Regulatory signals mediating down-regulation of the common beta chain of the IL-5, IL-3, and GM-CSF receptors

• Published in: Journal of allergy and clinical immunology 2004-02, Vol.113 (2), p.S334-S334
• Main Authors: Martinez-Moczygemba, M., Lei, J.T., Philip, B.
• Format: Article
• Language: English
• ISSN: 0091-6749; 1097-6825
• DOI: 10.1016/j.jaci.2004.01.709

Our previous studies revealed that IL-5 receptor (IL-5R) signaling is down-regulated by ubiquitin/proteasome degradation of the βc cytoplasmic domain, followed by endocytosis and lysosomal degradation of the remaining truncated IL-5R complex. Since the mechanisms that initiate and terminate IL-5R signaling are poorly understood, we investigated the events regulating βc ubiquitination, proteasome degradation, and endocytosis. Site-directed mutagenesis was used to generate βc mutants defective in tyrosine and serine phosphorylation. These mutants were transfected into HEK293 cells and assayed for their ability to support βc ubiquitination and proteasome degradation. To determine if βc ubiquitination was required for its proteasome degradation, transient transfection of a dominant-negative ubiquitin isoform (K48R Ub) was performed in TF-1 cells. In addition, transient transfection of the WT IL-5R into a temperature-sensitive ts20 cell line that is defective in ubiquitination at 42 oC was also performed. Studies with the βc mutants revealed that βc tyrosine phosphorylation was required for its ubiquitination, whereas serine phosphorylation was not. The K48R ubiquitin mutant reduced βc ubiquitination and proteasome degradation by approximately 50% compared to control conditions. Moreover, ligand-independent βc tyrosine phosphorylation was observed in ts20 cells defective in the ubiquitination process at 42 oC. These data suggest that βc tyrosine phosphorylation is an initiating signal for its ubiquitination. In addition, ubiquitination is required for βc proteasome degradation. Lastly, the ubiquitin conjugation machinery is required for down-regulation of βc tyrosine phosphorylation in the absence of ligand. Understanding the molecular mechanisms of IL-5-stimulated signal termination should provide novel targets for modulating IL-5-mediated inflammation.