Kalirin, a Cytosolic Protein with Spectrin-like and GDP/GTP Exchange Factor-like Domains That Interacts with Peptidylglycine α-Amidating Monooxygenase, an Integral Membrane Peptide-processing Enzyme
1997; Elsevier BV; Volume: 272; Issue: 19 Linguagem: Inglês
10.1074/jbc.272.19.12667
ISSN1083-351X
AutoresM. Rashidul Alam, Richard C. Johnson, Daniel N. Darlington, Tracey Hand, Richard E. Mains, Betty Eipper,
Tópico(s)Adenosine and Purinergic Signaling
ResumoAlthough the integral membrane proteins that catalyze steps in the biosynthesis of neuroendocrine peptides are known to contain routing information in their cytosolic domains, the proteins recognizing this routing information are not known. Using the yeast two-hybrid system, we previously identified P-CIP10 as a protein interacting with the cytosolic routing determinants of peptidylglycine α-amidating monooxygenase (PAM). P-CIP10 is a 217-kDa cytosolic protein with nine spectrin-like repeats and adjacent Dbl homology and pleckstrin homology domains typical of GDP/GTP exchange factors. In the adult rat, expression of P-CIP10 is most prevalent in the brain. Corticotrope tumor cells stably expressing P-CIP10 and PAM produce longer and more highly branched neuritic processes than nontransfected cells or cells expressing only PAM. The turnover of newly synthesized PAM is accelerated in cells co-expressing P-CIP10. P-CIP10 binds to selected members of the Rho subfamily of small GTP binding proteins (Rac1, but not RhoA or Cdc42). P-CIP10 (kalirin), a member of the Dbl family of proteins, may serve as part of a signal transduction system linking the catalytic domains of PAM in the lumen of the secretory pathway to cytosolic factors regulating the cytoskeleton and signal transduction pathways. Although the integral membrane proteins that catalyze steps in the biosynthesis of neuroendocrine peptides are known to contain routing information in their cytosolic domains, the proteins recognizing this routing information are not known. Using the yeast two-hybrid system, we previously identified P-CIP10 as a protein interacting with the cytosolic routing determinants of peptidylglycine α-amidating monooxygenase (PAM). P-CIP10 is a 217-kDa cytosolic protein with nine spectrin-like repeats and adjacent Dbl homology and pleckstrin homology domains typical of GDP/GTP exchange factors. In the adult rat, expression of P-CIP10 is most prevalent in the brain. Corticotrope tumor cells stably expressing P-CIP10 and PAM produce longer and more highly branched neuritic processes than nontransfected cells or cells expressing only PAM. The turnover of newly synthesized PAM is accelerated in cells co-expressing P-CIP10. P-CIP10 binds to selected members of the Rho subfamily of small GTP binding proteins (Rac1, but not RhoA or Cdc42). P-CIP10 (kalirin), a member of the Dbl family of proteins, may serve as part of a signal transduction system linking the catalytic domains of PAM in the lumen of the secretory pathway to cytosolic factors regulating the cytoskeleton and signal transduction pathways. Cytosolic proteins are involved in the formation of secretory granules (1Barr F.A. Leyte A. Huttner W.B. Trends Cell Biol. 1992; 2: 91-94Abstract Full Text PDF PubMed Scopus (57) Google Scholar, 2Ohashi M. Huttner W.B. J. Biol. Chem. 1996; 269: 24897-24905Google Scholar, 3Chen Y.-G. 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Chem. 1995; 270: 28397-29401Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). We have used one of the few integral membrane proteins known to be involved in the biosynthesis of neuropeptides, peptidylglycine α-amidating monooxygenase (PAM), 1The abbreviations used are: PAM, peptidylglycine α-amidating monooxygenase; TGN, trans-Golgi network; kb, kilobase pair(s); RHP, RACE hybrid primer; PCR, polymerase chain reaction; pBS, pBluescript II (SK−); aa, amino acids; nt, nucleotide(s); DH, Dbl homology; PH, pleckstrin homology; PAGE, polyacrylamide gel electrophoresis; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; Ab, antibody; mAb, monoclonal antibody; EST, expressed sequence tag; GST, glutathione S-transferase; CD, cytosolic domain; LAR, leukocyte common antigen-related; MCS, multiple cloning site. to search for cytosolic proteins involved in these processes (17Alam M.R. Caldwell B.D. Johnson R.C. Darlington D.N. Mains R.E. Eipper B.A. J. Biol. Chem. 1996; 271: 28636-28640Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). PAM is a bifunctional enzyme and integral membrane forms contain an NH2-terminal signal sequence, the two catalytic domains that catalyze the sequential reactions required for peptide amidation, a single transmembrane domain, and a short cytosolic domain (18Eipper B.A. Milgram S.L. Husten E.J. Yun H.-Y. Mains R.E. Protein Sci. 1993; 2: 489-497Crossref PubMed Scopus (231) Google Scholar). PAM is involved in the production of all α-amidated peptides and functions only after neuroendocrine-specific endoproteases and carboxypeptidases have exposed the COOH-terminal glycine residue that serves as the nitrogen donor for amide formation (19Bradbury A.F. Smyth D.G. Trends Biochem. Sci. 1991; 16: 112-115Abstract Full Text PDF PubMed Scopus (173) Google Scholar). Immunocytochemical evidence indicates that PAM begins to function in the trans-Golgi network (TGN), but most peptide amidation occurs in immature secretory granules (20Schnabel E. Mains R.E. Farquhar M.G. Mol. Endocrinol. 1989; 3: 1223-1235Crossref PubMed Scopus (148) Google Scholar). Using immunoelectron microscopy, integral membrane forms of PAM have been localized to the TGN, especially to distal tubuloreticular regions, and to large dense core vesicles (21Milgram S.L. Kho S.T. Martin G.V. Mains R.E. Eipper B.A. J. Cell Sci. 1997; 110: 695-706Crossref PubMed Google Scholar). When expressed independently in neuroendocrine cells, each lumenal catalytic domain of PAM is targeted to large dense core vesicles (22Milgram S.L. Johnson R.C. Mains R.E. J. Cell Biol. 1992; 117: 717-728Crossref PubMed Scopus (103) Google Scholar). Integral membrane forms of PAM are localized to the TGN region of both neuroendocrine and nonneuroendocrine cells (7Tausk F.A. Milgram S.L. Mains R.E. Eipper B.A. Mol. Endocrinol. 1992; 6: 2185-2196PubMed Google Scholar, 8Milgram S.L. Mains R.E. Eipper B.A. J. Cell Biol. 1993; 121: 23-35Crossref PubMed Scopus (74) Google Scholar). A small percentage of membrane PAM is present on the cell surface or in endosomes at steady state. Elimination of the distal 40 amino acids of the 86-amino acid cytosolic domain results in relocation of membrane PAM to the plasma membrane (8Milgram S.L. Mains R.E. Eipper B.A. J. Cell Biol. 1993; 121: 23-35Crossref PubMed Scopus (74) Google Scholar, 9Milgram S.L. Mains R.E. Eipper B.A. J. Biol. Chem. 1996; 271: 17526-17535Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). When transferred to a plasma membrane protein such as the interleukin 2 receptor α-chain (Tac), the cytosolic domain of PAM directs the majority of the protein to the TGN region and confers the ability to undergo internalization from the plasma membrane (9Milgram S.L. Mains R.E. Eipper B.A. J. Biol. Chem. 1996; 271: 17526-17535Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Mutation of a tyrosine residue in the COOH-terminal domain of PAM greatly diminishes internalization of PAM from the cell surface without dramatically altering its TGN localization (9Milgram S.L. Mains R.E. Eipper B.A. J. Biol. Chem. 1996; 271: 17526-17535Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). The TGN localization of membrane PAM is greatly compromised upon deletion of an 18-amino acid domain that includes the tyrosine residue essential for internalization. Integral membrane PAM proteins are phosphorylated, and mutagenesis studies indicate that phosphorylation affects routing (23Yun H.-Y. Milgram S.L. Keutmann H.T. Eipper B.A. J. Biol. Chem. 1995; 270: 30075-30083Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Using a rat hippocampal library and the yeast two-hybrid system, we recently identified partial cDNAs encoding two PAMCOOH-terminal interactor proteins (P-CIPs) (17Alam M.R. Caldwell B.D. Johnson R.C. Darlington D.N. Mains R.E. Eipper B.A. J. Biol. Chem. 1996; 271: 28636-28640Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). The biological relevance of these interactions is supported by the fact that the interactions are eliminated when the 18-amino acid segment identified as essential for proper routing of PAM is eliminated. P-CIP2 is similar to serine/threonine dual specificity protein kinases, while P-CIP10 contains at least five spectrin-like repeats (17Alam M.R. Caldwell B.D. Johnson R.C. Darlington D.N. Mains R.E. Eipper B.A. J. Biol. Chem. 1996; 271: 28636-28640Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). In this study we identify the full-length P-CIP10 protein as a member of the Dbl family of GDP/GTP exchange factors (24Cerione R.A. Zheng Y. Curr. Op. Cell Biol. 1996; 8: 216-222Crossref PubMed Scopus (466) Google Scholar) and establish the phenotype of stably transfected AtT-20 cell lines expressing PAM-1 and P-CIP10. The 2.0-kb P-CIP10 cDNA fragment identified using the yeast two-hybrid system (17Alam M.R. Caldwell B.D. Johnson R.C. Darlington D.N. Mains R.E. Eipper B.A. J. Biol. Chem. 1996; 271: 28636-28640Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar) was used to screen 1 × 106 plaque-forming units from a random primed rat hippocampal cDNA library in λ-ZAPII (Stratagene). Seven positive clones were plaque-purified, and the two largest cDNA inserts recovered were overlapping 4.7-kb (clone 10/28) and 4.1-kb (clone 10/34) fragments. Both strands of these cDNAs were sequenced completely; only the 5′ ends of clones 10/28 (upstream sequence from nucleotide 36) and 10/34 (upstream sequence from nucleotide 419) differed. Attempts to extend these sequences by 5′-rapid amplification of cDNA ends (RACE) were unsuccessful. The five shorter cDNAs were fragments of the larger pieces. DNA manipulations were carried out according to standard protocols. Since no in-frame stop codon was identified in clones 10/28 and 10/34, 3′ RACE was used to extend the 3′-end of the 10/28 cDNA (25Johnson R.C. Darlington D.N. Hand T.A. Bloomquist B.T. Mains R.E. Endocrinology. 1994; 135: 1178-1185Crossref PubMed Scopus (46) Google Scholar). Briefly, poly (A)+ RNA from adult rat parietal cortex (200 ng) or olfactory bulb (140 ng) was reverse transcribed with the Promega reverse transcription system (Madison, WI) using a RACE hybrid primer (RHP) 5′-GGAATTCGAGCTCATCGAT17-3′ (0.75 μm) and avian myeloblastosis virus reverse transcriptase (15 units). After the initial 35-cycle amplification using 5′-CAGGATGCCTTTCAAGTG-3′ (nt 4375–4392 of final P-CIP10a) and RHP, an aliquot of the PCR product was used in a nested PCR with 5′-CCTCTAGAGCACCCCATCCTCAGACAAT-3′ (nt 4592–4611 of P-CIP10a) and RHP. A 770-base pair fragment (674 nt of new sequence) from independent amplifications of both tissues was purified, subcloned into pBluescript II (SK−) (pBS), and sequenced. The additional 3′-sequence contained no in-frame stop codon, so 3′-RACE was repeated as above using sense primers 5′-CTGCTTCTTCCCCCTGGTGA-3′ (nt 5097–5116 of P-CIP10a) for the first round of amplification and 5′-GGTCTAGAATGGAGGCAAGTCTGAGT-3′ (nt 5144–5164 of P-CIP10a) for the second round of PCR reactions with the same RHP. The 650-base pair fragment (444 nt of new 3′ sequence) obtained in this second 3′-RACE reaction had an in-frame stop codon. Reverse transcriptase-PCR was used to verify that the novel sequence contained in the 3′-RACE products was contiguous to clone 10/28. Full-length P-CIP10a (10/28 cDNA at the 5′ end) and P-CIP10b (10/34 cDNA at the 5′ end) cDNAs were constructed using a PCR-generated pBS.P-CIP10–3′ (nt 4255–5724) intermediate. Full-length P-CIP10a cDNA (pBS.P-CIP10a) was created by a three-way ligation ofBsp106I/BamHI-digested pBS, a 4.45-kb fragment obtained from clone 10/28 by digestion with Bsp106I (5′-MCS)-AspEI (nt 4401) and a 1.32-kb fragment obtained from AspEI/BamHI-cut pBS.P-CIP10–3′. To create the full-length P-CIP10b cDNA (pBS.P-CIP10b), clone 10/34 was digested with BstBI (nt 2674) and XbaI (3′-MCS), and the 10/34 sequence downstream of BstBI was replaced with the 3.4-kb BstBI (nt 2291 of P-CIP10a)-XbaI (3′-MCS) fragment from pBS.P-CIP10a. Construction of pGEX-CIP10, an expression vector encoding a GST fusion protein containing P-CIP10a (aa 447–1138), was described (17Alam M.R. Caldwell B.D. Johnson R.C. Darlington D.N. Mains R.E. Eipper B.A. J. Biol. Chem. 1996; 271: 28636-28640Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). To construct pET-HisDH, a bacterial expression vector encoding all of the Dbl homology (DH) domain and most of the pleckstrin homology (PH) domain of P-CIP10a (P-CIP10a (aa 1254–1537)), the 857-base pair fragment from clone 10/28 was subcloned into pET28a (Novagen) in-frame with the histidine tag. A mammalian expression vector encoding full-length P-CIP10a (pSCEP.P-CIP10a) was constructed by inserting the full-length cDNA piece from pBS.P-CIP10a into pSCEP (26Paquet L. Massie B. Mains R.E. J. Neurosci. 1996; 16: 964-973Crossref PubMed Google Scholar). To construct pBS.Myc.P-CIP10, the c-Myc epitope (underlined) with a Gly5linker (MEQKLISEEDLNGGGGG) was joined in-frame to Gly5 of P-CIP10a using standard methods. The full-length cDNA insert was then transferred to pSCEP to generate p.SCEP.Myc.P-CIP10. All PCR-generated cDNA was confirmed by DNA sequencing. Truncated forms of P-CIP10 cDNAs were used as templates for in vitro transcription and translation reactions. pBS.10a (nt 1–1142) was generated by digesting pBS.P-CIP10a with BglII (cuts at nt 1142) and BamHI (cuts in 3′-MCS) and religating. pBS.10b (nt 1–1525) was generated in the same manner from pBS.P-CIP10b. Radiolabeled proteins ([35S]methionine, 40 μCi/40-μl reaction; Amersham Corp.) were synthesized using a rabbit reticulocyte lysate in vitro transcription and translation system (TNT; Promega Corp.) and analyzed by SDS-PAGE with or without prior immunoprecipitation. Total RNA (10 μg) prepared from different adult rat tissues was electrophoresed on 1% agarose gels containing formaldehyde (25Johnson R.C. Darlington D.N. Hand T.A. Bloomquist B.T. Mains R.E. Endocrinology. 1994; 135: 1178-1185Crossref PubMed Scopus (46) Google Scholar). Poly(A)+ RNA was prepared with the Promega PolyATtract® mRNA isolation system II. RNA transferred to nitrocellulose membranes was hybridized with a cDNA probe encompassing nt 1363–3398 of P-CIP10a using standard protocols (25Johnson R.C. Darlington D.N. Hand T.A. Bloomquist B.T. Mains R.E. Endocrinology. 1994; 135: 1178-1185Crossref PubMed Scopus (46) Google Scholar).In situ hybridization was carried out as described (17Alam M.R. Caldwell B.D. Johnson R.C. Darlington D.N. Mains R.E. Eipper B.A. J. Biol. Chem. 1996; 271: 28636-28640Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Monoclonal antibody 6E6 recognizes PAM COOH-terminal cytosolic domain (CD) (9Milgram S.L. Mains R.E. Eipper B.A. J. Biol. Chem. 1996; 271: 17526-17535Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Rabbit polyclonal antibody JH1764 was generated by immunization of adult female rabbits with purified recombinant rat PHM (aa 37–382) and recognizes native PHM (21Milgram S.L. Kho S.T. Martin G.V. Mains R.E. Eipper B.A. J. Cell Sci. 1997; 110: 695-706Crossref PubMed Google Scholar). Rabbit antibody JH2007 was raised to a synthetic polypeptide (P-CIP10a (aa 992–1013)) linked to keyhole limpet hemocyanin with glutaraldehyde (Hazleton HRP, Inc., Denver, PA) (27Husten E.J. Tausk F.A. Keutmann H.T. Eipper B.A. J. Biol. Chem. 1993; 268: 9709-9717Abstract Full Text PDF PubMed Google Scholar). Rabbit polyclonal antibody JH2000 was raised by immunization with GST.P-CIP10a (aa 447–1138). The fusion protein purified by SDS-PAGE and electroelution was used as antigen. Antibody JH2000 recognizes full-length P-CIP10 only after denaturation. Monoclonal antibody 4C9 was generated to recombinant P-CIP10a (aa 1254–1537) (His-DH) expressed inEscherichia coli and purified by adsorption to nickel-bound His-Bind resin (Novagen) (9Milgram S.L. Mains R.E. Eipper B.A. J. Biol. Chem. 1996; 271: 17526-17535Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Monoclonal antibody 9E10 to the Myc epitope (28Borjigin J. Nathans J. J. Biol. Chem. 1994; 269: 14715-14722Abstract Full Text PDF PubMed Google Scholar) was prepared from conditioned medium or ascites fluid (Hazleton, HRP). All cells were cultured in Dulbecco's modified Eagle medium/F-12 containing 10% fetal bovine serum (Hyclone, Logan, UT) and 10% Nu serum (Collaborative Research, Bedford, MA). Transfected cell lines were grown with appropriate drug selection: G418 (0.5 mg/ml) for pMt.Neo or pMc.Neo (Stratagene, La Jolla, CA) cotransfections; hygromycin (200 units/ml) for pCEP4 (Invitrogen) or pSCEP (26Paquet L. Massie B. Mains R.E. J. Neurosci. 1996; 16: 964-973Crossref PubMed Google Scholar) cotransfections. AtT-20/PAM-1 cells were transfected with pSCEP.P-CIP10a or pSCEP.Myc.P-CIP10a using lipofection. Cells resistant to both hygromycin and G418 were selected, expanded, and screened for expression of P-CIP10 or Myc.P-CIP10 transcript by Northern blot analysis. Cell lines were subcloned and again screened by Northern blot analysis. Cells plated on 15-mm culture dishes and grown to 70–90% confluency were incubated in methionine/cysteine-free complete serum-free medium for 10 min and then labeled with the same medium containing 1 mCi/ml [35S]methionine/cysteine (ProMix; Amersham) for 15 or 30 min followed by a nonradioactive chase in complete serum-free medium. Cells were either directly extracted into SDS buffer (1% SDS, 50 mm Tris-HCl, pH 7.5, 10 mm β-mercaptoethanol) by incubation at 95 °C for 5 min or were scraped into TMT buffer (10 mm sodium TES, pH 7.5, 20 mm mannitol, 1% Triton X-100) and subjected to three cycles of freezing and thawing and centrifugation to pellet insoluble material. For the subcellular fractionation experiment, cells were removed from the dishes by scraping into an isotonic buffer (50 mm HEPES-KOH, pH 7.5, 250 mm sucrose), disrupted using a ball bearing cell cracker (15-μm clearance), and subjected to differential centrifugation (23Yun H.-Y. Milgram S.L. Keutmann H.T. Eipper B.A. J. Biol. Chem. 1995; 270: 30075-30083Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Immunoprecipitation of P-CIP10 utilized Ab JH2000 or Myc mAb 9E10 (23Yun H.-Y. Milgram S.L. Keutmann H.T. Eipper B.A. J. Biol. Chem. 1995; 270: 30075-30083Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar,28Borjigin J. Nathans J. J. Biol. Chem. 1994; 269: 14715-14722Abstract Full Text PDF PubMed Google Scholar). PAM-1 was immunoprecipitated using Ab JH1764 (22Milgram S.L. Johnson R.C. Mains R.E. J. Cell Biol. 1992; 117: 717-728Crossref PubMed Scopus (103) Google Scholar). Immunoprecipitated proteins were resolved by SDS-PAGE and visualized by fluorography. Apparent molecular masses were determined using prestained molecular weight standards (Rainbow standards; Amersham). The capacities of Ab JH2000 and mAb 9E10 were determined by adding increasing amounts of AtT-20/P-CIP10a or AtT-20/Myc.P-CIP10 cell extract to a fixed amount of antibody plus radiolabeled in vitro translated Myc.P-CIP10. Live AtT-20 cells were photographed at low magnification using phase contrast optics. Coded images were analyzed using a BioQuant TCW (version 3.00; R & M Biometrics, Nashville, TN) to record the total number of cells, number of cells with processes (longer than 1 cell body length), number of round cells, number of giant cells (cell body bigger than 3 nuclei), length of each process, and number of bifurcations (branch points). Four or five images were analyzed for each cell line (215–460 cells); after decoding, data were analyzed for statistical significance using a t test. E. coli transformed with vectors encoding GST-Rac1, GST-RhoA, and GST-Cdc42Hs were obtained from Dr. Richard C. Cerione (Cornell University, Ithaca, NY). The purified fusion proteins were prepared, dialyzed, bound to glutathione-Sepharose beads, and depleted of bound nucleotide (29Hart M.J. Sharma S. elMasry N. Qiu R.-G. McCabe P. Polakis P. Bollag G. J. Biol. Chem. 1996; 271: 25452-25458Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 30Hart M.J. Eva A. Zangrilli D. Aaronson S.A. Evans T. Cerione R.A. Zheng Y. J. Biol. Chem. 1994; 269: 62-65Abstract Full Text PDF PubMed Google Scholar). AtT-20 cells expressing PAM-1 or PAM-1 with Myc.P-CIP10 were extracted into 20 mm Tris-HCl, pH 7.5, 50 mm NaCl containing 1% Triton X-100. After freezing and thawing three times, extracts were diluted with 3 volumes of the guanine-nucleotide depletion buffer (30Hart M.J. Eva A. Zangrilli D. Aaronson S.A. Evans T. Cerione R.A. Zheng Y. J. Biol. Chem. 1994; 269: 62-65Abstract Full Text PDF PubMed Google Scholar) and centrifuged. For each binding reaction, about 50 μg of GST-GTPase bound to 50 μl of glutathione-Sepharose beads was mixed with an aliquot of cell extract containing 2 mg (0.25 mg/ml) of protein from nonlabeled cells or 5 × 107 cpm/ml acid-precipitable protein from radiolabeled cells. After mixing for 3 h at 4 °C, the beads were washed with nucleotide depletion buffer. The beads incubated with nonlabeled extracts were eluted with Laemmli buffer, and bound proteins were subjected to SDS-PAGE and Western blot analysis using Ab JH2000. The beads incubated with radiolabeled cell extracts were eluted by boiling in 50 mm Tris-HCl, pH 7.5, 1% SDS, diluted, and subjected to immunoprecipitation (9Milgram S.L. Mains R.E. Eipper B.A. J. Biol. Chem. 1996; 271: 17526-17535Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) with Ab JH2000. P-CIP10 was identified in a hippocampal/cortical cDNA library prepared from 3-week-old rat pups that had been subjected to a single maximal electroconvulsive stimulus (17Alam M.R. Caldwell B.D. Johnson R.C. Darlington D.N. Mains R.E. Eipper B.A. J. Biol. Chem. 1996; 271: 28636-28640Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Before trying to clone a full-length P-CIP10 cDNA, we used Northern blot analysis to determine the size of the P-CIP10 transcript and the tissues expressing the highest levels of P-CIP10 (Fig. 1 A). A somewhat heterogeneous set of P-CIP10 transcripts was visualized in total RNA prepared from olfactory bulb, parietal cortex, and hippocampus, with lower levels detected in hypothalamus and none detected in cerebellum. P-CIP10 mRNA was detectable in total RNA prepared from kidney and spleen but was not visualized in anterior or neurointermediate pituitary or atrium, tissues that contain high levels of PAM mRNA. Multiple forms of P-CIP10 mRNA were apparent in all of the tissues examined; when poly(A)+ mRNA from olfactory bulb and parietal cortex was subjected to Northern blot analysis, distinct bands of 8.0 and 5.7 kb were detected (Fig. 1 A, inset). A similar distribution of P-CIP10 transcripts was observed whenin situ hybridization was performed on sections of adult rat brain (Fig. 1, B and D). P-CIP10 transcripts were prevalent in the olfactory bulb, including the internal granular layers, internal plexiform layer, mitral cell layer, and accessory olfactory bulb. P-CIP10 transcripts were also prevalent in all layers of the cerebral cortex, piriform cortex, and amygdala and in the dentate gyrus and CA1–3 regions of the hippocampus. P-CIP10 transcripts were present at lower levels in several hypothalamic structures, including the paraventricular, supraoptic, dorsomedial, and arcuate nuclei. Our partial cDNA was substantially shorter than the P-CIP10 mRNAs observed in tissues. We used the 2.0-kb P-CIP10 cDNA fragment to screen a rat hippocampal cDNA library. The two largest cDNA fragments isolated (10/28 and 10/34) were identical except at their 5′-ends (Fig.2 A). No in-frame stop codons were found at either end of either cDNA. The GC content of the 5′-ends of both clones was high, and attempts to extend the sequences by 5′-RACE were unsuccessful (25Johnson R.C. Darlington D.N. Hand T.A. Bloomquist B.T. Mains R.E. Endocrinology. 1994; 135: 1178-1185Crossref PubMed Scopus (46) Google Scholar); sequence and in vitro translation data (see below) indicated that an initiator Met was included in each clone. By sequentially employing 3′-RACE, we extended the sequence to include an in-frame stop codon (Fig. 2 A). No sequence diversity was found in the newly amplified 3′-fragments. The fragments were assembled to form two full-length P-CIP10 cDNAs, P-CIP10a (5′-end of 10/28) and P-CIP10b (5′-end of 10/34) (Fig. 2 A). A single long open reading frame with a stop codon near the 3′-end was found in both P-CIP10 cDNAs. The GC-rich nature of the 5′-end of both P-CIP10a and P-CIP10b and the presence of a single Met residue in both unique regions raised the possibility that a transcriptional start site was present in each clone. The nucleotide sequence surrounding each Met agreed with the consensus translational initiation sequence defined for higher eukaryotes (Fig. 2 B) (31Kozak M. J. Cell Biol. 1991; 115: 887-903Crossref PubMed Scopus (1451) Google Scholar, 32Kozak M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2662-2666Crossref PubMed Scopus (213) Google Scholar). To determine whether these potential translational initiation sites were functional, we performed coupled in vitrotranscription/translation reactions. We truncated P-CIP10a and P-CIP10b at a common site less than 1200 nt from the potential translational initiation sites so that the predicted 20-amino acid difference between the translation products of P-CIP10a and P-CIP10b would be detectable (Fig. 2 B). Each P-CIP10 cDNA yielded a protein of the size predicted if translation were initiated at the Met in each unique 5′-region (Fig. 2 C); for both P-CIP10a and P-CIP10b, the next Met is more than 80 amino acid residues downstream. The P-CIP10a transcription/translation reaction proceeded much more efficiently than the P-CIP10b reaction, and we used the P-CIP10a cDNA for all further studies. P-CIP10a encodes a protein of 1899 amino acids with a calculated molecular mass of 217 kDa and pI of 5.67 (Fig. 2 D). P-CIP10 is largely hydrophilic, with the characteristics of a cytosolic protein. The NH2 terminus of P-CIP10 lacks a hydrophobic signal sequence, and no hydrophobic stretches typical of transmembrane domains are present. By homology search and computer-based structural analysis, P-CIP10 can be divided into five regions: a short NH2-terminal region, a region of spectrin-like repeats, a DH domain, a PH domain, and the COOH-terminal region (Fig.2 E). The NH2-terminal 150 amino acids of P-CIP10 are homologous to Trio, a new member of the Dbl family of proteins identified by virtue of its interaction with the cytosolic domain of the leukocyte common antigen-related (LAR) transmembrane protein-tyrosine phosphatase (33Debant A. Serra-Pages C. Seipel K. O'Brien S. Tang M. Park S.-H. Streuli M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5466-5471Crossref PubMed Scopus (398) Google Scholar). The next 1000 amino acid residues of P-CIP10 are most homologous to Trio (33Debant A. Serra-Pages C. Seipel K. O'Brien S. Tang M. Park S.-H. Streuli M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5466-5471Crossref PubMed Scopus (398) Google Scholar), spectrin, and fodrin (34Bloom M.L. Birkenmeier C.S. Baker J.E. Blood. 1993; 82: 2906-2914Crossref PubMed Google Scholar, 35Winkelmann J.C. Chang J.-G. Tse W.T. Scarpa A.L. Marchesi V.T. Forget B.G. J. Biol. Chem. 1990; 265: 11827-11832Abstract Full Text PDF PubMed Google Scholar). Spectrin and fodrin are cytoskeletal proteins involved in the maintenance of plasma membrane structure by cross-linking to actin and to various integral and membrane-associated proteins (36Bennett V. Gilligan D.M. Annu. Rev. Cell Biol. 1993; 9: 27-66Crossref PubMed Scopus (444) Google Scholar, 37Viel A. Branton D. Curr. Op. Cell Biol. 1996; 8: 49-55Crossref PubMed Scopus (66) Google Scholar). Secondary structure predictions indicate that this region of P-CIP10 is almost entirely α-helical and that the NH2-terminal part of P-CIP10 can be arranged into nine spe
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