Surface Location and High Affinity for Calcium of a 500-Kd Liver Membrane Protein Closely Related To
Total Page：16
File Type：pdf, Size：1020Kb
The EMBO Journal vol.7 no.13 pp.4119-4127, 1988 Surface location and high affinity for calcium of a 500-kd liver membrane protein closely related to the LDL-receptor suggest a physiological role as lipoprotein 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 proteins 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, apolipoprotein 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 apolipoproteins 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: apolipoprotein E/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 cholesterol 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 chylomicron 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 gene, and not simply a mutant or alternatively protein sequence is composed of three types of short spliced product of the LDL-receptor gene.