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Identification and Characterization of a Membrane Receptor for Proteolysis-Inducing Factor on Skeletal Muscle

Nutritional Biomedicine, School of Life and Health Sciences, Aston University, Birmingham, United Kingdom

Requests for reprints: Michael J. Tisdale, Nutritional Medicine, School of Life and Health Sciences, Aston University, Birmingham, B4 7ET, United Kingdom. Phone: 44-1213593611; Fax: 44-1213333172; E-mail: m.j.tisdale{at}aston.ac.uk.

Proteolysis-inducing factor (PIF) is a sulfated glycoprotein produced by cachexia-inducing tumors, which induces atrophy of skeletal muscle. PIF has been shown to bind specifically with high affinity (K_d, in nanomolar) to sarcolemma membranes from skeletal muscle of both the mouse and the pig, as well as murine myoblasts and a human muscle cell line. Ligand binding was abolished after enzymatic deglycosylation, suggesting that binding was mediated through the oligosaccharide chains in PIF. Chondroitin sulfate, but not heparan or dermatan sulfate, showed competitive inhibition (K_d, 1.1 x 10^–7 mol/L) of binding of PIF to the receptor, suggesting an interaction with the sulfated oligosaccharide chains. Ligand blotting of [³⁵S]PIF to triton solublized membranes from C₂C₁₂ cells provided evidence for a binding protein of apparent M_r of 40,000. Amino acid sequence analysis showed the PIF receptor to be a DING protein. Antisera reactive to a 19mer from the N-terminal amino acid residues of the binding protein attenuated protein degradation and activation of the ubiquitin-proteasome pathway induced by PIF in murine myotubes. In addition, the antisera was highly effective in attenuating the decrease in body weight in mice bearing the MAC16 tumor, with a significant increase in muscle wet weight due to an increase in the rate of protein synthesis, together with a reduction in protein degradation through attenuation of the increased proteasome expression and activity. These results confirm that the PIF binding protein has a functional role in muscle protein atrophy in cachexia and that it represents a potential new therapeutic target. [Cancer Res 2007;67(23):11419–27]