Expression of novel "LOCGEF" isoforms of ARHGEF18 in eosinophils

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.