Expression of novel "LOCGEF" isoforms of ARHGEF18 in eosinophils

Abstract

Genomic, transcriptomic and proteomic databases indicate that the N-terminal 322 residues encoded by the presumptive LOC100996504 gene, which is adjacent to the ARHGEF18 guanine nucleotide exchange factor gene on chromosome 19, constitute the N-terminal portion of a 1361-residue isoform of ARHGEF18, dubbed LOCGEF-X3. LOCGEF-X3 arises from the use of a leukocyte-specific alternative transcriptional start site and splicing that bypasses the initial noncoding exon of the canonical 1015-residue ARHGEF18 isoform, p114. Eosinophil LOCGEF-X3 was amplified and cloned, recombinant LOCGEF-X3 was expressed, and anti-ARHGEF18 antibody was found to recognize a band in immunoblots of eosinophil lysates that co-migrates with recombinant LOCGEF-X3. PCR of eosinophils revealed minor amounts of transcripts for X4 and X5 isoforms of LOCGEF that arise from differential splicing and differ from the X3 isoform at their extreme N-termini. No p114 transcript or protein band was detected in eosinophils. Immunostaining with anti-ARHGEF18 antibody revealed relocalization of LOCGEF and RHOA from the periphery of round unstimulated eosinophils to the 2 poles of eosinophils polarized by treatment with IL5, CCL11, or IL33 in suspension. Canonical p114 ARHGEF18 has been implicated in maintenance of epithelial cell polarity. We suggest that the "LOC" portion of LOCGEF, which is unlike any other protein domain, has unique functions in control of polarity in activated eosinophils and other leukocytes.

Keywords: ARHGEF18; LOC100996504; alternative transcriptional start; granulocyte; guanine nucleotide exchange factor; p114-GEF.

Figure 1. Schematic of determinants of ARHGEF18 isoforms

(A and B) Transcription factors predicted by ChIP-Seq to bind at initiating exons of LOCGEF (A) and p114 (B) are mapped onto genomic segments comprising the exons for p114, LOCGEF and theoretical Isoform 1 (predicted transcriptional start site indicated by a black arrowhead). (C) Sashimi plots mapping sequence-spanning reads from two Illumina BodyMap 2.0 datasets: white blood cells (red) and breast tissue (blue). Number of reads for selected splice events are shown. Plots have been sized to correspond to the genomic segments shown above. (D) Blow-up of exon L2 and percentage spliced in values for exon L2’s splice sites. (E) Initiating methionines and following sequences corresponding to p114, LOCGEF-X3, LOCGEF-X4 and LOCGEF-X5 are given along with sequence for LOC100996504 annotated in UniProtKB. The numbering above is in reference to residues of LOCGEF-X3. “MTVS…” of p114 is encoded in exon 2. “MGGD…” and “MTTV…” of LOCGEF-X3 and LOCGEF-X4 are encoded in exon L2. Use of an alternative initiating codon in exon L3 leads to the LOCGEF-X5 isoform beginning “MEDL….”

Figure 2. Eosinophils have transcripts encoding LOCGEF-X3, -X4, and -X5

(A) Sequencing of LOCGEF amplicons demonstrating splicing of exon L10 exon 2 in eosinophil cDNA. (B) PCR of eosinophil cDNA demonstrating presence of nX5 and nX7/nX8 transcripts in eosinophils. Lane 1, exACTGene DNA ladder; lane 2, nX7/nX8 amplicons; lane 3, nX5/6/7/8 amplicons. The band in lane 2 is of the expected size for primers that amplify nX7, 358 bp; or nX8, 362 bp. The major band in lane 3 is of the expected amplicon size for nX5, 248 bp; and the minor band is of the expected amplicon size for nX7, 297 bp; or nX8, 301 bp. No band of the size expected for nX6, 411bp, was found. (C) Sequencing of the nX7/nX8 amplicons demonstrating presence of both nX7 and nX8, which cannot be distinguished by gel electrophoresis.

Figure 3. LOCGEF protein is present in eosinophils

Immunoblotting with rabbit (A) or goat (B) anti-ARHGEF18 antibodies demonstrated a ~185 kDa band in eosinophils lysate (EOS) that co-migrates with recombinant LOCGEF-X3 and not recombinant p114. (C) Immunoblotting by rabbit anti-ARHGEF18 of sets of lysates from unstimulated (-) and IL5-stimulated (+) eosinophils from three separate donors. (D) Ponceau stain of the same transferred proteins, with ratios of levels of LOCGEF in IL5/untreated, determined by densitometric analysis adjusted according to total protein loading.

Figure 4. Immunofluorescence of LOCGEF in relation to RHOA, RAC2 or STAT3 in unstimulated (A), IL5- (B), IL33- (C) or CCL11-stimulated (D) eosinophils

Cells were fixed in suspension, cytospun, stained, and examined by confocal microscopy. DAPI was used to stain the eosinophil nuclei. For STAT3 staining, single 0.3-μm slices taken 0.9-μm above the surface of the coverslip are shown. All other images are maximum intensity projections of 11-15 z-series slices. The arrow points to the nucleopod tip of an IL5-activated eosinophil, the arrowhead to the opposite pole. The scale bar is 10 μm.