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• W2108809782 abstract "The CRMP (collapsinresponse mediator protein) family is thought to play key roles in growth cone guidance during neural development. The four members (CRMP1–4) identified to date have been demonstrated to form hetero-multimeric structures through mutual associations. In this study, we cloned a novel member of this family, which we call CRMP5, by the yeast two-hybrid method. This protein shares relatively low amino acid identity with the other CRMP members (49–50%) and also with dihydropyrimidinase (51%), whereas CRMP1–4 exhibit higher identity with each other (68–75%), suggesting that CRMP5 might be categorized into a third subfamily. The mouse CRMP5 gene was located at chromosome 5 B1. Northern blot and in situ hybridization analyses indicated that CRMP5 is expressed throughout the nervous system similarly to the other members (especially CRMP1 and CRMP4) with the expression peak in the first postnatal week. Association experiments using the yeast two-hybrid method and co-immunoprecipitation showed that CRMP5 interacts with dihydropyrimidinase and all the CRMPs including itself, except for CRMP1, although the expression profile almost overlaps with that of CRMP1 during development. These results suggest that CRMP complexes in the developing nervous system are classifiable into two populations that contain either CRMP1 or CRMP5. This indicates that different complexes may have distinct functions in shaping the neural networks. The CRMP (collapsinresponse mediator protein) family is thought to play key roles in growth cone guidance during neural development. The four members (CRMP1–4) identified to date have been demonstrated to form hetero-multimeric structures through mutual associations. In this study, we cloned a novel member of this family, which we call CRMP5, by the yeast two-hybrid method. This protein shares relatively low amino acid identity with the other CRMP members (49–50%) and also with dihydropyrimidinase (51%), whereas CRMP1–4 exhibit higher identity with each other (68–75%), suggesting that CRMP5 might be categorized into a third subfamily. The mouse CRMP5 gene was located at chromosome 5 B1. Northern blot and in situ hybridization analyses indicated that CRMP5 is expressed throughout the nervous system similarly to the other members (especially CRMP1 and CRMP4) with the expression peak in the first postnatal week. Association experiments using the yeast two-hybrid method and co-immunoprecipitation showed that CRMP5 interacts with dihydropyrimidinase and all the CRMPs including itself, except for CRMP1, although the expression profile almost overlaps with that of CRMP1 during development. These results suggest that CRMP complexes in the developing nervous system are classifiable into two populations that contain either CRMP1 or CRMP5. This indicates that different complexes may have distinct functions in shaping the neural networks. semaphorin-3A polymerase chain reaction digoxigenin expressed sequence tag dihydropyrimidinase rapid amplification of cDNA ends embryonic dayn kilobase(s) postnatal dayn group of overlapping clones It is now established that growing axons respond to a complex balance of guidance cues, attractive or repulsive factors acting at short range or long range. Well known guidance cues are collapsins/semaphorins, netrins, and ephrins (1Tessier-Lavigne M. Goodman C.S. Science. 1996; 274: 1123-1133Crossref PubMed Scopus (2706) Google Scholar). The semaphorins are a large group of axonal guidance molecules with at least 30 members (2Chen H. Chedotal A. He Z. Goodman C.S. Tessier-Lavigne M. Neuron. 1997; 19: 547-559Abstract Full Text Full Text PDF PubMed Scopus (572) Google Scholar). They serve as repulsive guidance cues, influencing growth cone guidance not only in a contact-dependent way but also from a distance (3Messersmith E.K. Leonardo E.D. Shatz C.J. Tessier-Lavigne M. Goodman C.S. Kolodkin A.L. Neuron. 1995; 14: 949-959Abstract Full Text PDF PubMed Scopus (458) Google Scholar, 4Shepherd I.T. Luo Y. Lefcort F. Reichardt L.F. Raper J.A. Development. 1997; 124: 1377-1385PubMed Google Scholar, 5Varela-Echavarria A. Tucker A. Püschel A.W. Guthrie S. Neuron. 1997; 18: 193-207Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). Using a COS cell expression cloning approach, a transmembrane protein called neuropilin-1 was identified as a collapsin-1/semaphorin-3A (Sema3A)1 receptor (6He Z. Tessier-Lavigne M. Cell. 1997; 90: 739-751Abstract Full Text Full Text PDF PubMed Scopus (973) Google Scholar, 7Kolodkin A.L. Levengood D.V. Rowe E.G. Tai Y.-T. Giger R.J. Ginty D.D. Cell. 1997; 90: 753-762Abstract Full Text Full Text PDF PubMed Scopus (1003) Google Scholar). Both neuropilin-1 and Sema3A knockout mice exhibited defasciculation and spreading of cranial nerves over a large area (8Kitsukawa T. Shimizu M. Sanbo M. Hirata T. Taniguchi M. Bekku Y. Yagi T. Fujisawa H. Neuron. 1997; 19: 995-1005Abstract Full Text Full Text PDF PubMed Scopus (560) Google Scholar, 9Taniguchi M. Yuasa S. Fujisawa H. Naruse I. Saga S. Mishina M. Yagi T. Neuron. 1997; 19: 519-530Abstract Full Text Full Text PDF PubMed Scopus (477) Google Scholar). Dorsal root ganglion growth cones from the neuropilin-1 knockout mice did not collapse in response to Sema3A, proving that the receptor neuropilin is necessary for inducing collapse (8Kitsukawa T. Shimizu M. Sanbo M. Hirata T. Taniguchi M. Bekku Y. Yagi T. Fujisawa H. Neuron. 1997; 19: 995-1005Abstract Full Text Full Text PDF PubMed Scopus (560) Google Scholar). In contrast to the rapid progress in identification and characterization of axon guidance molecules and their receptors, much remains to be explored about the intracellular mechanism by which signals are transduced into the eventual response of the growth cone. Since chick CRMP62 (now designated CRMP2) was first isolated as a factor required for collapsin-1 (Sema3A)-mediated signaling (10Goshima Y. Nakamura F. Strittmatter P. Strittmatter S.M. Nature. 1995; 376: 509-514Crossref PubMed Scopus (641) Google Scholar), a new family of cytoplasmic proteins that may be involved in such signal transduction for semaphorins has been identified through the work of several groups (11Quinn C.C. Gray G.E. Hockfield S. J. Neurobiol. 1999; 41: 158-164Crossref PubMed Scopus (193) Google Scholar). They are independently referred to as CRMP (collapsin response mediatorprotein; Refs. 10Goshima Y. Nakamura F. Strittmatter P. Strittmatter S.M. Nature. 1995; 376: 509-514Crossref PubMed Scopus (641) Google Scholar and 12Wang L.-H. Strittmatter S.M. J. Neurosci. 1996; 16: 6197-6207Crossref PubMed Google Scholar, 13Yoshida H. Watanabe A. Ihara Y. J. Biol. Chem. 1998; 273: 9761-9768Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 14Kamata T. Subleski M. Hara Y. Yuhki N. Kung H.-F. Copeland N.G. Jenkins N.A. Yoshimura T. Modi W. Copeland T.D. Mol. Brain Res. 1998; 54: 219-236Crossref PubMed Scopus (49) Google Scholar, 15Kamata T. Daar I.O. Subleski M. Copeland T.D. Kung H.-F. Xu R.-H. Mol. Brain Res. 1998; 57: 201-210Crossref PubMed Scopus (15) Google Scholar), TOAD (turnedon after division; Refs. 16Minturn J.E. Geschwind D.H. Fryer H.J.L. Hockfield S. J. Comp. Neurol. 1995; 355: 369-379Crossref PubMed Scopus (106) Google Scholar and17Minturn J.E. Fryer H.J.L. Geschwind D.H. Hockfield S. J. Neurosci. 1995; 15: 6757-6766Crossref PubMed Google Scholar), Ulip (Unc-33-likephosphoprotein; Refs. 18Byk T. Dobransky T. Cifuentes-Diaz C. Sobel A. J. Neurosci. 1996; 16: 688-701Crossref PubMed Google Scholar, 19Byk T. Ozon S. Sobel A. Eur. J. Biochem. 1998; 254: 14-24Crossref PubMed Scopus (103) Google Scholar, 20Gaetano C. Matsuo T. Thiele C.J. J. Biol. Chem. 1997; 272: 12195-12201Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar), or DRP (DHPase-related protein; Ref. 21Hamajima N. Matsuda K. Sakata S. Tamaki N. Sasaki M. Nonaka M. Gene ( Amst. ). 1996; 180: 157-163Crossref PubMed Scopus (203) Google Scholar). To date, four different CRMP (TOAD/Ulip/DRP) genes have been described in rats, mice, and humans, with exclusive expression in the developing nervous system (12Wang L.-H. Strittmatter S.M. J. Neurosci. 1996; 16: 6197-6207Crossref PubMed Google Scholar, 19Byk T. Ozon S. Sobel A. Eur. J. Biochem. 1998; 254: 14-24Crossref PubMed Scopus (103) Google Scholar); CRMP2 mRNA is detectable in lung (12Wang L.-H. Strittmatter S.M. J. Neurosci. 1996; 16: 6197-6207Crossref PubMed Google Scholar), and CRMP4 is detectable in heart and adult testis (18Byk T. Dobransky T. Cifuentes-Diaz C. Sobel A. J. Neurosci. 1996; 16: 688-701Crossref PubMed Google Scholar, 22Kato Y. Hamajima N. Inagaki H. Okumura N. Koji T. Sasaki M. Nonaka M. Mol. Reprod. Dev. 1998; 51: 105-111Crossref PubMed Scopus (31) Google Scholar). We have been searching for topographically expressed molecules in the embryonic chick retina, using a cDNA subtraction technique and a novel cDNA display system called Restriction Landmark cDNA Scanning (23Yuasa J. Hirano S. Yamagata M. Noda M. Nature. 1996; 382: 632-635Crossref PubMed Scopus (114) Google Scholar, 24Suzuki H. Yaoi T. Kawai J. Hara A. Kuwajima G. Watanabe S. Nucleic Acids Res. 1996; 24: 289-294Crossref PubMed Scopus (27) Google Scholar) to identify molecules that are involved in regional specificity in the retina, including those responsible for the topographic retinotectal projection. Among a number of molecules thus isolated, we found that CRMP3 was included in those asymmetrically expressed along the nasotemporal (anteroposterior) axis, although this expression pattern was transient during the retinal development. In the present study, we performed a yeast two-hybrid screen of a mouse brain cDNA library using chick CRMP3 as bait to identify CRMP3-interacting molecules and found a novel CRMP. This molecule, which we refer to as CRMP5, was nearly equally divergent from other CRMP members and DHPase but exclusively expressed in the nervous system. CRMP5 interacted with DHPase and other CRMPs except CRMP1, suggesting that CRMP5 is involved in axon outgrowth and guidance together with the other CRMP members. The cDNA fragment encoding the full-length coding region of chick CRMP3 (GenBankTMaccession number AF249294) was amplified by PCR with specific primers containing restriction sites using the isolated cCRMP3 clone as a template. After confirming the sequence identity, this fragment was inserted into the EcoRI and BamHI sites of the bait vectors, pBTM116 (kindly provided by Drs. P. Bartel and S. Fields) containing the LexA-coding sequence and pGBT9 (CLONTECH, Palo Alto, CA) containing GAL4 DNA-binding domain, to generate pLexA-cCRMP3 and pGAL4BD-cCRMP3, respectively. pLexA-cCRMP3 was then transformed intoSaccharomyces cerevisiae strain L40, which harbors reporter genes HIS3 and LacZ under the control of upstream LexA-binding sites. The library screening was performed as described (25Fields S. Song O.-K. Nature. 1989; 340: 245-246Crossref PubMed Scopus (4880) Google Scholar, 26Kawachi H. Tamura H. Watakabe I Shintani T. Maeda N. Noda M. Mol. Brain Res. 1999; 72: 47-54Crossref PubMed Scopus (50) Google Scholar). Approximately 1.0 × 106 clones were screened using a mouse 17-day embryo MATCHMAKER cDNA library (CLONTECH). Positive clones were selected with 1 mm 3-aminotriazole on plates lacking leucine, tryptophan, and histidine. To confirm the positive interaction using another two-hybrid system, each positive clone thus obtained was again transformed into yeast strain SFY526 (CLONTECH) harboring reporter geneLacZ together with pGAL4BD-cCRMP3 and assayed for β-galactocidase activity according to the manufacturer's protocol (MATCHMAKER Library User Manual; CLONTECH). The nucleotide sequences of positive clones thus isolated were determined on both strands. To obtain a longer 5′-flanking region, 5′-rapid amplification of cDNA ends (5′-RACE) was performed using total RNA prepared from embryonic day 17 (E17) mouse brains. Briefly, total RNA was reverse-transcribed with ThermoScript reverse transcriptase (Life Technologies, Inc.) in the presence of 0.6 m trehalose (Wako, Osaka, Japan) (27Carninci P. Nishiyama Y. Westover A. Itoh M. Nagaoka S. Sasaki N. Okazaki Y. Muramatsu M. Hayashizaki Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 520-524Crossref PubMed Scopus (203) Google Scholar). After two rounds of PCR, amplified DNAs were cloned into the pGEM-T Easy vector (Promega, Madison, WI) and subjected to sequence analysis. The direct R-banding fluorescence in situ hybridization method was used for chromosomal assignment of the mouse CRMP5 gene. Preparation of R-banded chromosomes and fluorescence in situ hybridization were performed as described (28Matsuda Y. Harada Y.-N. Natsume-Sakai S. Lee K. Shiomi T. Chapman V.M. Cytogenet. Cell Genet. 1992; 61: 282-285Crossref PubMed Scopus (231) Google Scholar, 29Matsuda Y. Chapman V.M. Electrophoresis. 1995; 16: 261-272Crossref PubMed Scopus (249) Google Scholar). Mitogen-stimulated splenocyte culture was synchronized by thymidine blockage, and the incorporation of 5-bromodeoxyuridine during the late replication stage was made for differential replication staining after the release from excessive thymidine. R-band staining was performed by exposure of chromosome slides to UV light after staining with Hoechst 33258. The chromosome slides were hardened at 65 °C for 2 h and then denatured at 70 °C in 70% formamide in 2× SSC and dehydrated in a 70, 85, and 100% ethanol series at 4 °C. Genomic fragments covering the mouse CRMP5 gene were screened from mouse genomic library using SalI (linker site)-NcoI cDNA fragment (nucleotide residues −42 to +249) as probe. One of the genomic fragments (22 kb long) was labeled by nick translation with biotin 16-dUTP (Roche Molecular Biochemicals) following the manufacturer's protocol. The labeled DNA fragment was ethanol-precipitated with 10 times volume of mouse Cot-1 DNA (Life Technologies, Inc.) and then denatured at 75 °C in 100% formamide. After hybridization, the slides were washed with 50% formamide in 2× SSC at 37 °C for 20 min, 2× SSC, and 1× SSC for 20 min each at room temperature. They were incubated with Cy2-labeled streptavidin (Amersham Pharmacia Biotech) at a 1:500 dilution in 1% bovine serum albumin, 4× SSC for 1 h at 37 °C. The slides were washed with 4× SSC, 0.1% Nonidet P-40 in 4× SSC, and 4× SSC for 10 min each on the shaker and then stained with 0.75 μg/ml propidium iodide. Nikon filter set B-2A (see Fig. 2 C) and UV-2A (data not shown) were used for observation. Kodak Ektachrome ASA100 films were used for microphotography. Total RNAs were prepared from various tissues of mice at postnatal day 0 (P0), and from whole embryos at E11, E13, and E15, whole heads at E17, and whole brains of postnatal mice at P0, P3, P7, and P14 by using ULTRASPECTM RNA (Biotecx Laboratories, Houston, TX). As for staging of mice, the day on which a vaginal plug was detected was considered E0, and the day of birth was considered P0. Poly(A)+ RNA was isolated from E17 whole brains using Dynabeads Oligo(dT)25 (DYNAL, Oslo, Norway). RNA separated on a 1% agarose gel containing 7.4% formaldehyde was transferred to a Hybond-N nylon membrane (Amersham Pharmacia Biotech). The blot was prehybridized at 68 °C in a solution containing 5× SSC, 0.1% SDS, 2% blocking solution (Roche Molecular Biochemicals), and 50% formamide. Hybridization was carried out overnight at 68 °C in the same solution containing the DIG-11-UTP-labeled antisense riboprobe transcribed from theApaI-SpeI cDNA fragment (nucleotide residues 1951–2828: noncoding probe) of CRMP5 (clone 1-8-7) (for nucleotide numbers, see Fig. 1) and detected using anti-DIG-alkaline phosphatase-conjugated antibody according to the manufacturer's instruction (Roche Molecular Biochemicals). To verify the amount of RNA loaded, the membrane was rehybridized with DIG-labeled cRNA probe for mouse glycelaldehyde-3-phosphate dehydrogenase. In situhybridization was performed using DIG-riboprobes transcribed from the cDNA fragment (nucleotide residues 286–2066) of Ulip1/CRMP4 (GenBankTM accession number X87817) and the SalI (linker site)-BamHI cDNA fragment (−42 to +1652: coding probe) of CRMP5 (clone 1-8-7) as templates (23Yuasa J. Hirano S. Yamagata M. Noda M. Nature. 1996; 382: 632-635Crossref PubMed Scopus (114) Google Scholar). E17 mouse embryos were fixed in 4% paraformaldehyde at 4 °C overnight. Cryosections were made at 14 μm. Hybridization was carried out overnight at 50 °C in 10 mm Tris-HCl (pH 7.6) containing 1 mm EDTA, 0.6 m NaCl, 0.1% SDS, 1× Denhardt's solution, 50% formamide, 400 μg/ml yeast tRNA, and 10% dextran sulfate and then incubated with anti-DIG alkaline phosphatase-conjugated antibody. Excess antibody was washed away, and the color substrates (nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate) were added to develop in the dark. To examine mutual associations among the CRMP family members and DHPase, DNA fragments covering the coding regions of mouse CRMP1–5 and DHPase were amplified by PCR using specific primers from the respective clones or RT-PCR (for CRMP2) to make restriction sites for cloning. After confirming the sequence, these fragments were inserted into the EcoRI andSalI sites of the pGBT9 bait vector. The CRMP1 and CRMP5 fragments were inserted also into the EcoRI andXhoI sites of the pACT2 prey vector (CLONTECH). The bait and prey constructs in various combinations were transformed into yeast strain Y190 (CLONTECH) harboring reporter genes HIS3and LacZ, and transformants were cultivated on plates lacking leucine and tryptophan. Assay was performed as described (26Kawachi H. Tamura H. Watakabe I Shintani T. Maeda N. Noda M. Mol. Brain Res. 1999; 72: 47-54Crossref PubMed Scopus (50) Google Scholar). For quantitative analysis of the mutual association, liquid β-galactosidase assay was performed using o-nitrophenyl β-d-galactopyranoside as substrate according to the manufacturer's protocol (Yeast Protocols Handbook;CLONTECH). Myc-CRMP5, FLAG-CRMP1–5, and FLAG-DHPase expression vectors were constructed in the following way: Oligo nucleotides encoding c-Myc or FLAG peptide including the first methionine were ligated to mammalian expression vector pcDNA3.1 (Invitrogen, Carsbad, CA), which were digested withNheI and EcoRI, to yield pcDNA-Myc and pcDNA-FLAG, respectively. Full-length CRMP1–5 and DHPase cDNA were cut out from the various pGBT9 bait vectors with EcoRI and SalI and inserted into pcDNA-Myc or pcDNA-FLAG digested with EcoRI and XhoI. Myc-CRMP5 and FLAG-CRMPs/FLAG-DHPase thus prepared were co-transfected into COS-7 cells by using LipofectAMINE Plus Reagent (Life Technologies, Inc.). At 48 h after transfection, cell lysates were prepared by sonication in RIPA buffer (50 mm Tris-HCl (pH 7.6), 150 mmNaCl, 1 mm dithiothreitol, 1% Triton X-100, 10 μg/ml leupeptin (Sigma), 10 μg/ml pepstatin A (Sigma), and 1 mmphenylmethylsulfonyl fluoride), followed by centrifugation at 12,000 × g for 20 min. The lysates were incubated with agarose beads conjugated with 9E10 anti-Myc monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for 3 h with agitation. The beads were collected by low speed centrifugation and washed four times by resuspension in 1 ml of RIPA buffer. Proteins eluted from the beads with sample buffer were subjected to SDS-polyacrylamide gel electrophoresis and analyzed by immunoblotting with M2 anti-FLAG monoclonal antibody (Sigma) or 9E10 anti-Myc monoclonal antibody (Santa Cruz Biotechnology). To check the expression levels of FLAG-CRMPs and FLAG-DHPase, cell lysates were also analyzed using M2 anti-FLAG monoclonal antibody. To know the signal transduction cascade of CRMPs upstream and downstream, we attempted to identify CRMP3-interacting molecules by the yeast two-hybrid screening of a mouse embryo cDNA library using chick CRMP3 as bait (pLexA-cCRMP3). Sixty-one clones were isolated by surveying approximately 1.0 × 106 transformants. Sequence analysis showed that CRMP1, CRMP3, CRMP4, and the mouse homologue of DHPase were included among them: six clones for CRMP1, one clone for CRMP3, 29 clones for CRMP4, and six clones for DHPase. This result supports the claim by Wang and Strittmatter (30Wang L.-H. Strittmatter S.M. J. Neurochem. 1997; 69: 2261-2269Crossref PubMed Scopus (141) Google Scholar) that CRMP isoforms associate with one another. The sequence of mouse DHPase, determined for the first time in the present study, shared 94 and 88% amino acid identity with rat and human DHPase, respectively (GenBankTM accession number AF249296). Besides the already identified CRMP isoforms described above, we found one clone, 1-8-7, encoding a novel protein sequence that shows significant but relatively low homology with CRMP1–4 and DHPase. Interaction of this novel CRMP isoform with chick CRMP3 was verified by the two-hybrid method using another bait construct pGAL4BD-cCRMP3 (data not shown). We here designate this new CRMP member as CRMP5. Moreover, two clones with no homology to these CRMP isoforms, which are supposed to be novel CRMP-interacting molecules, were also identified among them (to be reported elsewhere). The CRMP5 cDNA clone, 1-8-7, contained a putative open reading frame that was in frame with the GAL4 activation domain sequence and therefore expected to be expressed as a fusion protein. Because clone 1-8-7 included no in-frame stop codon located upstream of the first methionine (nucleotides 1–3 in Fig.1), we performed 5′-RACE to identify further the upstream sequence. By reverse transcriptase reaction with trehalose (27Carninci P. Nishiyama Y. Westover A. Itoh M. Nagaoka S. Sasaki N. Okazaki Y. Muramatsu M. Hayashizaki Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 520-524Crossref PubMed Scopus (203) Google Scholar), we obtained longer RACE-PCR products and found two in-frame stop codons in clone 4-1. Thus, we concluded that the initially isolated CRMP5 cDNA clone, 1-8-7, has the full-length open reading frame. In Fig. 1, the contig nucleotide sequence composed of 1-8-7 and 4-1, together with the deduced amino acid sequence, is presented. Our search of the mouse EST data base identified 12 overlapping mouse cDNAs matching to the 3′-noncoding region of 1-8-7. The resultant contig sequence had an additional poly(A) signal that is located about 400 bp downstream of the nucleotide sequence shown in Fig. 1 (data not shown; for GenBankTM accession numbers, see the legend). Therefore, two types of CRMP5 mRNA transcripts that are different only in the length of the 3′-noncoding region are probably generated. We screened EST data bases for homologues of CRMP5 in other species. This analysis identified six human EST sequences (GenBankTMaccession numbers N51749, AA350414, AA058664, AA351100, AA488145, and AI36969). They showed significant, although still partial, homology with mouse CRMP5; the N-terminal (corresponding to amino acid residues 1–184 of mCRMP5) and C-terminal (residues 482–564) amino acid sequences were predicted from the EST clones. Compared with mouse CRMP5, the deduced amino acid identity was 88% for the N-terminal part and 98% for the C-terminal part. The CRMP5 cDNA encoded a protein of 564 amino acids whose calculated molecular mass is 61,516 Da. It shares approximately 50% amino acid identity both with the members of the CRMP family (CRMP1–4; 49–50%) and with DHPase (51%) through the entire length (Fig. 2 A). Because the already known CRMP family members exhibit approximately 70% identity with each other (68–75%), CRMP5 might be categorized to another subfamily (12Wang L.-H. Strittmatter S.M. J. Neurosci. 1996; 16: 6197-6207Crossref PubMed Google Scholar,19Byk T. Ozon S. Sobel A. Eur. J. Biochem. 1998; 254: 14-24Crossref PubMed Scopus (103) Google Scholar). As shown in Fig. 2 B, a phylogenetic tree of CRMP isoforms indicated that CRMP5 is relatively close to DHPase and suggested that CRMP1–4 diverged first from a common ancestor with DHPase, followed after a lag by CRMP5. Amino acid sequences of CRMP isoforms were aligned (Fig.3). The N-terminal three-quarters is more conserved than the C-terminal region. When CRMP5 is compared with the other CRMPs, it is evident that some amino acid substitutions are identical or similar to those of DHPase, together with the gap at the N-terminal region. On the other hand, the sequence at aligned positions 54–57 is absent, and the C-terminal region after 530 is present, similar to other CRMP members. It is notable that some regions (at aligned positions 279–298, 313–339, 508–536, and 572–576) were different from all the other CRMP isoforms including DHPase. CRMP isoforms are known as phosphoproteins (18Byk T. Dobransky T. Cifuentes-Diaz C. Sobel A. J. Neurosci. 1996; 16: 688-701Crossref PubMed Google Scholar, 19Byk T. Ozon S. Sobel A. Eur. J. Biochem. 1998; 254: 14-24Crossref PubMed Scopus (103) Google Scholar) and bear several consensus sequences for phosphorylation sites that are conserved among the members. CRMP5 contains 17 such potential phosphorylation sites (Fig. 1): a single potential site for tyrosine phosphorylation, two potential sites for protein kinase A, six sites for protein kinase C, and eight sites for casein kinase II. Among them, three protein kinase C target sites are unique to CRMP5 (see legend to Fig. 1). This suggests that the function of CRMP5 could also be regulated by phosphorylation, specifically and/or commonly. Recently, it has been proposed that the amidohydrolases including DHPase share the same active site architecture (31Holm L. Sander C. Proteins. 1997; 28: 72-82Crossref PubMed Scopus (424) Google Scholar). Notably, four histidine residues (at aligned positions 77, 79, 202, and 258) and one aspartatic acid residue (at position 336) involved in metal binding are highly conserved (Fig. 3). Because CRMP5 has substitutions in two histidine residues, it is unlikely that it bears DHPase activity just as the other CRMP members do not (10Goshima Y. Nakamura F. Strittmatter P. Strittmatter S.M. Nature. 1995; 376: 509-514Crossref PubMed Scopus (641) Google Scholar, 18Byk T. Dobransky T. Cifuentes-Diaz C. Sobel A. J. Neurosci. 1996; 16: 688-701Crossref PubMed Google Scholar, 30Wang L.-H. Strittmatter S.M. J. Neurochem. 1997; 69: 2261-2269Crossref PubMed Scopus (141) Google Scholar). The chromosomal location of the mouse CRMP5 gene was determined by the direct R-banding fluorescence in situ hybridization using a mouse CRMP5 genomic DNA fragment as probe. The CRMP5 gene was localized to R-positive B1 band of chromosome 5 (32Evans E.P. Lyon M.F. Rastan S. Brown S.D.M. Genetic Variants and Strains of the Laboratory Mouse. Oxford University Press, Oxford1996: 1446-1449Google Scholar, 33Somssich I.E. Hameister H. Lyon M.F. Rastan S. Brown S.D.M. Genetic Variants and Strains of the Laboratory Mouse. Oxford University Press, Oxford1996: 1450-1451Google Scholar) (Fig. 2 C). The CRMP family members are known to be expressed in the nervous system, whereas DHPase is present in liver and kidney (21Hamajima N. Matsuda K. Sakata S. Tamaki N. Sasaki M. Nonaka M. Gene ( Amst. ). 1996; 180: 157-163Crossref PubMed Scopus (203) Google Scholar). Because CRMP5 is almost equally divergent from the other CRMPs and DHPase, we next addressed the issue of whether CRMP5 expression is neural-tissue specific. Northern blot analysis using P0 mouse tissues (Fig.4 A), clearly showed that CRMP5 mRNA is present only in the brain but not in the other tissues. Next, the expression profile of CRMP5 mRNA during development was examined. CRMP5 mRNA was already expressed at E11, as early as CRMP4/Ulip1 (data not shown), peaking in the first postnatal week when neural network formation and its refinement culminates and subsequently declining to a lower level during the second week (Fig. 4 B). By longer exposure, another minor hybridization band with an estimated mRNA size of 10.7 kb was detected. The hybridization band of about 4.2 kb is likely to be an artifact caused by overlap with 28 S ribosomal RNA. These results suggest that CRMP5 expression is neural-tissue specific and developmentally regulated as already reported for some CRMP members (10Goshima Y. Nakamura F. Strittmatter P. Strittmatter S.M. Nature. 1995; 376: 509-514Crossref PubMed Scopus (641) Google Scholar, 12Wang L.-H. Strittmatter S.M. J. Neurosci. 1996; 16: 6197-6207Crossref PubMed Google Scholar, 14Kamata T. Subleski M. Hara Y. Yuhki N. Kung H.-F. Copeland N.G. Jenkins N.A. Yoshimura T. Modi W. Copeland T.D. Mol. Brain Res. 1998; 54: 219-236Crossref PubMed Scopus (49) Google Scholar, 15Kamata T. Daar I.O. Subleski M. Copeland T.D. Kung H.-F. Xu R.-H. Mol. Brain Res. 1998; 57: 201-210Crossref PubMed Scopus (15) Google Scholar, 16Minturn J.E. Geschwind D.H. Fryer H.J.L. Hockfield S. J. Comp. Neurol. 1995; 355: 369-379Crossref PubMed Scopus (106) Google Scholar, 17Minturn J.E. Fryer H.J.L. Geschwind D.H. Hockfield S. J. Neurosci. 1995; 15: 6757-6766Crossref PubMed Google Scholar, 18Byk T. Dobransky T. Cifuentes-Diaz C. Sobel A. J. Neurosci. 1996; 16: 688-701Crossref PubMed Google Scholar, 19Byk T. Ozon S. Sobel A. Eur. J. Biochem. 1998; 254: 14-24Crossref PubMed Scopus (103) Google Scholar, 20Gaetano C. Matsuo T. Thiele C.J. J. Biol. Chem. 1997; 272: 12195-12201Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). When poly(A)+ RNA was analyzed, the 5.0-kb band for CRMP5 turned out to be double bands of 4.8 and 5.2 kb (Fig. 4 C). When the expression level of CRMP5 mRNA was compared with that of CRMP4 (18Byk T. Dobransky T. Cifuentes-Diaz C. Sobel A. J. Neurosci. 1996; 16: 688-701Crossref PubMed Google Scholar) by Northern blot hybridization, it was lower by approximately one order of magnitude, and this difference was retained during development up to adulthood (data not shown). To elucidate the expression pattern of CRMP5, we conductedin situ hybridization on sections of E17 embryos with probes for CRMP" @default.
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