Separate endocytic pathways regulate IL-5 receptor internalization and signaling.
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.
Lei
Jonathan T
JT
Biology of Inflammation Center, Baylor College of Medicine, Houston, TX 77030-3411, USA.
Martinez-Moczygemba
Margarita
M
D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology; D002772:Cholera Toxin / Q000494:pharmacology; D002966:Clathrin / Q000502:physiology; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D004804:Eosinophils / Q000502:physiology*; D005434:Flow Cytometry; D006801:Humans; D015848:Interleukin-5 / Q000494:pharmacology; D004915:Leukemia, Erythroblastic, Acute; D053649:Receptors, Interleukin-5 / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D014168:Transferrin / Q000494:pharmacology / Q000502:physiology*
D002966:Clathrin; D015848:Interleukin-5; D053649:Receptors, Interleukin-5; D014168:Transferrin; D002772:Cholera Toxin