Alternative Splicing in the Ligand Binding Domain of Mouse ApoE Receptor-2 Produces Receptor Variants Binding Reelin but Not α2-Macroglobulin
2001; Elsevier BV; Volume: 276; Issue: 25 Linguagem: Inglês
10.1074/jbc.m102662200
ISSN1083-351X
AutoresChristian Brandes, Larissa Kahr, Walter Stockinger, Thomas Hiesberger, Wolfgang J. Schneider, Johannes Nimpf,
Tópico(s)Signaling Pathways in Disease
ResumoLR7/8B and ApoER2 are recently discovered members of the low density lipoprotein (LDL) receptor family. Although structurally different, these two proteins are derived from homologous genes in chicken and man by alternative splicing and contain 7 or 8 LDL receptor ligand-binding repeats. Here we present the cDNA for ApoER2 cloned from mouse brain and describe splice variants in the ligand binding domain of this protein, which are distinct from those present in man and chicken. The cloned cDNA is coding for a receptor with only five LDL receptor ligand-binding repeats,i.e. comprising repeats 1–3, 7, and 8. Reverse transcriptase-polymerase chain reaction analysis of mRNA from murine brain revealed the existence of two additional transcripts. One is lacking repeat 8, and in the other repeat 8 is substituted for by a 13-amino acid insertion with a consensus site for furin cleavage arising from an additional small exon present in the murine gene. None of the transcripts in the mouse, however, contain repeats 4–6. In murine placenta only the form containing repeats 1–3 and 7 and the furin cleavage site is detectable. Analysis of the corresponding region of the murine gene showed the existence of 6 exons coding for a total of 8 ligand binding repeats, with one exon encoding repeats 4–6. Exon trapping experiments demonstrated that this exon is constitutively spliced out in all murine transcripts. Thus, the murineApoER2 gene codes for receptor variants harboring either 4 or 5 binding repeats only. Recombinant expression of the 5-repeat and 4-repeat variants showed that repeats 1–3, 7, and 8 are sufficient for binding of β-very low density lipoprotein and reelin, but not for recognition of α2-macroglobulin, which binds to the avian homologue of ApoER2 harboring 8 ligand binding repeats. LR7/8B and ApoER2 are recently discovered members of the low density lipoprotein (LDL) receptor family. Although structurally different, these two proteins are derived from homologous genes in chicken and man by alternative splicing and contain 7 or 8 LDL receptor ligand-binding repeats. Here we present the cDNA for ApoER2 cloned from mouse brain and describe splice variants in the ligand binding domain of this protein, which are distinct from those present in man and chicken. The cloned cDNA is coding for a receptor with only five LDL receptor ligand-binding repeats,i.e. comprising repeats 1–3, 7, and 8. Reverse transcriptase-polymerase chain reaction analysis of mRNA from murine brain revealed the existence of two additional transcripts. One is lacking repeat 8, and in the other repeat 8 is substituted for by a 13-amino acid insertion with a consensus site for furin cleavage arising from an additional small exon present in the murine gene. None of the transcripts in the mouse, however, contain repeats 4–6. In murine placenta only the form containing repeats 1–3 and 7 and the furin cleavage site is detectable. Analysis of the corresponding region of the murine gene showed the existence of 6 exons coding for a total of 8 ligand binding repeats, with one exon encoding repeats 4–6. Exon trapping experiments demonstrated that this exon is constitutively spliced out in all murine transcripts. Thus, the murineApoER2 gene codes for receptor variants harboring either 4 or 5 binding repeats only. Recombinant expression of the 5-repeat and 4-repeat variants showed that repeats 1–3, 7, and 8 are sufficient for binding of β-very low density lipoprotein and reelin, but not for recognition of α2-macroglobulin, which binds to the avian homologue of ApoER2 harboring 8 ligand binding repeats. low density lipoprotein receptor very LDLR apolipoprotein LDLR relative with 7 or 8 LA repeats apoE receptor 2 receptor-associated protein α2-macroglobulin polymerase chain reaction type A binding repeats type B repeats reverse transcriptase base pair rapid amplification of cDNA ends horseradish peroxidase glutathioneS-transferase The low density lipoprotein receptor (LDLR)1 family consists of a growing number of structurally related composite cell surface receptors with partially overlapping ligand specificity (1Schneider W.J. Nimpf J. Bujo H. Curr. Opin. Lipidol. 1997; 8: 315-319Crossref PubMed Scopus (62) Google Scholar, 2Schneider W.J. Nimpf J. Curr. Opin. Lipidol. 1993; 4: 205-209Crossref Scopus (30) Google Scholar). For example, the LDLR harbors structurally and functionally defined modules, corresponding to distinct exons in the gene (3Schneider W.J. Biochim. Biophys. Acta. 1989; 988: 303-317Crossref PubMed Scopus (57) Google Scholar). These modules are as follows: (i) the "type A-binding repeats" (LA repeats) of ∼40 residues each, displaying a triple disulfide bond-stabilized and negatively charged surface mediating receptor/ligand interactions; (ii) "type B repeats" (EG repeats), also containing six cysteines each; EG repeats are homologous to regions in the epidermal growth factor precursor; (iii) modules of ∼50 residues with a consensus tetrapeptide, Tyr-Trp-Thr-Asp (YWTD); (iv) a so-called "O-linked sugar domain"; (v) a short transmembrane domain of ∼20 amino acids; and (vi) the cytoplasmic region with a signal for receptor internalization via coated pits. LR7/8B (4Novak S. Hiesberger T. Schneider W.J. Nimpf J. J. Biol. Chem. 1996; 271: 11732-11736Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 5Brandes C. Novak S. Stockinger W. Herz J. Schneider W.J. Nimpf J. Genomics. 1997; 42: 185-191Crossref PubMed Scopus (51) Google Scholar) and its human homologue called ApoER2 (6Kim D.-H. Iijima H. Goto K. Sakai J. Ishii H. Kim H.-J. Suzuki H. Kondo H. Saeki S. Yamamoto T. J. Biol. Chem. 1996; 271: 8373-8380Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar, 7Kim D.-H. Magoori K. Inoue T.R. Mao C.C. Kim H.-J. Suzuki H. Fujita T. Endo Y. Saeki S. Yamamoto T.T. J. Biol. Chem. 1997; 272: 8498-8504Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar) belong to the close relatives of the LDLR made up of exactly the same domains in the same order as in the LDLR. The occurrence of distinct splice variants adds yet another level of complexity to this family of proteins. LR7/8B expression in chicken is highly restricted to the brain, where the protein resides in large neurons and Purkinje cells, and in cells constituting brain barrier systems such as the epithelial cells of the choroid plexus and the arachnoidea and the endothelium of blood vessels (8Stockinger W. Hengstschläger-Ottnad E. Novak S. Matus A. Hüttinger M. Bauer J. Lassmann H. Schneider W.J. Nimpf J. J. Biol. Chem. 1998; 273: 32213-32221Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). The finding that chicken LR8B acts as receptor for α2-macroglobulin (8Stockinger W. Hengstschläger-Ottnad E. Novak S. Matus A. Hüttinger M. Bauer J. Lassmann H. Schneider W.J. Nimpf J. J. Biol. Chem. 1998; 273: 32213-32221Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) suggests a role in the clearance of α2-macroglobulin-proteinase complexes from the cerebrospinal fluid. Ligand binding studies with two splice variants of human ApoER2 demonstrated high affinity of the receptor to β-VLDL, indicating that in mammals the receptor might be involved in apoE-mediated transport processes in the brain as well (7Kim D.-H. Magoori K. Inoue T.R. Mao C.C. Kim H.-J. Suzuki H. Fujita T. Endo Y. Saeki S. Yamamoto T.T. J. Biol. Chem. 1997; 272: 8498-8504Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). This is an interesting aspect because a genetic link between certain alleles of the ApoE gene and the development of late onset Alzheimer's disease has been established (9Strittmatter W.J. Roses A.D. Annu. Rev. Neurosci. 1996; 19: 53-77Crossref PubMed Scopus (392) Google Scholar). However, no differences in the splice variant patterns of ApoER2 between control and Alzheimer's patients exist (10Clatworthy A.E. Stockinger W. Christie R.H. Schneider W.J. Nimpf J. Hyman B.T. Rebeck G.W. Neuroscience. 1999; 90: 903-911Crossref PubMed Scopus (57) Google Scholar). Targeted disruption of the apoER2 gene alone or in combination with that for the VLDL receptor gene revealed a key function of both receptors during embryonic brain development (11Trommsdorff M. Gotthardt M. Hiesberger T. Shelton J. Stockinger W. Nimpf J. Hammer R. Richardson J.A. Herz J. Cell. 1999; 97: 689-701Abstract Full Text Full Text PDF PubMed Scopus (1096) Google Scholar). Absence of functional ApoER2 and VLDL receptor leads to an inversion of cortical layers and absence of cerebellar foliation. This phenotype is indistinguishable from that seen in animals carrying either a mutation in the reeler gene or in the disabled gene (for review see Refs. 12Cooper J.A. Howell B.W. Cell. 1999; 97: 671-674Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 13Curran T. D'Arcangelo G. Brain Res. Rev. 1998; 26: 285-294Crossref PubMed Scopus (216) Google Scholar, 14Walsh C.A. Goffinet A.M. Curr. Opin. Genet. & Dev. 2000; 10: 270-274Crossref PubMed Scopus (84) Google Scholar). Both ApoER2 and VLDL receptor bind reelin, which is secreted by Cajal-Retzius cells (15D'Arcangelo G. Homayoundi R. Keshvara L. Rice D.S. Sheldon M. Curran T. Neuron. 1999; 24: 471-479Abstract Full Text Full Text PDF PubMed Scopus (696) Google Scholar, 16Hiesberger T. Trommsdorff M. Howell B.W. Goffinet A. Mumby M.C. Cooper J.A. Herz J. Neuron. 1999; 24: 481-489Abstract Full Text Full Text PDF PubMed Scopus (799) Google Scholar), and do so apparently together with cadherin-related neuronal receptors (17Senzaki K. Ogawa M. Yagi T. Cell. 1999; 99: 635-647Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar) and/or α3β1 integrin (18Dulabon L. Olson E.C. Taglienti M.G. Eisenhuth S. McGrath B. Walsh C.A. Kreidberg J.A. Anton E.S. Neuron. 2000; 27: 33-44Abstract Full Text Full Text PDF PubMed Scopus (505) Google Scholar), which may act as co-receptors for reelin to transmit the signal into migrating neurons. Upon reelin stimulation the intracellular adapter protein disabled-1 (Dab1), which interacts with the cytoplasmic domains of apoER2 and VLDLR (19Howell B.W. Lanier L.M. Frank R. Gertler F.B. Cooper J.A. Mol. Cell. Biol. 1999; 19: 5179-5188Crossref PubMed Scopus (336) Google Scholar, 20Trommsdorff M. Borg J.-P. Margolis B. Herz J. J. Biol. Chem. 1998; 273: 33556-33565Abstract Full Text Full Text PDF PubMed Scopus (491) Google Scholar), becomes tyrosine-phosphorylated (21Howell B.W. Herrick T.M. Cooper J.A. Genes Dev. 1999; 13: 643-648Crossref PubMed Scopus (359) Google Scholar, 22Howell B.W. Herrick T.M. Hildebrand J.D. Zhang Y. Cooper J.A. Curr. Biol. 2000; 10: 877-885Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). These data suggest that ApoER2 and VLDL receptor directly relate the extracellular reelin signal into a cellular response via Dab1, leading to the ultimate cell responses required for the correct positioning of newly generated neurons during brain development. In addition, interaction screens with apoER2 and other members of the receptor family have resulted in the identification of many other candidate proteins interacting with the cytoplasmic domains of the respective receptors, suggesting that they may be part of an intricate network of signaling pathways (23Gotthardt M. Trommsdorff M. Nevitt M.F. Shelton J. Richardson J.A. Stockinger W. Nimpf J. Herz J. J. Biol. Chem. 2000; 275: 25616-25624Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar, 24Stockinger W. Brandes C. Fasching D. Hermann M. Gotthardt M. Herz J. Schneider W.J. Nimpf J. J. Biol. Chem. 2000; 275: 25625-25632Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). Here we present the full-length cDNA and the partial characterization of the mouse apoER2 gene and show that differential splicing events in the ligand binding domain produce functional receptor variants that are distinct from those expressed in chicken and man. Splicing is tissue-specific and is regulated during brain development. The 5′-end of the mouse cDNA for apoER2 was cloned by 5′-RACE using the MarathonTM cDNA amplification kit (CLONTECH) according to the manufacturer's protocol. First and second strand cDNA was synthesized from 5 μg of poly(A)+ RNA and the poly(A) synthesis primer provided by the kit. 5′-RACE was done in two successive rounds of PCR amplification, with the sense primer located in an adapter ligated to the cDNA. The antisense primer was located in the known sequence from a partial cDNA clone (4Novak S. Hiesberger T. Schneider W.J. Nimpf J. J. Biol. Chem. 1996; 271: 11732-11736Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). The first round, which resulted in 334 bp of new sequence, was done using primer ASR1 (5′-CCGCTCCTGGTTGCACCGTTTGATG). The second round was performed with primer ASR2 (5′-GTCCTCGTCGCTGTTGTCCG) and resulted in additional 174 bp of new sequence exceeding the start codon. Total RNA was produced from 200 mg of frozen tissue (total brain and placenta from mature female Balb/c mice, and total chicken brain from female Brown Derco) using TriReagent (Molecular Research Center, Inc.). Poly(A)+ RNA was prepared from 500 μg of total RNA using the Micro-Fast Track mRNA Isolation Kit (Invitrogen) according to the manufacturer's protocol. First strand cDNA synthesis was performed with 1 μg of poly(A)+ RNA and random hexamer primers using SuperScript Reverse Transcriptase (Life Technologies, Inc.). One-tenth of the cDNA was used for subsequent PCR in the presence of 1.5 mm MgCl2, 0.2 mmdNTPs, 2 units of Taq DNA polymerase (PerkinElmer Life Sciences), and 1 μm of the appropriate primers on a GeneAmp 2400 (PerkinElmer Life Sciences). PCR conditions are as follows: 5 min initial denaturation at 94 °C, 1 min denaturation at 94 °C; 1 min annealing (for specific annealing temperatures, see primers), and 1 min extension at 72 °C for 40 cycles. The following primers have been used (numbers of the primers refer to the respective primers shown in Fig. 2: mouse 1, 5′-CGAGAATGAGTTCCAGTGTGG; mouse 2, 5′-CGTGAAGATCAGAGATGGGC, 1/2 annealing at 60 °C; mouse 3, 5′-CTGCCGAGAAGTTAAGCTGC; mouse 4, 5′-CCGCTCCTGGTTGCACCGTTTGATG, 3/4 annealing at 58 °C; mouse 5, 5′-GAGTTCCAGTGCAGCAACCG; mouse 6, 5′-CCGCTGCGGCAGGTGAACTGG, 5/6 annealing at 60 °C; chicken 7, 5′-CCAAGCAAGTATGCCCAGC; chicken 8, 5′-TTGGACACAGCCTGCCTCG, 7/8 annealing at 57 °C. PCR products were analyzed by agarose gel electrophoresis. Bands were isolated from the gel using the QIAEX II gel extraction kit (Qiagen) and subcloned into the pCR2.1 vector (Invitrogen). Several clones from each fragment were isolated, and positive clones were identified by PCR analysis and sequenced. A mouse ES P1 library (Genome Systems, Inc.) was screened by PCR using the following primer pair: A, 5′-TGTCTGCACAATAACGGCGGCTGTTC, and B, 5′-ACAGGTCTTCTGGTCCAAGAGCTGGAAG. PCR conditions are as follows: 2 min initial denaturation at 94 °C, 1 min denaturation at 94 °C, 1 min annealing at 56 °C, 1 min extension at 72 °C for 35 cycles. Using 1 μg of genomic DNA, a 105-bp amplicon derived from exon 9 (EG repeat A) was produced under these conditions. P1 plasmid DNA from positive clones was prepared from 150-ml cultures (Escherichia colistrain NS 3529, Genome Systems, Inc.) using the plasmid MidiKit (Qiagen) according to manufacturer's instructions including an additional phenol extraction step before loading on the column. P1 DNA was digested with BglII and shotgun-subcloned into theBamHI site of pBluescriptII (sK+). Clones were screened by PCR using two independent primer combinations (pair 1, 5′-GCGCGGACGGCGACTTCACC, 5′-GCCTTCGATTCGTCAGAGCC; pair 2, 5′-CCGCCGTCCACGCACTCGCC, 5′-CGAGAATGAGTTCCAGTGTGG) located within the 5′-part and the 3′-part of the ligand binding domain, respectively. The resulting clones were G14AS13 and 8AS-3.5 (see Fig. 3 A). The rest of the gene covering the ligand binding domain of the receptor was covered by PCR clones derived from the undigested P1 clone with the following primer combinations (see Fig. 3 A): 1, 5′-TGCAGCTTCAGCATCTCTCC and 5′-GTCCTCGTCGCTGTTGTCCG; 2, 5′-CCTTGGTGTGGAGATGCGATGAGG and 5′-AGGTGAGCATGGCGGCCTGCC; 3, 5′-GCGCGGACGGCGACTTCACC and 5′-GCAGCTTAACTTCTCGGCAGG; 4, 5′-GACGGAGAGAAGGACTGTGAGG and 5′-TGGTCCACTCAGTCAACCTGTCC; 5, 5′-AGTTCTATGAGAACTCATCCACC and 5′-GTGCTCATGACTTAGTGATGTGG; 6, 5′-AGTGGCGAGTGCGTGGACGGC and 5′-TGAGGTCAGTGCAGATGTGG. PCR products were isolated from the gel using the QIAEX II gel extraction kit (Qiagen), subcloned into the pCR2.1 vector (Invitrogen), and sequenced. For the exon trap experiment two constructs were made. One contained the exon coding for repeats 4–6 including 89 bp of the upstream and 563 bp of the downstream intron, respectively (corresponding to genomic clone 5, Fig. 3). For this, entire clone 5 was cloned into filled in SalI sites of the exon trap vector pet01 (MoBiTec GmbH, Göttingen) resulting in pEx4-6. The second construct contained genomic clone 5 plus clone G14A13B3 (see Fig. 3), thus harboring two exons, one for repeat 2 and the other for repeats 4–6. This construct (pEx2, 4–6) was made by cloning the entire genomic clone G14A13B3 into pEx4-6 upstream of clone 5 using XbaI and NotI sites in the polylinker of pet01. 3 μg of DNA was transfected into 293 cells using Lipofectin reagent (Life Technologies, Inc.), and cells were grown on a 90-mm plate. 48 h after transfection, cells were harvested, and mRNA was prepared using the Micro-fast track from Invitrogen. 1 μg of mRNA was taken and reverse-transcribed using 1 μl of Superscript (Life Technologies, Inc.) and the cDNA primer provided by the exon trap kit. RT-PCR was performed using 1/10th of the cDNA reaction using two primers from the exon trap kit (5′-primer 2 and 3′-primer 3) located 5′ and 3′ of the insert of pet01, respectively. PCR products were analyzed on a 1.5% agarose gel and sequenced. Chicken LR8B and mouse apoER2Δ4-6 and apoER2Δ4-6,8 were expressed in the human embryonic kidney cell line 293. The full-length cDNA of mouse apoER2Δ4-6 and apoER2Δ4-6,8 were assembled by joining a partial cDNA for the mouse protein (4Novak S. Hiesberger T. Schneider W.J. Nimpf J. J. Biol. Chem. 1996; 271: 11732-11736Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar) with the appropriate PCR products derived from mouse brain cDNA with primers 5RS1 (5′-GGAGCCCCGGGCCCGCTATGG) and 5RAS6 (5′-GCTCATCAATGAGGACCACC) via an internal BglII site. The products of the PCR reaction with 5RS1, 5RAS6 containing repeats 1–3, 7, and 8 and repeats 1–3 and 7 were purified on a 1% agarose gel, cloned into pCR2.1, and sequenced. The partial cDNA (see above) was cloned into the eukaryotic expression vector pCI-Neo. In order to produce the full-length cDNAs, fragments containing 4 repeats and 5 repeats were cut out from the pCR2.1 constructs via an internalBglII and the XhoI site (present in the polylinker of pCR2.1) and cloned into the pCI-Neo construct containing the rest of the cDNA. The construct used for expression of chicken LR8B in 293 cells has been described (8Stockinger W. Hengstschläger-Ottnad E. Novak S. Matus A. Hüttinger M. Bauer J. Lassmann H. Schneider W.J. Nimpf J. J. Biol. Chem. 1998; 273: 32213-32221Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Transfection of the cells was performed using Lipofectin Reagent (Life Technologies, Inc.) according to the manufacturer's protocol. Stable transformants were selected by the addition of 500 mg/liter G418 to the medium (Dulbecco's modified Eagle's medium (Life Technologies, Inc.), 10% fetal calf serum, 584 mg/liter glutamine). Total cell extracts from 293 cells expressing LR8B, ApoER2Δ4-6, or ApoER2Δ4-6,8 were prepared as described for chicken embryo fibroblasts (25Hayashi K. Nimpf J. Schneider W.J. J. Biol. Chem. 1989; 264: 3131-3139Abstract Full Text PDF PubMed Google Scholar). Electrophoresis, transfer to nitrocellulose membranes, and Western blotting were performed as described previously (26Hiesberger T. Hermann M. Jacobsen L. Novak S. Hodits R.A. Bujo H. Meilinger M. Hüttinger M. Schneider W.J. Nimpf J. J. Biol. Chem. 1995; 270: 18219-18226Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). The polyclonal antibodies against the cytoplasmic domains of chicken LR8B (8Stockinger W. Hengstschläger-Ottnad E. Novak S. Matus A. Hüttinger M. Bauer J. Lassmann H. Schneider W.J. Nimpf J. J. Biol. Chem. 1998; 273: 32213-32221Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) and mouse ApoER2 (24Stockinger W. Brandes C. Fasching D. Hermann M. Gotthardt M. Herz J. Schneider W.J. Nimpf J. J. Biol. Chem. 2000; 275: 25625-25632Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar) are described in the respective references. Rabbit βVLDL was prepared from the plasma of animals fed a 2% cholesterol, 10% corn oil diet for 3 weeks (27Kovanen P.T. Brown M.S. Basu S.K. Bilheimer D.W. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 1396-1400Crossref PubMed Scopus (215) Google Scholar) and was radiolabeled with 125I by the iodine monochloride method as described earlier (25Hayashi K. Nimpf J. Schneider W.J. J. Biol. Chem. 1989; 264: 3131-3139Abstract Full Text PDF PubMed Google Scholar). Lipoprotein concentration is expressed in terms of protein content that was measured by a modified Lowry procedure as described previously (28Schneider W.J. Goldstein J.L. Brown M.S. J. Biol. Chem. 1980; 255: 11442-11447Abstract Full Text PDF PubMed Google Scholar). α2M was isolated from chicken plasma as described (29Jacobsen L. Hermann M. Vieira P.M. Schneider W.J. Nimpf J. J. Biol. Chem. 1995; 270: 6468-6475Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Native α2M was radiolabeled using Iodo-Gen pre-coated iodination tubes according to the manufacturer's recommendation (Pierce, catalog number 28601) to specific activities of 300–400 cpm/ng. Labeled α2M-trypsin complexes (α2M*) were generated as described (29Jacobsen L. Hermann M. Vieira P.M. Schneider W.J. Nimpf J. J. Biol. Chem. 1995; 270: 6468-6475Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Complete activation of labeled α2M by trypsin was monitored by native gel electrophoresis using the Tris borate system described by van Leuven (30Leuven F.V. Cassiman J.J. Berge H.V.D. J. Biol. Chem. 1981; 256: 9016-9022Abstract Full Text PDF PubMed Google Scholar). Recombinant human RAP was produced as a glutathioneS-transferase (GST) fusion protein using a pGEX 2T-derived (Amersham Pharmacia Biotech) expression plasmid in DH5α bacteria (31Herz J. Goldstein J.L. Strickland D.K. Ho Y.K. Brown M.S. J. Biol. Chem. 1991; 266: 21232-21238Abstract Full Text PDF PubMed Google Scholar). Internalization of 125I-βVLDL and125I-α2M* by LR8B-, ApoER2Δ4-6-, and ApoER2Δ4-6,8-expressing 293 cells was measured according to the standard protocol for uptake of LDL (32Goldstein J.L. Basu S.K. Brown M.S. Methods Enzymol. 1983; 98: 241-259Crossref PubMed Scopus (1287) Google Scholar). In brief, 293 cells were cultured in poly-l-lysine-treated 3-cm dishes and grown to a 70% confluency. After three washes with phosphate-buffered saline, cells were incubated with Dulbecco's modified Eagle's medium containing 2% bovine serum albumin, 584 mg/liter glutamine, and125I-labeled ligand at the indicated concentrations for 3 h at 37 °C. After washing, the cells were lysed by addition of 1 ml of 0.1 n NaOH and incubation at 23 °C for 10 min. The radioactivity in the lysate was determined with a γ-counter (COBRA II, Packard Instrument Co.), and the protein concentration was determined. Kd values were determined by Scatchard analysis and expressed in nm using aMr = 350.000 for βVLDL. 293 cells expressing ApoER2Δ4-6 and ApoER2Δ4-6,8 and mock-transfected cells were washed and resuspended in phosphate buffer and incubated with conditioned media from reelin-expressing 293T cells as described (15D'Arcangelo G. Homayoundi R. Keshvara L. Rice D.S. Sheldon M. Curran T. Neuron. 1999; 24: 471-479Abstract Full Text Full Text PDF PubMed Scopus (696) Google Scholar). In brief, 293T cells (60–80% confluent) were transfected with 7 μg of the reelin construct pCRL. The following day, the culture medium was replaced with a serum-reduced medium (Opti-MEM). After 2 more days the conditioned medium was collected and used for incubation of ApoER2-expressing cells. For ApoER2 expression, 293 cells were transiently transfected with 10 μg of ApoER2Δ4-6 and ApoER2Δ4-6,8 constructs. After 48 h the cells were harvested, washed once with 1 ml of phosphate-buffered saline, and incubated with 1 ml of the reelin supernatant at 4 °C for 4 h in the absence or presence of GST-RAP (30 μg/ml), EDTA (20 mm), or β-VLDL (50 μg/ml). CaCl2 was added to a final concentration of 0.5 mm. The cells were washed 2× with 1.5 ml of phosphate-buffered saline and lysed with 50 μl buffer containing 50 mm Tris, 150 mm NaCl, 1% Triton X-100, 1 mm EDTA, 20 mm sodium fluoride, 1 mm sodium orthovanadate, and proteinase inhibitors. After 30 min lysis on ice, the cell extracts were centrifuged at 13,000 × g for 30 min. 6–10 μl of the supernatant was loaded on a 7.5% gel. Transfer to nitrocellulose was done as described previously (16Hiesberger T. Trommsdorff M. Howell B.W. Goffinet A. Mumby M.C. Cooper J.A. Herz J. Neuron. 1999; 24: 481-489Abstract Full Text Full Text PDF PubMed Scopus (799) Google Scholar). The blot was incubated for 3 h with G10 antibody (provided by Dr. Andre Goffinet, Namur Med. School, Namur, Belgium) at a concentration of 1:1000. Goat anti-mouse HRP was used as a secondary antibody at a concentration of 1:10,000. The blots were developed using an ECL kit. Alternatively, reelin binding to both splice variants of the receptor was tested by a cell-independent assay described by Hiesberger et al. (16Hiesberger T. Trommsdorff M. Howell B.W. Goffinet A. Mumby M.C. Cooper J.A. Herz J. Neuron. 1999; 24: 481-489Abstract Full Text Full Text PDF PubMed Scopus (799) Google Scholar). For this assay, cDNAs coding for the ligand binding domains of ApoER2Δ4-6 and ApoER2Δ4-6,8, respectively, were amplified by PCR using the respective full-length cDNAs as templates and fused to the constant human IgG domain as described (16Hiesberger T. Trommsdorff M. Howell B.W. Goffinet A. Mumby M.C. Cooper J.A. Herz J. Neuron. 1999; 24: 481-489Abstract Full Text Full Text PDF PubMed Scopus (799) Google Scholar) resulting in ApoER2Δ4-6-Fc and ApoER2Δ4-6,8-Fc. Recombinant fusion proteins were expressed in 293 cells, and the secreted proteins (1 ml of cell supernatant) were bound to protein-A Sepharose (30 μl slurry) as described. The protein-A Sepharose was then incubated with 500 μl of conditioned media from reelin-expressing 293T cells in the presence of 30 μg/ml RAP-GST or 20 mm EDTA or medium for 4 h at 4 °C. Detection of bound reelin was performed as described (16Hiesberger T. Trommsdorff M. Howell B.W. Goffinet A. Mumby M.C. Cooper J.A. Herz J. Neuron. 1999; 24: 481-489Abstract Full Text Full Text PDF PubMed Scopus (799) Google Scholar) using G10 as primary antibody and goat anti-mouse IgG conjugated to HRP as second antibody. The amounts of receptor-Fc fusion proteins present on the blot were analyzed by Western blotting using an HRP-coupled anti-V5 antibody (Invitrogen). When we reported the cDNA cloning of chicken LR8B, we also presented the amino acid sequence derived from a partial cDNA for the corresponding murine homologue (4Novak S. Hiesberger T. Schneider W.J. Nimpf J. J. Biol. Chem. 1996; 271: 11732-11736Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). The 5′-end of that cDNA defined the carboxyl-terminal part of the last LA-binding repeat of the mouse receptor. By using this sequence information for RT-PCR experiments, we were able to assign this sequence to repeat number 7 of a mouse variant of LR7/8B that lacks repeat 8 due to differential splicing (5Brandes C. Novak S. Stockinger W. Herz J. Schneider W.J. Nimpf J. Genomics. 1997; 42: 185-191Crossref PubMed Scopus (51) Google Scholar). By using two rounds of 5′-RACE, we now have obtained the full-length cDNA for mouse ApoER2 (Fig.1). As outlined below, the murine receptor appears to be a highly heterogeneous family of proteins due to multiple alternative splicing events. Therefore we refer to the murine protein as apoER2 as originally suggested for the human protein by Yamamoto and colleagues (6Kim D.-H. Iijima H. Goto K. Sakai J. Ishii H. Kim H.-J. Suzuki H. Kondo H. Saeki S. Yamamoto T. J. Biol. Chem. 1996; 271: 8373-8380Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). According to the rule of Van Heijne (33Heijne G.V. Eur. J. Biochem. 1983; 133: 17-21Crossref PubMed Scopus (1606) Google Scholar), we assigned the cleavage site for the signal peptide to amino acids 28/29, producing a mature receptor in which the first LA repeat is 7 amino acids longer than that of human apoER2 and 8 amino acids longer than that of chicken LR7/8B. Surprisingly, the murine cDNA codes for a receptor harboring only 5 LA repeats. Upon computer-assisted sequence alignment (Geneworks) with human apoER2 and chicken LR8B, it became evident that repeats 4–6 are missing from this transcript. As reported recently, such a variant does exist for human ApoER2 (7Kim D.-H. Magoori K. Inoue T.R. Mao C.C. Kim H.-J. Suzuki H. Fujita T. Endo Y. Saeki S. Yamamoto T.T. J. Biol. Chem. 1997; 272: 8498-8504Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Furthermore, the cloned murine transcript contains the eighth repeat but not a 13-amino acid insertion that harbors a furin consensus cleavage site at its carboxyl-terminal end. Alternative transcripts containing such an insertion exist in man and mice but not in chicken (5Brandes C. Novak S. Stockinger W. Herz J. Schneider W.J. Nimpf J. Genomics. 1997; 42: 185-191Crossref PubMed Scopus (51) Google Scholar). Taken
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