Journal of Leukocyte Biology
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Published online before print May 29, 2008
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© by The Society for Leukocyte Biology
Journal of Leukocyte Biology, doi:10.1189/jlb.1207828

Received for publication December 12, 2007.
Revised April 3, 2008.
Accepted for publication April 25, 2008.

Article

Separate endocytic pathways regulate IL-5 receptor internalization and signaling

Jonathan T. Lei and Margarita Martinez-Moczygemba @

Biology of Inflammation Center, and the Immunology Allergy and Rheumatology Section, Departments of Medicine and Immunology, Baylor College of Medicine, Houston, Texas, USA

@ To whom correspondence should be addressed. E-mail: mxm{at}bcm.edu.


  Abstract

Eosinophils are critically dependent on IL-5 for their activation, differentiation, survival, and augmentation of cytotoxic activity. We previously showed that the cytoplasmic domain of the hematopoietic receptor, {beta}c, which is shared by IL-5, IL-3, and GM-CSF, is directly ubiquitinated and degraded by the proteasomes in a JAK2-dependent manner. However, studies describing the spatial distribution, endocytic regulation, and trafficking of {beta}c-sharing receptors in human eosinophils are currently lacking. Using deconvolution microscopy and biochemical methods, we clearly demonstrate that IL-5Rs reside in and are internalized by clathrin- and lipid raft-dependent endocytic pathways. Microscopy analyses in TF1 cells and human eosinophils revealed significant colocalization of {beta}c, IL-5R{alpha}, and Cy3-labeled IL-5 with transferrin- (clathrin) and cholera toxin-B- (lipid raft) positive vesicles. Moreover, whereas internalized IL-5Rs were detected in both clathrin- and lipid raft-positive vesicles, biochemical data revealed that tyrosine phosphorylated, ubiquitinated, and proteasome-degraded IL-5Rs partitioned to the soluble, nonraft fractions (clathrin-containing). Lastly, we show that optimal IL-5-induced signaling requires entry of activated IL-5Rs into the intracellular compartment, as coimmunoprecipitation of key signaling molecules with the IL-5R was completely blocked when either endocytic pathway was inhibited. These data provide the first evidence that IL-5Rs segregate and traffic into two distinct plasma membrane compartments, and they further establish that IL-5R endocytosis regulates signaling both positively and negatively.

Key Words: endocytosis • receptor trafficking • ubiquitin/proteasome degradation pathway • eosinophils • signal transduction






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