosis/ the complex (Tschopp et al., 1986; Stanley, 1988). epidermal growth factor/LDL-receptor We therefore decided to search for proteins containing the highly conserved acidic region of the class A cysteine-rich motif using a complementary oligonucleotide, since proteins Introduction interacting specifically with positively charged apolipo- proteins could be expected to contain these structural and Studies of the structure and function of the low density functional units. Because of the high abundance of comple- lipoprotein (LDL) receptor have contributed a great deal to ment proteins in liver we first searched a mouse lymphocyte the understanding of homeostasis in normal and cDNA library and found a partial cDNA clone which diseased states, and provided an early insight into the cell contained eight of these motifs arranged in a similar manner biology of endocytic pathways (for review see Brown and to the apo B, E-binding domain of the LDL receptor. Using Goldstein, 1986). The LDL-receptor cannot be the only this partial mouse cDNA clone as an initial hybridization receptor with specificity for apolipoprotein E (apo E) since probe for cDNA walking, eight clones coding for a 15-kb the levels of remnants, HDLc and fl-VLDL cDNA were then isolated from human liver cDNA libraries particles (which also contain apo E) are not elevated in constructed in plasmid and lambda vectors (Figure 1). One

tIRL Press Limited, Oxford, England 4119 J.Herz et al.

LRP-5

LRP-2 LRP-4 LRP-8 ;~ LRP-3 LRP-1 LRP-6

LRP-9 LRP-7

Sal 11 Xba Kpn Bsm I Hind IlI I I BpiI I I IIl I -I 0 14896 bp 359p ...ACTGTG/gtgagca...... tcctcag/AGCCC... Intron

Fig. 1. Overlapping cDNA clones coding for the putative lipoprotein receptor. Several rounds of screening of the human liver library resulted in a total of seven overlapping clones (LRP-1 to LRP-7) which accounted for 11 kb of cDNA. The 3' clone, LRP-8, was isolated from a different oligo(dT) primed human liver cDNA library in XgtlO and contained an unspliced intron interrupting amino acid residue 3713. The large 5' clone, LRP-9, was isolated in a later screen of the total plasmid library with LRP-2. Unique restriction enzyme sites are shown. clone at the 3'-end contained what appeared to be an intron cysteine residues. A large shift in apparent mobility was of 359 bp interrupting two class A repeats and containing observed when the immunoprecipitated protein was electro- stop codons in all reading frames (Figure 1). Rescreening phoresed in a reduced as compared with non-reduced state the plasmid library with oligonucleotide probes on either side on SDS-PAGE (data not shown), thus showing that the of the intron resulted in the isolation of four clones covering cysteine residues are disulphide linked and therefore probably this same region, none of which contained the inserted located on the extracellular side of the plasma membrane. sequence. The position of the inserted sequence exactly be- This is also supported by the prediction of a cleavable signal tween two class A cysteine-rich motifs, and the presence sequence at the amino terminus. On the carboxy-terminal of consensus splice sites (Mount, 1982) at the junctions side of the membrane-spanning segment a sequence of 100 (Figure 1), suggest that this sequence is an unspliced intron. amino acids is found. This is twice the length of the The assembled cDNA sequence contains an open reading cytoplasmic tail in the LDL-receptor and contains a dupli- frame coding for 4544 amino acids with a 5'-untranslated cation of the sequence around the tyrosine (residue 807 in region of467 bp and a 3'-non-coding region of 798 bp with Figure 4) which has previously been shown to be essential a polyadenylation signal (AAATAAAA) 4 bp upstream of for clustering of the LDL-receptor into coated pits (Davis the oligo(dT) priming site. This gives a total cDNA length et al., 1987). The conservation of some of the residues of 14 896 bp, agreeing with the observed mRNA size of adjacent to the tyrosine indicates that they also may con- about 15 kb (see Figure 7a). From the predicted amino acid tribute to the signal for clustering into coated pits. Secondary sequence of the protein (Figure 2) a mol. wt of 503 kd is structure prediction (Gibrat et al., 1987) indicates that the expected for the unglycosylated polypeptide chain. If all 52 asparagine and proline residues of this NPVY sequence are extracellular sites for attachment of N-linked oligosaccharides very likely to be present in a surface turn structure of the were used (asterisks, Figure 2) the mol. wt would rise by protein and could therefore easily participate in an interaction - 150 kd. Although a high level of homology was observed with components of the coat structure. The high conservation between the predicted amino acid sequence of this molecule of this one region in the cytoplasmic tail suggests that this and those of the LDL-receptor and EGF-precursor (up to protein is also clustered into coated pits and endocytosed in 40% in the class A regions) there is little overall homology a similar manner to the LDL-receptor. at the nucleotide level showing that this clone is encoded The entire extracellular portion of the predicted mature by a separate , and not simply a mutant or alternatively protein sequence is composed of three types of short spliced product of the LDL-receptor gene. repeating segments which are also found in the LDL-receptor and EGF-precursor (Gray et al., 1983; Scott et al., 1983). Domain structure of LDL-receptor related protein The first of these contains six cysteine residues in a span (LRP) of40 amino acids and has so far only been found in the LDL- A hydropathy plot (Kyte and Doolittle, 1982) ofthe predicted receptor and a number of complement components. The amino acid sequence demonstrated the presence of two characteristic feature of this 'class A motif' is a pattern of hydrophobic regions (underlined in Figure 2 and identified cysteine spacing plus a number of other conserved amino with arrows in Figure 3a), one at the extreme amino ter- acids (Figure 5). In both the terminal complement com- minus, and the other close to the carboxy terminus. The ponents and the LDL-receptor the carboxy terminus of the former corresponds to a cleavable signal peptide of probably motif is negatively charged, but the terminal complement 19 amino acids in length (von Heijne, 1986), while the latter components may be distinguished from the LDL-receptor strongly resembles the membrane-spanning segment of class A motifs by the presence of a highly-conserved membrane proteins (Rao and Argos, 1986), having a positively-charged segment in the centre of the motif (Figure histidine residue on the amino-terminal side of the 5). In this respect the class A repeats of the LRP are membrane-spanning segment in the plane of the bilayer essentially indistinguishable from those of the LDL-receptor and three positive charges on the carboxy-terminal side. The in charge distribution and conserved amino acids arguing portion of the molecule on the amino-terminal side of the for a lipoprotein-binding function rather than a complement- membrane-spanning segment is very large and rich in like interaction. Furthermore the class A motifs of the LRP

4120 Molecular cloning of an LDL-receptor related protein

-19 MLTPP LL7LLP0LLSA&VAIDAPKTCSPKQFACRDQITCISKGWRCDGERDCPDGSDEAPEICPQSKAQRCQPNEHNCLGTELCVPMS70 71 RLCNGVQDCMDGSDEGPHCRE LQGNCSRLGCQHHCVPTLDGPTCYCNSSFQLQADGKTCKDFDECSVYGTCSQLCTNTDGSFICGCVEGYLLQPDNRSCK 170 171 AKNEPVDRPPVLLIANSQNILATYLSGAQVSTITPTSTRQTTAMDFSYANETVCWVHVGDSAAQTQLKCARM70PGLKGFVDEHTINISLSLHHVEQMAIDW270 271 LTGNFYFVDDIDDRIFVCNRNGDTCVTLLDLELYNPKGIALDPAMGKVFFTDYGQIPKVERCDMDGQNRTKLVDSKIVFPHGITLDLVSRLVYWADAYLD 370 371 YIEVVDYEGKGRQTIIQGILIEHLYGLTVFENYLYATNSDNANAQQKTSVIRVNRFNSTEYQVVTRVDKGGALHIYHQRRQPRVRSHACENDQYGKPGGC 470 471 SDICLLANSHKARTCRCRSGFSLGSDGKSCKKPEHELFLVYGKGRPGIIRGMDMGAKVPDEMIPIENLMNPRALDFHAETGFIYFADTTSYLIGRQKID 570 571 GTERET LKDGIHNVEGVAVDWMGDNLYWTDDGPKKT SVARLEKAAQTRKTLIEGKMTHPRAIVVDPLNGWMYWTIDWEEDPKDSRRGRLERAWMGSHR 670 671 DIFVTSKTVLWPNGLSLD IPAGRLYWVDAFYDR IETILLNGTDRKIVYEGPELNHAFGLCHHGNYLFWTEYRSGSVYRLERGVGGAPPTVTLLRSERPPI 770 771 FE IRMYDAQQQQVGTNKCRVNNGGC SSLCATPGSRQCACAEDQVLDADGVTCLANPSYVPPPQCQPGEFACANSRCIQERWKCDGDNDCLDNSDEAPAL 870 871 CHQHTCPSDRFKCENNRCIPNRWLCDGDNDCGNSEDESNATCSARTCPPNQFSCASGRCIPISWTCDLDDDCGDRSDESASCAYPTCFPLTQFTCNNGRC 970 971 IN INWRCDNDNDCGDNSD1AGCSHSCSSTQFKCNSGRCIPEHWTCDGDNDCGDYSDETHANCTNQATRPPGGCHTDEFQCRLDGLCIPLRWRCDGDTDCM1070 1071 DS SDEKSCEGVTHVCDPSVKFGCKDSARCISKAWVCDGDNDCEDNSDEENCESLACRPPSHPCANNTSVCLPPDKLCDGNDDCGDGSDEGELCDQCSLNN 1170 1171 GGCS HNCSVAPGEGIVCSCPLGMELGPDNHTCQIQSYCAKHLCSQKCDQNKFSVKCSCYEGWVLEPDGES1CRSLDPFKPFIIFSNRHEIRRIDLHKGDY127^ 1271 SVLVPGLRNT IALDFHLSQSALYWDVVEDKIYRGKLLDNGALTSFEVVIQYGLATPEGLAVDWIAGNIYWVESNLDQIEVAKLDGTLRTTLIAGDIEHP 1370 1371 RAIALDPRDGILFWTDWDASLPRIEAASMSGAGRRTVHRETGSGGWPNGLTVDYLEKRILWIDARSDAIYSARYDGSGHMEVLRGHEFLSPHPFAVTLYGG 1470 1471 EVYWTDWRTNTAANKTGHNVTVQRTNTQPFDLQVYHPSRQPMAPNPCEANGGQGPCSHLCLINYNRTVSCACPHIMKLHKDNTTCYEFKKFLLYAR 1570 1571 QMEI RGVDLDAPYYNYIISFTVPDIDNVVLDYDAREQRVYWSDVRTQAIKRAFINGTGVETVVSADLPNAHGIAVDWVSRNLFWTSYDTNKKQINVARL 1670 1671 DGSFKNA1QGLEQPHGLVVHPLRGKLYWTDGDNISMANMDGSNRTLLFSGQKGPVGIAIDFPESKLYWISSGNHTINRCNLDGSGLEVIDAMRSQLGKA1770 1771 TAAIMGDKLWWADQVSEKMGTCSKADGSGSVVLRNS8TTLV7DESIQLDHKGTNPCSVNNGDCSQLCLPTSETTRSCMCTAGYSLRSGQQACEGV1870 1871 GSFLLYSVHEG RGIPLDPNDKSDALVPVSGTSIAVGIDFHAENDTIYWVDMGLSTISRAKRDQTWREDVVTNGIGRVEGIAVDWIAGNIYWTDQGFDVI 1970 1971 EVARLNGSFRYV VSQGLDKPRAITVHPEKGYLFWTEWGQYPRIERSRLDGTERWLVNVSISWPNGISVDYQDGKLYWCDARTDKIERIDLETGENREV 2070 2071 VLSSNNMDMFSVSVFEDFIYWSDRTHANGSIKRGSKDNATDSVPLRTGIGVQLKDIKVFNRDRQKGTNVCAVANGGCQQLCLYRGRGQRACACAHGMLAE 2170 2171 DGASCREYAGYLLYSERT LKSIHLSDERNLNAPVQPFEDPEHNKNVIALAFDYRAGTSPGTPNRIFFSDIHFGNIQQINDDGSRRITIVENVGSVEGLA 2270 2271 YHRGWDTLYWTS YTTSTITRHTVDQTRPGAFERETVI2TMSGDDHPRAFVLDECQNI0FWNWNEQHPSIMRAALSGANVLTLIEKDIRTPNGLAIDHRAE2370 2371 KLYFSDATLDKIERCEYDGSHRYVILKSEPVHPFGLAVYGEHIFWTDWVRRAVQRANKHVGSNMKLLRVDIPQQPMG IIAVANDTNSCELSPCRINNGGC 2470 2471 QD LCLLTHQGHVNCSCRGGRILQDDLTCRAVNSSCRAQDEFECANGECINFSLTCDGVPHCKDKSDEKPSYCNSRRCKKTFRQCSNGRCVSNMLWCNGAD 2570 2571 DCGDGSDE PCNKTACGVGEFRCRDGTC GNSSRCNQFVDCEDASDEMNCSATDC SSYFRLGVKGVLFQPCERTSLCYAPSWVCDGANDCGDYSDERDCP 2670 2671 GVKRPRCP LNYFACPSGRCIPMSWTCDKEDDCEHGEDETHCNKFCSEAQFECQNHRCISKQWLCDGSDDCGDGSDEAAHCEGKTCGPSSFSCPGTHVCVP 2770 2771 ERWLCDGDKDCADGADES ... IAAGCLYNSTCDDREFMCQNRQC IPKHFVCDHDRDCADGSDESPECE YPTCGPSEFRCANGRCLSSRQWECDGENDCHDQSD 2870 2871 EAPKNPHCTSPE HKCNASSQFLCSSGRCVAEALLCNGQDDCGDSSDERGCHINECLSRKLSGCSQDCEDLKIGFKCRCRPGFRLKDDGRTCADVDECSTT 2970 2971 FPCSQRCINTHGSYKCLCVEGYAPRGGDPHSCKAVTDEEPFLIFANRYYLRKLNLDGSNYTLLKQGLNNAVALDFDYREQMIYWTDVTTQGSMIRRMHLN 3070 3071 GSNVQVLHRTGLSNPDGLAVDWVGGNLYWCDKGRDTI3EVSKLNGAYRTVLVSSGLREPRALVVDVQNGYLYWTDWGDHSLIGRIGMDGSSRSVIVDTKIT3170 3171 WPNGLTLDYVTERIYWADAREDYIEFASLDGSNRHVVLSQDIPHIFALTLFEDYVYWTDWETKSINRAHKTTGTNKTLLI STLHRPMDLHVFHALRQPDV 3270C 3271 PNHPCKVNNGGCSNLCLLSPGGGHKCACPTNFYLGSDGRTCVSNCTASQFVCKNDKCIPFWWKCDTEDDCGDHSDEPPDCPEFKCRPGQFQC STGICTNP 3370 3371 AFICDGDNDCQDNSDEANCDIHVCLPSQFKCTNTNRCIPGIFRCNGQDNCGDGEDERDCPEVTCAPNQFQCSITKRCIPRVWVCDRDNDCVDGSDEPANC 3470 3471 TQM3CGVDEFRCKDSGRCIPARWKCDGEDDCGDGSDEPKEECDERTCEPYQFRCKNNRCVPGRWQCDYDNDCGDNSDEESCTPRPCSESEFSCANGRCIA357C 3571 GRWKCDGDHDCADG SDEKDCTPRCDMDQFQCKSGHCIPLRWRCDADADCMDGSDEEACGTGVRTCPLDEFQCNNTLCKPLAWKCDGEDDCGDNSDENPEE 3670 3671 CARFVCPPNRPFRCKNDRVCLWI GRQCDGTDNCGDGTDEEDCEPPTAHTTHCKDKKEFLCRNQRCLS SSLRCNMFDDCGDGSDEEDCSIDPKLTSCATNA 3770 3771 SICGDEARCVRTEKAAYCACRSGFHTVPGQPGCQD INECLRFGTCSQLCNNTKGGHLCSCARNF KTHNTCKAEGSEYQVLYIADDNE IRSLFPGHPHSA 3870 3871 YEQAFQGDESVRIDAMDVHVKAGRVYWTNWHTGTI SYRSLPPAAPPTTSNRHRRQIDRGVTHLNISGLKSRGIAIDWVAGNVYWTDSGRDVIEVAQMKG 3970 3971 ENRKTL SGMIDEPHAIVVDPLRGTMYWSDWGNHPKIETAAMDGTLRETLVQDNIQWPTGLAVDYHNERLYWADAKLSVIGSIRLNGTDPIVAADSKRGL 4070 4071 SHPF IDVFEDYIYGVTYINNRVFKIHKFGHSPLVNLTGGLSHASDVVLYHQHKQPEVTNPCDRKKCEWLCLLSPSGPVCTCPNGKRLDNGTCVPVPSPT 4170 4171 PPPDAPRPGTCNLQCFNGGSCFLNARRQPKCRCQPRYTGDKCE LDQCWEHCRNGGTCAASPSGMPTCRCPTGFTGPKCTQQVCAGYCANNSTCTVNQGNQ 4270 4271 PQCRCLPGFLGDRCQYRQCSGYCENFGTCQMAADGSRQCRCTAYFEGSRCEVNKCSRCLEGACVVNKQSGDVTCNCTDGRVAPSCLTCVGHCSNGGSCTM 4370 4371 L NSKMMPECQCPPHMTGPRCEEHVFSQQQPGHI 4I4LLLLLLLVLVAGVFWYKRRVQGAKGFQHQRMTNGAMNVEIGNPTYKMYEGGEPDDVGGLLD447C 4471 ADFALDPDKPTNFTNPVYATLYMGGHGSR4HSLASTDEKRELLGRGPEDEIGDPLA4525 Fig. 2. Amino acid sequence of the putative lipoprotein receptor. The open reading frame shown here extends from base 467 to base 14098. Leader peptide sequence and hydrophobic membrane-spanning sequence are shown underlined. Possible attachment sites for shown with asterisks. N-linked oligosaccharide are The sequence has been numbered from the consensus cleavage site of signal peptidase which most cleaves acids before the first cysteine of likely seven amino the first class A motif. The DNA sequence has been submitted to the EMBL nucleotide sequence data library. occur in clusters of two, eight, 1O and 11 repeats similar patterns (B. 1 and B.2 in Table I) occur, the most character- to the cluster of seven repeats in the LDL-receptor. istic feature being the sequence between the fifth and the Class B (or epidermal growth factor) motifs also contain sixth cysteine residue. In the LRP the first 16 cysteine class B repeats six residues in a span of 40 amino acids but the are of the B.2 pattern and occur in pairs following each of pattern of spaces between the cysteine residues is different the four clusters of class A motifs and in a single copy from that of the class A motifs (Table I). Two distinguishable separating groups of 50 amino acid 'YWTD' repeats (Figure 4121 J.Herz et al.

a 4 i ---t 2 z IEI &il.L_._,i I 1 1 I m II. lIai lhi I I i,it.lL JII_i_Ii a iL 1 l - A. .fj;likiiii all. li HPhobic 1~~~~~~~~~~~~~~~ HPhilic

, !N I^L m - * -I - - L L ~ L L LL S 1 XSJ w w L 2 1^] b

0

r.

.1 F. JIk -. .I/"/ ,/ /,"'///X / *_- ///w ",/. t._ ~/I/I/ ;/ /,, '/~/ /

i tylrTrrcLte~nt CiII Dxi| ir,i1n n 1 receptor related proteItn-rrs^pt

ZtLV--.F------t .) t -----7-

L1)L receptorjhJII..

--.A4AI JA A A IA -IIR

( I Precursor

--t-+- 1 ----tI~ ----- Xi I

El] El I 1". ]IC t' .,' II d )I,-[, II ri , -, ) (2 L I I . rI'r)L'nT. tp11l(.; I i ''.

Fig. 3. Structure of the putative receptor. (a) Hydropathy plot according to Kyte and Doolittle (1982), arrows indicate hydrophobic segments. (b) Dot matrix comparison with the LDL-receptor using a stringency of 16 out of 30 residues and the parameter table of Gribskov and Burgess (Gribskov and Burgess, 1986). Along the axes the location of homologous repeats is shown. (c) Alignment of the putative receptor with the LDL- receptor and EGF-precursor. The LRP has been broken into four pieces to emphasize the similarity with the other molecules.

3c). This is the same subclass of B-motif as is found in the LRP 4448-4460 E I G K - M Y E LDL-receptor (Sudhof et al., 1985) and the first five class PlijY LRP B motifs of the EGF precursor (Table I). 4482-4495 F T N P V YIA T L YM G G In contrast, the last six class B repeats of the LRP, which LDLR 801- 814 NFDI|N P V YQ K T T E D E replace the 0-linked sugar domain of the LDL-receptor (LRP17 to LRP22 in Table I) are all of the B. 1 pattern. These Fig. 4. Homology in the cytoplasmic tail region with the LDL- repeats are separated from the sixteenth B.2 repeat by a receptor. Residues 806 and 807 in the LDL-receptor were shown to be cluster of eight proline residues in 17 amino acids (residues critical for rapid endocytosis (Davis et al., 1987). 4164-4180 in Figure 2) suggesting an important break in the folding of the protein. In exactly the same position which growth factor is released by proteolytic activities in relative to a putative membrane-spanning region in the EGF- the extracellular environment (Pfeffer and Ullrich, 1985). precursor four identifiable class B repeats are found (Figure Using transfected cells it has been confirmed that these 3b), which are also of the B. 1 class. The last of these B molecules do indeed insert into membranes (Mrockowski motifs forms the biologically active growth factor after et al., 1988; Gentry et al., 1987) and extracellular processing cleavage of the precursor molecule. Indeed all the class B has been demonstrated for a-TGF (Gentry et al., 1987) and sequences with growth factor activity [EGF, vaccinia virus VVGF (Stroobant et al., 1985). growth factor (VVGF) and a-transforming growth factor The striking similarity between the B. 1 repeats with (TGF)] fall into the B. 1 class (Table I) and are derived from growth factor activity and the last six class B repeats of the inactive membrane-bound precursors. This led to the LRP, both with regard to amino acid sequence and position suggestion that these molecules are cell surface proteins in with respect to a membrane-spanning segment, raises the 4122 Molecular cloning of an LDL-receptor related protein

--C----F-C---RCIP--W-CDG--DC-D-SDE--C-- Taking into account the high degree of conservation of the extracellular domain between the partial mouse cDNA sequence (to be published in detail elsewhere) and the corresponding human sequence we went on to test whether LRP the conservation also included the cytoplasmic tail. Mouse liver and brain membranes were prepared by standard procedures and aliquots containing - 400 yg of protein each were separated on a 3-10O% PAGE. Following transfer to nitrocellulose the filters were probed with anti-tail antibody in the absence and presence of competing peptide (Figure 6b). An abundant protein in liver membranes, of the same size as the one immunoprecipitated from metabolically 4+ labelled HepG2 cells, reacts strongly with the antiserum as did a broader less homogeneous band between 75 and 80 kd. LDLr Reaction to both bands could be blocked by the addition of competing peptide suggesting that the group of lower mol. wt proteins could be derived by proteolysis from the LRP although we cannot entirely exclude the possibility that the low mol. wt bands are derived from a different protein also --CG--DFQC-TGRCIKRRL-CNGD-DCGD-SDEDDCE- containing the same epitope. If both bands are indeed the + same protein it must have a different stability in the two tissues.

El Pl , TCC 0E Tissue distribution We investigated the pattern of LRP expression to see if it might parallel the expected tissue distribution of any known lipoprotein receptor having ligands containing apo B or E (Figure 7b). For this purpose we used a mouse cDNA probe and mouse tissues since these were more readily available. Fig. 5. Consensus amino acids and charge distribution in the class A nucleotide homology with the sequence motifs. 31 sequences from the LRP, seven from the LDL- The mouse probe had 90% receptor and seven from the terminal complement components (four human sequence and encoded the region of the protein species of C9, human C8M, C8,B and C7) were aligned by their between residues 657 and 1378. Many tissues contained cysteine pattern and scored for the total number of positively or detectable amounts of the mnRNA (including spleen, thymus, negatively charged amino acids at each position. Amino acids heart, kidney and bone not shown in Figure 6b), but the occurring at the indicated position in >50% of the repeats are shown in the consensus. molecule was most highly expressed in liver, lung and brain with significant amounts also in intestine and muscle. The determined from these hybridiza- that the LRP could also release growth factor concentration of message possibility tions in mouse liver was between 1 in 103 and 1 in 104. and have a secondary peptides after proteolytic degradation This agrees reasonably well with the frequency of positive role in growth control. clones found in the human liver cDNA library, taking into as their name implies are character- The YWTD-repeats, account the short clone length in a random primed library a conserved Tyr-Trp-Thr-Asp sequence ized by highly and the possible differences between mouse and human liver. These repeat sequences are also in a span of 50 amino acids. It is, however, rather more abundant than the LDL receptor and EGF-precursor in groups of found in the LDL-receptor (Russell et al., 1983). About one order of magnitude more five flanked by class B-motifs. It has been shown that this LRP cDNA were found the EGF- clones hybridizing with an probe region is present in both the LDL-receptor and in the human liver cDNA library than clones hybridizing a exons one complete precursor as set of each encoding with an LDL-receptor cDNA probe. motif (Sudhof et al., 1985b). The occurrence of the same sequence pattern in the same grouping in the LRP (Figure Calcium-binding properties 3c) suggests that all three genes are closely related. For the LDL-receptor the first Class A motif has been shown to mediate Ca2'-binding of the receptor (van Driel et al., Surface location 1987) and the presence of this divalent cation is necessary A protein of the expected mol. wt for the LRP is made in for the binding of LDL (Brown and Goldstein, 1986). We the human hepatoma cell line HepG2 and located on the cell therefore looked for Ca-2-binding activity of the LRP using surface. This was shown by immune precipitation of proteins blots of non-reduced mouse liver and brain membrane from surface-iodinated and from metabolically labelled cells proteins separated on low percentage polyacrylamide gels. with an antibody raised against the carboxy-terminal 13 After incubation with 45Ca and exposure to X-ray film the amino acids of the predicted protein sequence. The LRP and one other high mol. wt protein were the major precipitated protein displays a slightly slower mobility on Ca-2'-binding proteins that could be detected in blots of SDS-PAGE than apo B-100 (calculated mol. wt 513 kd) crude membrane extracts of liver. In brain membranes only suggesting that some of the potential oligosaccharide attach- one band, corresponding to the LRP, bound Ca2+. The ment sites are used. Competition with the peptide completely position of LRP on the blot was determined by reprobing abolished the ability of the antiserum to precipitate the protein the same filter with anti-tail antibody, followed by binding (Figure 6a and b). of second antibody and enzymatic detection. One of the two 4123 J.Herz et al.

Table I. Cysteine-rich class B motifs B.1 motif

HEGF 5 EC P . . . LSHDGYCLHDGVCMY I EALDKYACNCVVGY I .... GERCQY VVGF 44 L C G. PEGDGYCL.HGDCIHARDIDGMYCRCSHGYT. .... GGIRCQH TGF 7 DC P DSHTQFCF.HGTCRFLVQEDKPACVCHSGYV. ... GARCEH LRP17 4180 T C N. .... LQCFNGGSCFLNARRQ.PKCRCQPRYT. .... GDKCEL LRP18 4216 QCW ... .EHCRNGGTCAASPSGM.PTCRCPTGFT. .... GPKCTQ LRP19 4252 V C A. .... GYCANNSTCTVNQGNQ.PQCRCLPGFL. .... GDRCQY LRP20 4288 QC S . GGYCENFGTCQMAADGS.RQCRCTAYFE. ... GSRCEV LRP21 4324 KC S . .RCL.EGACVVNKQSGDVTCNCTDGRV . . .APSCL LRP22 4357 T C V. .... GHCSNGGSCTMNSKMM.PECQCPPHMT. ... GPRCEE Consensus - C - G-C-N-G-C------P-CRC--G-- G-R C--

B.2 motif

LDL1 296 ECL. . ... DNNGGCS. HVCNDLKIG. YECLCPDGFQ. LVAQ. RRCED LDL2 336 ECQ ..... DPDTCS. QLCVNLEGG. .YKCQCEEGFQ. LD PHTKACKK LDL3 645 WCE.RTTLSNGGCQ.Q YLCLPAPQSPKFTCACPDGML. LA RDMR SC L T

LRPI 95 NCS ...... RLGCQ. HHCVPTLDG. . PTCYCNSSFQ. LQADGKTCKD LRP2 134 ECS .. VYGTCS. QLCTNTDGS. .FICGCVEGYL. LQ PDNR S CKA LRP3 458 ACENDQYGKPGGCS DICLLANSHKARTCRCRSGFS. LG S DGK S CKK LRP4 787 KCR .... VNNGGCS. SLCLATPGS. .RQCACAEDQV. LDADGVT C LA

LRP5 1165 QCSS . ... LNNGGCS. HNCSVAPGEG.IVCSCPLGME. LGPDNHTCQ I

LRP6 1207 YCA .. KHLKCS. QKCDQNKF S . VKCSCYEGWV. L E PDGE SCR S LRP7 1520 PCE ..ANGGQGPCS.S HLCL I NYNR'T VSCACPHLMK. LHKDNTTCYE LRP8 1830 PCS .... VNNGDCS. QLCLPT S ET T RSCMCTAGYS. L R S GQQAC EG LRP9 2139 VCA .... VANSSCQ. QLC LYRGRG(Q. RACACAHGM. LA EDGA SCR E LRP1O 2462 PCR .. INNGGCQ.Q DLC L LTHQGIH.VNCSCRGGR.. I LQDDLTCRA LRPI 1 2924 ECL .. SRKLSGCS. Q D C ED L K I G FKCRCRPGFR.F LKDDGRTCAD LRP12 2966 ECS ..... TTFPCS. QRC I NTHGS YKCLCVEGYAPY RGGDPH S CKA LRP13 3274 PCK. ... VNNGGCS.S NLCLLS PUGG G. HKCACPTNFY. LG S DGR TCV S LRP14 3767 SCA .... TNASICGDE ARCVRT EKA ..AYCACRSGFH. TV PGQPGCQD LRP15 3808 ECL ..... RFGTCS. QLCNNTKGGG ..HLCSCARN ... FMKTHNT CKA LRP16 4131 PCD . RKKCE. WL CL L S PS G ..PVCTCPNGKR. L. .DNGTCVP Consensus -C- -N G G C S -L C L----G - - C - C - - G - - L--D--T C--

Class B cysteine-rich motifs are shown for the LRP, the LDL-receptor (LDL, Yamamoto et al., 1984), human epidermal growth factor (HEGF; Gregory and Preston, 1977), the 19-kd early protein of vaccinia virus (VVGF; Blomquist et al., 1984) and human ca-transforming growth factor (TGF; Derynck et al., 1984). Consensus amino acids are shown if they occur in at least 50% of the sequences. Class B repeats fall into two sub- classes and have been drawn with their cysteine residues aligned in order to emphasize their different consensus sequences. Padding spaces are indicated with dots. Residues 665-668 have been omitted from the third LDL-receptor sequence. major Ca2+-binding proteins from liver membranes and the particles to their receptors. Its unusually high apparent single protein from brain comigrate with LRP (Figure 6c), mobility of -600 kd on SDS - PAGE and the lack of although it seems to have a slightly higher mobility in brain. reactivity on ligand blots could explain why this molecule This could indicate different post-translational modification has not been described previously. in different tissues. The concentration of the LDL-receptor The amino acid sequence derived from cDNA clones in these fractions was too low to be detected by this technique shows that the entire protein is made up of sequence motifs on the blot thus indicating higher abundance or affinity in found principally in the LDL-receptor and EGF-precursor. liver and brain of LRP for calcium. We do not yet know The molecule appears to have four functional regions: (i) an which of the 31 class A repeats of the LRP contain(s) the extracellular domain resembling four copies of the LDL calcium binding activity. receptor; (ii) a region of growth factor repeats similar to that found in EGF; (iii) a transmembrane segment; and (iv) a Discussion cytoplasmic tail which contains two copies of a possible In this paper we have described an abundant liver protein signal for clustering into coated pits. of 503 kd whose structural resemblance to the LDL-receptor In the extracellular domain of the LRP are found four strongly suggests a similar function for the uptake of lipopro- clusters of class A cysteine-rich motifs similar to those which tein particles. Apart from the structural similarity this are responsible for binding apo B and apo E in the LDL- conclusion is supported by three further lines of evidence. receptor (Yamamoto et al., 1984). Together these motifs (i) The finding of a striking homology in the cytoplasmic contain a net negative charge of 158 with a consensus tail within a region shown by detailed in vitro mutagenesis sequence more like that of the LDL-receptor than the experiments of the LDL-receptor to be essential for clustering terminal complement components. This strongly suggests into coated pits and rapid internalization; (ii) the location that the molecule is a surface receptor capable of binding of the LRP on the cell surface; and (iii) the fact that LRP positively-charged ligands such as apo B or apo E within binds Ca2+ with high affinity, a cation which is absolutely lipoprotein particles. Particles containing apoproteins without required for the binding of apolipoprotein (B, E) containing high positive charge like acetyl LDL (Brown and Goldstein,

4124 Molecular cloning of an LDL-receptor related protein

b 1 2 C 2 3 4 a 1 2 5

Fig. 6. Surface location and Ca2+-binding of LRP. (a) Immunoprecipitation of extract from surface iodinated HepG2 cells Lane 1, 5 M1 of total cell lysate; lane 2, 150 il cell lysate immunoprecipitated with anti-tail antiserum; lane 3, 150 uI cell lysate immunoprecipitated with anti-tail antiserum in the presence of 10 1tg competing peptide. Arrowhead indicates LRP. (b) Immunoblotting of LRP. Mouse liver membrane proteins (-400 jig) were separated on a non-reducing 3-10% SDS-PAGE and transferred to nitrocellulose. The track was cut in half and incubated in the absence (lane 1) and presence (lane 2) of competing peptide (5 ug/ml) with anti-tail antiserum at a dilution of 1:500. (c) Ca2+-binding. Mouse liver (-400 yg; lanes 1 and 3) and brain (lanes 2 and 4) membranes were separated on non-reducing 3-1I0% SDS - PAGE and transferred to nitrocellulose. The same filter was first used for Ca2'-blotting and after exposure reprobed with anti-tail antibody. Lanes 1 and 2; Ca2'-blot; lanes 3 and 4, Western blot.

1983) and lipoprotein(a) (McLean et al., 1987) are unlikely could lie in the complex structure of LRP. The arrangement therefore to bind to this molecule. Two that of the clusters containing the class A motifs might be very contain apo E, which may not yet have molecularly defined sensitive to SDS-denaturation and binding of a ligand to LRP receptors are chylomicron remnants and HDLc. If these could require positive cooperativity between multiple binding particles have receptors that are different from the LDL sites. We are therefore constructing a full length cDNA receptor, then they would be expected to be found pre- molecule in order to be able to determine the ligand dominantly in liver. Northern blot analysis showed that LRP specificity by transfection of cultured cells. expression was indeed greatest in liver although measurable The fact that LRP binds calcium ions further supports the amounts were found in most tissues, with high levels also hypothesis that LRP is a lipoprotein receptor with apo E in brain and lung. This is of interest since apo E is specificity. Apo E binding to liver membranes has been synthesized in a broad range of tissues, and is the principal found to be Ca2+ dependent just like LDL- and HDLc- apolipoprotein made in lung and brain (Lenich et al., 1988). binding to the LDL-receptor (Hui et al., 1986). As the LDL- Apo E takes part in the clearance of -rich lipo- receptor has been shown to bind Ca2+ (van Driel et al., proteins and in reverse cholesterol transport in these tissues. 1987), one would expect the LRP to also bind Ca2+ if it In addition, specialized roles for apo E have been postulated is to interact with apo E in a similar fashion. Only a few for modulating cell growth in brain, lymphocytes and other proteins in liver membranes are found to bind this cation, peripheral tissues (Mahley, 1988). A receptor with apo E LRP being one of them. specificity might therefore have a broad tissue distribution, An additional feature of interest in the LRP is the presence similar to that of the LRP. Preliminary attempts to show of class B sequence motifs bearing strong homology to the binding to lipoprotein particles by ligand blot have not so class B sequences which have growth factor activity. A far succeeded in our hands, although we could readily detect second possible function of the molecule could therefore be binding of LDL to the LDL-receptor from mouse brain the release of peptides with growth factor activity by membranes (data not shown). However, a protein of similar proteolytic processing. A similar dual function has been size as LRP was shown to bind LDL by Hoeg et al. (1986). proposed for the EGF-precursor (Pfeffer and Ullrich, 1985), One explanation for our being unable to reproduce this result and the homeotic gene products Notch from Drosophila

4125 J.Herz et al.

cannot be accounted for by defects of the LDL-receptor (Brown and Goldstein, 1984). Two of these result in elevated J-4 _I plasma lipoproteins which contain apo E and might therefore be due to defects in the LRP molecule. Characterization of I I I LDL-receptor like molecules is therefore of importance to the understanding of other familiar disorders leading to atherosclerosis. Materials and methods Cell culture and immunoprecipitation HepG2 cells were grown to 50% confluency in the presence of 10% FCS, incubated for 2 h in serum free medium and then scraped off the plate with a rubber policeman into PBS. Cells were washed twice in PBS at 4°C and then labelled on ice for 1 h with 125iodine using the lodogen method (Fraker and Speck, 1978). After labelling, the cells were washed again twice and then lysed in buffer containing 20 mM Tris, 120 mM NaCI, 4 mM MgCI2, 1% NP 40 at pH 7.6 in the presence of 400jug/ml PMSF. The lysate was cleared by centrifuging for 5 min at 4°C in an Eppendorf centrifuge and one-quarter of the supernatant incubated with 10 a1 of antiserum in the absence or presence of competing peptide. The immune complexes were isolated on Protein A-Sepharose, dissociated by boiling in SDS + DTT and separated on a 2-10% PAGE. The gel was dried and labelled proteins were visualized by exposure to Kodak XAR 5 film at -70°C for 48 h. Peptide coupling and immunization Peptide was coupled to keyhole limpet hemocyanin (KLH) as described (Kreis, 1986) using glutaraldehyde as the coupling agent. Carrier-peptide Fig. 7. Tissue distribution of the putative receptor in mouse tissues complex (200 /Ag) emulsified in complete Freund's adjuvant were used for using a homologous mouse cDNA probe. (a) Northern blot of liver initial immunization and boost of rabbits. Initial immunization RNA size of the mRNA, RNA mol. wt marker in kb. injections showing was performed by popliteal lymph node injection of half the material. The Slot blots of total RNA from five tissues of mouse (1, liver; (b) remainder was injected intradermally on five to six sites in the neck. Boost 2, intestine; 3, 4, skeletal muscle; 5, brain) in three different lung; injections were performed at 3-week intervals. Seven days after the third animals A, B and C. Amounts shown are in pg of double-stranded boost the animals were bled and the serum used without further purification. DNA from which the probe was made. The range of values for relative abundance in the measured tissue of three animals was: liver Library construction (normalized to 1.0) > lung (0.5-1.4) > brain (0.5 -0.9) > intestine cDNA libraries from the mouse T-cell line B 6.1 and from human liver (0.3-0.7) > skeletal muscle (0.2-0.3). were constructed in the vectors pEX and pGEM4 respectively essentially as described using the adaptor cloning strategy (Haymerle et al., 1986). (Wharton et al., 1985) and Lin from Caenorhabditis elegans Poly(A+) RNA was prepared by poly(U)-chromatography on MAP-paper 1985), all of which could require proteolytic (Medac, Hamburg) according to the manufacturer's recommendations. The (Greenwald, mouse T-cell library and the human liver library both contained <5% clones on the in to cleavage cell surface order release growth factor without inserts, 80% of the recombinants had inserts of > 1000 bp as judged peptides. Some support for this hypothesis comes from from small scale plasmid preparations. Western blots experiments of LRP using an anti-peptide antibody. These showed that the molecule could be more Screening procedures stable in liver than in brain, and that the major breakdown In situ colony hybridization of the human liver library with a partial mouse cDNA clone was performed at 65°C in 6 x SSC/0.1% SDS on colony product was of a size which could expose the growth factor- blots (50 000 colonies/plate) and washed in 1 x SSC/0. 1% SDS at 220C. like sequences. The resulting positive clone (LRPI) was used in the next screening round. A third feature of importance are two homologous All further hybridizations were done at 65°C in 6 x SSC/0. 1% SDS and in the tail of the molecule that contain washed in 0.1 x SSC/0.1% SDS at 65°C. All probes were prepared by sequences cytoplasmic random primed extension (Feinberg and Vogelstein, 1983). a possible signal for 'clustering' of receptors into coated pits. These sequences show a high homology to the LDL-receptor Sequencing methods extending over seven amino acids of the cytoplasmic tail and The complete cDNA sequence was obtained using the dideoxy chain termination method (Sanger et al., 1977) and the Sequenase enzyme (United including the tyrosine residue which is essential for clustering States Biochemical) by a combination of random fragment sequencing in of the LDL-receptor. The conservation of enough amino M13, end sequencing of restriction fragments and double-stranded DNA acids to give a predicted turn in the molecule at this point sequencing from synthetic oligonucleotide primers. for a structural feature in the clustering signal as well argues Western blot as the essential tyrosine residue, and possibly indicates that other amino Crude plasma membranes from liver and brain were prepared by differential acids may be important in the recognition site. centrifugation at 1000 g x 10 min, 10 000 g x 10 min, and 150 000 g We are at present testing this by site-directed mutagenesis. x 40 min. Pelleted membranes from the latter fraction were solubilized Cloning of the LDL-receptor has led to the molecular in SDS-sample buffer at 37°C and the proteins were transferred onto 0.22 Am description of familial disorders of hypercholesterolaemia. nitrocellulose filters (Schleicher and Schull) for 18 h as described by Burnette This has been of importance to the understanding of (1981) at 7 V/cm. Blotted proteins were visualized by staining with Ponceau great S and cut into strips. Prior to immunological detection or incubation with endocytosis, as well as leading to the possibility of gene calcium the filters were destained with PBS. Incubation with primary therapy. We believe, from its closely related structural and antiserum was carried out at room temperature for I 2 h in PBS containing biochemical properties that the LRP is another member of 0.1% Triton and 0.25% Gelatin. The filters were washed 3 x 5 min in a family of lipoprotein receptors with specificity for different the same buffer and then incubated with a dilution of 1:1000 goat anti-rabbit IgG (horse-radish peroxidase conjugated) for 30 min. The filters were again ligands containing apo E and apo B. Several lipid metabolic washed three times, rinsed in 50 mM Tris pH 7.6 and then developed us- disorders from a single genetic defect are known which ing either 3,3'-diaminobenzidine or 4-chloro-1-naphthol as substrate. 4126 Molecular cloning of an LDL-receptor related protein

Ca2+ blot Kita,T., Goldstein,J.L., Brown,M.S., Watanabe,Y., Hornick,C.A. and The calcium binding blots were performed essentially as described by Havel,R.J. (1982) Proc. Natl. Acad. Sci. USA, 79, 3623-3627. Maruyama et al. (1984) except that 50 mM NaCl instead of 60 mM KCI Kreis,T. (1986) EMBO J., 5, 931-941. and 15 mM Tris-HCI pH 7.0 instead of 10 mM imidazol-buffer at pH 6.8 Kyte,J. and Doolittle,R.F. (1982) J. Mol. Biol., 157, 105-132. was used. The filters were washed for 5 min by rinsing them several times Lalazar,A., Weisgraber,K.H., Rall,S.C., Giladi,H., Innerarity,T.L., in 25% ethanol (total volume 400 ml), air dried and exposed to Kodak XAR Levanon,A.Z., Boyles,J.K., Amit,B., Gorecki,M., Mahley,R.W. and film at -70°C for 24 h. Vogel,T. (1988) J. Biol. Chem., 263, 3542-3545. Lenich,C., Brecher,P., Makrides,S., Chobanian,A. and Zannis,V.I. (1988) RNA blots J. Lipid Res., 29, 755-764. RNA was extracted with urea and precipitated using lithium chloride (Auffrey Mahley,R.W. (1988) Science, 240, 622-630. and Rougon, 1980). Samples (2 tLg) were treated with formaldehyde and Maruyama,K., Mikawa,T. and Ebashi,S. (1984) J. Biochem., 95, 511-519. loaded on Biotrace Nylon membranes (Gelman) after dilution in 500 Al of McLean,J.W., Tomlinson,J.E., Kuang,W.-J., Eaton,D.L., Chen,E.Y., 10 x SSC. tRNA (5 Ag) was added to dilutions of the standards (mouse Fless,G.M., Scanu,A.M. and Lawn,R.M. (1987) Nature, 300,132-137. cDNA insert from which the probe was made) to simulate similar binding Mount,S.M. (1982) Nucleic Acids Res., 10, 459-472. conditions. After binding, blots were baked and hybridized at 65°C in 5 Mroczkowski,B., Reich,M., Whittaker,J., Bell,G.I. and Cohen,S. (1988) x SSC containing 100 itg salmon sperm DNA and 100 yg of yeast RNA Proc. Natl. Acad. Sci. USA, 85, 126-130. per ml. Washing was performed in 0.1 x SSC at 65°C. Total RNA was Pfeffer,S. and Ullrich,A. (1985) Nature, 313, 184. estimated by hybridizing with 1 yg of 32P-labelled oligo(dT)12- 18 in the Rao,A.G., Howard,O.M.Z., Ng,S.C., Whitehead,A.S., Colten,H.R. and presence of 10 Ag of unlabelled oligo(dT) at 37°C in 5 x SSC and washing Sodetz,J.M. (1987) Biochemistry, 26, 3556-3564. at the same stringency. Autoradiographs were scanned and the concentra- Rao,J.K.M. and Atgos,P. (1986) Biochim. Biophys. Acta, 869, 197-2 14. tion of specific signal calculated (i.e. the ratio of LRP signal to oligo(dT) Russell,D.W., Yamamoto,T., Schneider,W.J., Slaughter,C.J., Brown,M.S. signal). and Goldstein,J.L. (1983) Proc. Natl. Acad. Sci. USA, 80, 7501-7505. Sanger,F., Nicklen,S. and Coulson,A.R. (1977) Proc. Natl. Acad. Sci. USA, Acknowledgements 74, 5463-5467. Scott,J., Urdea,M., Quiroga,M., Sanchez-Pescador,R., Fong,N., Selby,M., We thank John Dickson for technical assistance, Philippe Neuner for Rutter,W.J. and Bell,G.I. (1983) Science, 221, 236-240. oligonucleotide synthesis and Dr G.Manfioletti for kindly providing the Stanley,K.K. (1988) Current Topics Microbiol. Immunol., 140, 82-104. human liver library in XgtlO. J.H. was supported by a fellowship from the Stanley,K.K., Kocher,H.-P., Luzio,J.P., Jackson,P. and Tschopp,J. (1985) Fonds Boehringer Ingelheim, S.R. was supported by an EMBO fellowship EMBOJ., 4, 375-382. and O.M. was supported by the Norwegian Cancer Society. Stanley,K.K., Page,M., Campbell,A.K. and Luzio,J.P. (1986) J. Mol. Immunol., 23, 451-458. Stroobant,P., Rice,A.P., Gullick,W.J., Cheng,D.J., Kerr,I.M. and References Waterfield,M.D. (1985) Cell, 42, 383-393. Sudhof,T.C., Goldstein,J.L., Brown,M.S. and Russell,D.W. (1985a) Auffray,C. and Rougon,F. (1980) Eur. J. Biochem., 107, 303-314. Science, 228, 815-822. Beisiegel,U., Catapano,A.L. and Soutar,A.K. (1987) In European Sudhof,T.C., Russell,D.W., Goldstein,J.L., Brown,M.S., Sanchez- Lipoprotein Club: the First Ten Years. European Lipoprotein Club, Pescador,R. and Bell,G.I. (1985b) Science, 228, 893-895. Glasgow, pp. 27- 37. and Nature, 322, 831-834. Proc. Acad. Tschopp,J., Masson,D. Stanley,K.K. (1986) Blomquist,M.C., Hunt,L.T. and Barker,W.C. (1984) Natl. van Driel,I.R., Goldstein,J.L., Sudhof,T.C. and Brown,M.S. (1987) J. Biol. Sci. USA, 81, 7363-7367. Chem., 262, 17443-17449. Brown,M.S. and Goldstein,J.L. (1983) Annu. Rev. Biochem., 52, 223 -261. von Heijne,G. (1986) Nucleic Acids Res., 14, 4683-4690. Brown,M.S. and Goldstein,J.L. (1984) In Petersdorf,R.G., Adams,R.D., Wharton,K.A., Johansen,K.M., Xu,T. and Artavanis-Tsakonas,S. (1985) Braunwald,E., Isselvacher,K.J., Martin,J.B. and Wilson,J.D. (eds), 7he Cell, 43, 567-581. Principles of Internal Medicine. McGraw-Hill, New York, 10th edn., Yamamoto,T., Davis,C.G., Brown,M.S., Schneider,W.J., Casey,M.L., pp. 547-559. Goldstein,J.L. and Russell,D.W. (1984) Cell, 39, 27-38. Brown,M.S. and Goldstein,J.L. (1986) Science, 232, 34-47. Burnette,W.N. (1981) Anal. Biochem., 112, 195-203. Received on August 31, 1988 Catterall,C.F., Lyons,A., Sim,R.B., Day,A.J. and Harris,T.J.R. (1987) Biochem. J., 242, 849-856. Davis,C.G., van Driel,I.R., Russell,D.W., Brown,M.S. and Goldstein,J.L. (1987) J. Cell. Biol., 262, 4075-4082. Derynck,R., Roberts,A.B., Winkler,M.E., Chen,E.Y. and Goeddel,D.V. (1984) Cell, 38, 287-297. DiScipio,R.G., Chakravarti,D.N., Muller-Eberhard,H.J. and Fey,G.H. (1988) J. Biol. Chem., 263, 549-560. Feinberg,A.P. and Vogelstein,B. (1983) Anal. Biochem., 132, 6- 13. Fraker,P.J. and Speck,J.C.,Jr. (1978) Biochem. Biophys. Res. Commun., 80, 849-857. Gentry,L.E., Twardzik,D.R., Lim,G.J., Ranchalis,J.E. and Lee,D.C. (1987) Mol. Cell. Biol., 7, 1585-1591. Gibrat,J.-F., Garnier,J. and Robson,B. (1987) J. Mol. Biol., 198, 425-443. Gray,A., Dull,T.J. and Ullrich,A. (1983) Nature, 303, 722-725. Greenwald,I. (1985) Cell, 43, 583-590. Gregory,H. and Preston,B.M. (1977) Int. J. Peptide Protein Res., 9, 107- 111. Gribskov,M. and Burgess,R.R. (I1986) Nucleic Acids Res., 14, 6745 -6763. Haefliger,J., Tschopp,J., Nardelli,D., Wahli,W., Kocher,H.-P., Tosi,M. and Stanley,K.K. (1987) Biochemistry, 26, 3551 -3556. Haymerle,H., Herz,J., Bressan,G., Frank,R. and Stanley,K.K. (1986) Nucleic Ac ids Res.. 14, 8615-8624. Hoeg,J.M., Demosky,S.J., Lackner,K.J.. Osborne,J.C.. Oliver,C. and Brewer,H.B. (1986) Biochim. Biophys. Acta, 876, 13-21. Hoffman,S.L., Russell.D.W., Goldstein,J.L. and Brown,M.S. (1987) Proc. Natl. Acad. Sci. USA, 84, 6312-6316. Hoffman,S.L., Russell,D.W., Brown,M.S., Goldstein,J.L. and Hammer,R.E. (1988) Science, 239, 1277-1281. Hui,D.Y., Brecht,W.J., Hall,E.A., Friedman,G., Innerarity,T.L. and Mahley,R.W. (1986) J. Biol. Chem., 261, 4256-4267. 4127