Discovery and characterization of a novel splice variant of the GM-CSF receptor alpha subunit.
OBJECTIVE
To characterize a novel splice variant of the alpha subunit of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (GMRalpha), which we discovered in human neutrophils.


METHODS
We used reverse transcriptase polymerase chain reaction to identify, characterize, and examine the expression of a novel splice variant of the GMRalpha transcript. At the protein level, surface plasmon resonance was used to measure the affinity of a recombinant soluble form of the novel GMRalpha protein for GM-CSF ligand. The full-length novel GMRalpha protein was expressed in a recombinant cell culture system, and its expression and localization were examined using Western blotting, I(125) GM-CSF binding assays, flow cytometry, and a soluble GMRalpha enzyme-linked immunosorbent assay.


RESULTS
The novel GMRalpha transcript identified herein contains a previously undescribed exon of the GMRalpha gene; this exon derives from an Alu DNA repeat element, and is alternatively spliced in the novel GMRalpha transcript. Inclusion of this 102 nucleotide exon results in translation of a protein product, which we have named Alu-GMRalpha. Alu-GMRalpha is identical to cell surface GMRalpha, but additionally contains a 34 amino-acid insert in the juxtamembrane region of the extracellular domain of GMRalpha. Functionally, the Alu-GMRalpha-specific epitope does not modify the ability of the protein to bind GM-CSF, but rather appears to be preferentially targeted by ectodomain proteases to mediate the release of a third soluble GM-CSF receptor into the extracellular space.


CONCLUSIONS
This study provides the first example of a cytokine receptor system in which soluble receptors are produced by three distinct mechanisms. Our results highlight the importance of soluble GMRalpha proteins in regulation of GM-CSF signaling.
Department of Biochemistry and Molecular Biology and Southern Alberta Cancer Research Institute, University of Calgary, Calgary, Alberta, Canada.
Jennifer L
JL
Pelley
Chris D
CD
Nicholls
Tara L
TL
Beattie
Christopher B
CB
Brown
D017398:Alternative Splicing / Q000502:physiology*; D020087:Alu Elements / Q000502:physiology*; D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D003001:Cloning, Molecular; D005091:Exons / Q000502:physiology*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism; D006801:Humans; D008024:Ligands; D051379:Mice; D016254:Mutagenesis, Insertional / Q000502:physiology; D041681:NIH 3T3 Cells; D009504:Neutrophils / Q000166:cytology / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D017434:Protein Structure, Tertiary / Q000235:genetics; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D020349:Surface Plasmon Resonance
D008024:Ligands; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; D011994:Recombinant Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D010447:Peptide Hydrolases