Mass Spectrometry Identifies Interleukin-34 and Protein-tyrosine Phosphatase ζ Interactions

Interleukin-34 (IL-34) is highly expressed in the brain. After signaling occurs through its cognate receptor, IL-34 signals colony-stimulating factor-1 receptor (CSF-1R) leading to the development of microglia. Researchers under the direction of E. Richard Stanley hypothesized that IL-34 may use an alternate receptor.¹Receptor-type protein-tyrosine phosphatase ζ (PTP-ζ) was of particular interest to the Stanley group. PTP-ζ is a cell surface receptor and a chondroitin sulfate proteoglycan. It is highly abundant in the brain, and known to bind and signal through multiple ligands.² PTP-ζ also has multiple downstream effects and is upregulated in gliablastoma.³

In their recent publication, the Stanley group obtained 8-10 week old mouse brain tissue for their experiments.¹ After preparing the tissue, they performed nanoelectrospray liquid chromatography and tandem mass spectrometry using a nano-HPLC system (LC Packings) connected on line to an LTQ linear ion trap mass spectrometer (Thermo Scientific). DTA files were generated from the raw data files. The files were merged and searched against all species of the NCBInr database (July 2, 2010) using Mascot (version 2.3). 9 proteins were identified with a 95% probability, and of those, 2 proteins had the highest Mascot protein score. With a 99% accuracy, the final analysis identified PTP-ζ and TN-R( an extracellular matrix ligand associated with PTP-ζ) as IL-34 associated proteins.

The Stanley group also analyzed the expression patterns of IL-34, PTP-ζ, and TN-R, using immunofluorescence. PTP-ζ expression was present in all areas of the cerebrum and cerebellum where IL-34 expression is also observed. IL-34 was also co-localized with TN-R in the cerebral cortical later 4 and in mature neurons of cortical layer 5. The expression of PTP-ζ was also localized to neural progenitors and glial cells. The authors also performed additional experiments using immunoprecipitation, western blot, flow cytometry, and cell proliferation assays using the human glioblastoma cell lines U251, SNB19, and U87MG. The combined results of these experiments, in addition to mass spectrometry identified ζ (PTP-ζ), as a novel IL-34 receptor.

The Stanley group determined that the affinity of PTP-ζ for IL-34 was lower for other ligands present, which the research group suggests may indicate involvement of an auxiliary receptor(s) and/or receptor clustering and cooperative signaling at the cell surface, and/or additional downstream signaling at low occupancy.

Because there are multiple ligands associated with PTP-ζ , the precise role of IL-34 needs to be investigated further. Based on the involvement of IL-34 in the glioblastoma cell line, and the expression of PTP-ζ in neuroblastoma and other tumors, IL-34 involvement with PTP-ζ may hold great relevance to translational studies involving tumorigenesis.

1. Nandi, S, (2013) ‘Receptor-type protein tyrosine phosphatase zeta is a functional receptor for interleukin-34.’, Journal of Biological Chemistry, 2013 Jun 20. [Epub ahead of print] doi: 10.1074/jbc.M112.442731

2. Peles E., Schlessinger J., Grumet M. (1998) ‘Multi-ligand interactions with receptor-like protein tyrosine phosphatase β: implications for intercellular signaling.’ Trends in Biochemical Science, 23 (4) (pp. 121–124)

3. Fujikawa A., et al. (2011) ‘Consensus substrate sequence for protein-tyrosine phosphatase receptor type Z.’ Journal of Biological Chemistry, 286 (43) (pp. 37137–37146)