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Neuron

Neuron. does not bind to DRG neurons treated with phosphatidylinositol-specific phospholipase C (PI-PLC) or to sympathetic neurons that do not communicate Ntm or additional members of the IgLON family at significant levels. Ntm promotes the outgrowth of DRG neurons, even after PI-PLC treatment, suggesting that its effects on outgrowth are mediated by heterophilic relationships. Of particular notice, both membrane-bound and soluble Ntm inhibit the outgrowth of sympathetic neurons. These results strongly suggest that Ntm, and other users of SDZ 205-557 HCl the IgLON family, regulate the development of neuronal projections via attractive and repulsive mechanisms that are cell type specific and are mediated by homophilic and heterophilic interactions. A c-myc epitope tag (EQKLISEEDL) was added between the third Ig-like domain name of Ntm and the site of GPI attachment (i.e., between amino acids 323 and 324 of rat Ntm) by the patch PCR method (Squinto et al., 1990). The plasmid pBSK-392B made up of the complete coding region of neurotrimin (Struyk et al., 1995) was used as a template. Primers included a 3 vector-specific oligonucleotide, a 5 primer (5-GCGCGTCAACGAGCAAAAGCTTATTTCTGAGGAGGATCTG-3), and the patch primer (5-ATTTCTGAGGAGGATCTGAATGGGACGTCAAGGAGG3). This resultant PCR product was gel-purified and religated back into the HinC II site of the SDZ 205-557 HCl neurotrimin coding sequence in pBSK-392B. The modified Ntm cDNA was verified by sequencing and subcloned into the eukaryotic expression vector pRC-CMV (Invitrogen, San Diego, CA). The final Ntm-pRC construct was introduced into the Chinese Hamster Ovary (CHO) cell SDZ 205-557 HCl line LR73 (Pollard and Stanners, 1979) by Lipofection, following the manufacturers instructions (Life Technologies, Gaithersburg, MD). Transfected cells were selected with 800 g/ml and maintained with 400 g/ml of G418 (Life Technologies); multiple colonies of transfected cells were pooled and expanded. CHO-Ntm expressors were isolated from pooled cells by fluorescence-activated cell sorting (FACS) after staining with a monoclonal antibody specific for the c-myc decapeptide tag (Evan et al., 1985) and fluorescein-conjugated donkey anti-mouse IgG antiserum (Chemicon, Temecula, CA). Expressors were expanded and subsequently subjected to a second round of FACS. The two populations of cells, i.e., those sorted one or two times, will be referred to as low expressor (LE) and high expressor (HE) cells, respectively. Control CHO-CMV cells were generated by pooling multiple clones transfected with the pRC-CMV plasmid alone. In some experiments, transfected cell lines SDZ 205-557 HCl were surface-biotinylated by incubation of cell monolayers with NHS-sulfo-biotin (Pierce, Rockford, IL) as described previously (Rosen et al., 1992) to facilitate subsequent identification of the myc-tagged neurotrimin. Monolayers were lysed with 1% SDS in 20 mm Tris, pH 7.4, 6 mm NaCl, and 15 mm DTT. After they were boiled, lysates were diluted in 3 vol of 2.5% Triton X-100, 50 mmTris, pH Rabbit Polyclonal to Ku80 7.4, 190 mm NaCl, and 6 mm EDTA and incubated with anti-myc ascites (containing 5 g of IgG), and immune complexes were collected with protein G-coupled Sepharose (Pharmacia Biotech, Piscataway, NJ). Precipitated proteins were separated by SDS-PAGE and electroblotted onto nitrocellulose. Blots were probed with streptavidin conjugated to alkaline phosphatase and incubated with 5-bromo-4-chloro-3-indolyl-phosphate/nitroblue tetrazolium (BCIP-NTP; Kirkegaard and Perry Laboratories, Gaithersburg, MD). To confirm that this neurotrimin expressed by the CHO-Ntm was indeed GPI-anchored, monolayers were incubated with PI-PLC (Oxford Glycosystems, Rosedale, NY) at a concentration of 1 1 U/ml for 1 hr at 37C in PBS made up of protease inhibitors (PMSF, aprotinin, and leupeptin, all at a concentration of 0.1%). Supernatants were collected and precipitated by the addition of 0.01 vol of 2% deoxycholic acid and 0.10 vol of TCA. Cell lysates and precipitated proteins from culture supernatants were fractionated by SDS-PAGE, electroblotted, and probed with the anti-myc antibody. CHO cell-surface proteins were cross-linked by incubating monolayers with the homobifunctional reagent Bis (sulfosuccinimidyl) suberate (BS3) (Pierce) at a concentration of 0.25 mm for 20 min, followed by a 10 min incubation with Leibovitzs L-15 media (Life Technologies) to stop the reaction. After three washes in PBS, monolayers were biotinylated, and Ntm was immunoprecipitated with the anti-myc monoclonal antibody as described above. In some cases, cross-linking was followed by a 1 hr incubation with PI-PLC to determine whether the cross-linked products were GPI-anchored. Immunoprecipitates were then prepared from the cell lysates and supernatants. The effect of cell density around the cross-linking of neurotrimin was investigated by plating 1 105 cells on either 35, 60, or 100 mm tissue culture dishes. After SDZ 205-557 HCl 24 hr of growth, cell-surface proteins were cross-linked and immunoprecipitated as.