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First published online 29 March 2005
doi: 10.1242/jcs.02310

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A novel isoform of human Golgi complex-localized glycoprotein-1 (also known as E-selectin ligand-1, MG-160 and cysteine-rich fibroblast growth factor receptor) targets differential subcellular localization

Jongcheol Ahn, Maria Febbraio^* and Roy L. Silverstein^*,

Department of Medicine, Division of Hematology and Medical Oncology, Weill Medical College of Cornell University, New York, NY 10021, USA

View larger version (46K): [in a new window]   Fig. 1 . Sequence comparison of GLG1 and GLG2. The nucleotide and predicted amino acid sequences of the human monocyte-derived GLG2 are aligned with the published sequence of GLG1 (GenBank accession #NM012201). For convenience, the putative transmembrane and cytoplasmic domains and 3'-UTR of the transcripts for each isoform are shown. The first nucleotide here corresponds to nucleotide #3453 of the registered sequence. The sequences and the direction of the primers used in the present study were also included. The vertical arrow indicates a putative 5'-splicing site. Primers used in the present study were shown by arrows. The amino acid sequence in bold represents the 24-amino-acid extension of GLG2.

View larger version (90K): [in a new window]   Fig. 2 . Subcellular localization of CD8/GLG1 and CD8/GLG2 chimeric molecules. Human embryonic kidney epithelial 293 cells were stably transfected with pcDNA3.1 containing DNA sequences for the extracellular and transmembrane domain of CD8 (a), the extracellular domain of CD8 and transmembrane and intracellular domain of GLG1 (CD8/GLG1, b), or the extracellular domain of CD8 and transmembrane and intracellular domain of GLG2 (c-f). Transfected cells were fixed on coverslips, permeabilized with Triton X-100, and analyzed by immunofluorescence with FITC-conjugated monoclonal anti-human CD8. Giantin, a marker for Golgi apparatus, was detected with a rabbit polyclonal serum followed by Rhodamine-conjugated goat anti-rabbit immunoglobulin. In panel (d), CD8/GLG2 cells were treated with brefeldin A for 1 hour before fixation. In panel (e), Golgi structures were allowed to recover after brefeldin A treatment by incubation with media for 1 hour after washing out the brefeldin A. CD8/GLG2 cells in panel (f) were incubated with 5 µg/ml of cycloheximide for 4 hours before fixation. Images were collected with a Zeiss LSM510 laser-scanning confocal microscope. Data shown are the projection of scanned images of transfected cells.

View larger version (34K): [in a new window]   Fig. 3. Transcripts for the GLG isoform are products of a single gene. (a) A 10 kb cloned genomic DNA fragment was digested either with EcoRI (E) or EcoRI plus BamHI (E/B), and analyzed with GLG isoform-specific probes. Those primers were amplified from the cDNA sequence for GLG1 and GLG2 using either primers 5 and 6 (for GLG1) or primers 3 and 4 (for GLG2), as shown in Fig. 1. DNA molecular weight markers for 4, 3, 2 and 1 kb were also shown. (b) Nucleotide sequences of 5' and 3' alternative splicing sites for GLG2 in the GLG genomic clones. The consensus sequences for spicing are indicated by asterisks. The translation stop codon for GLG1 is underlined. (c) A model for the production of GLG1 and GLG2 by alternative splicing. Isoform-specific probes were represented by lines on the 3'-UTR of GLG1 and 24 amino acids and its 3'-UTR of GLG2 transcripts.

View larger version (33K): [in a new window]   Fig. 4. Transcripts for GLG1 and GLG2 in various cells. (a) RT-PCR analysis identifies transcripts for both GLG1 and GLG2 in human peripheral blood monocytes. Total RNA from human monocytes was reverse transcribed with primer 2 (for GLG1) or primer 3 (for GLG2). Reverse transcription reaction mixture (1:100) was used for the amplification of isoform-specific sequences from transmembrane domain to the 3'-UTR sequence of transcripts of GLG1 and GLG2 (b) Cellular specificity of splicing activity for the production of GLG2 transcript. Poly (A)+ RNAs from various tumor lines (1, melanoma G361; 2, lung carcinoma A549; 3, colorectal adenocarcinoma SW480; 4, Burkitt's lymphoma Raji; 5, lymphoblastic leukemia MOLT-4; 6, chronic myelogenous leukemia K-562; 7, HeLa cell S3; 8, promyelocytic leukemia HL-60) were analyzed with GLG isoform-specific probes as described in the Materials and Methods. RNA molecular markers for 9.49, 7.46 and 4.40 kb were also shown. (c) Species specificity for the alternative splicing activity. Total RNAs from African Green monkey COS-2 (lane 1), human melanoma cell lines BOWES (lane 2), murine RAW cells (lane 3 and 4), CHO cells (lane 5) and murine SP2/0 (lane 6) were analyzed with the originally cloned 1.8 kb fragment. Data from two different experiments are shown.

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