Separate endocytic pathways regulate IL-5 receptor internalization and signaling

J Leukoc Biol. 2008 Aug;84(2):499-509. doi: 10.1189/jlb.1207828. Epub 2008 May 29.


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, betac, 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 betac-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 betac, IL-5Ralpha, 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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line, Tumor
  • Cell Membrane / drug effects
  • Cell Membrane / physiology
  • Cholera Toxin / pharmacology
  • Clathrin / physiology
  • Endocytosis / drug effects
  • Endocytosis / physiology*
  • Eosinophils / physiology*
  • Flow Cytometry
  • Humans
  • Interleukin-5 / pharmacology
  • Leukemia, Erythroblastic, Acute
  • Receptors, Interleukin-5 / physiology*
  • Signal Transduction / drug effects
  • Signal Transduction / physiology
  • Transferrin / pharmacology
  • Transferrin / physiology*


  • Clathrin
  • Interleukin-5
  • Receptors, Interleukin-5
  • Transferrin
  • Cholera Toxin