Characterization of the interleukin 3 receptor.
A variety of homobifunctional crosslinking agents have been used to gain insight into the nature of the murine interleukin 3 (mIL-3) receptor. When [125I]mIL-3 was cross-linked to receptor sites on the surfaces of intact B6SUtA1 cells with disuccinimidyl suberate (DSS), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed the existence of two radiolabeled species with molecular weights of 140 (p140) and 70 (p70) kd (after subtraction of [125I]mIL-3). The relative intensities of the two bands did not change when the [125I]mIL-3 concentration was varied, confirming Scatchard results which suggested only one affinity class. However, when [125I]mIL-3 was crosslinked to intact cells and then incubated at 37 degrees C, the intensity of p140 decreased relative to p70, suggesting a conversion of p140 to p70. This conversion could be inhibited by sodium azide, methylamine, and bacitracin and could also be prevented by first boiling for 1 min in 2% SDS and 5% 2-mercaptoethanol. The putative protease that carried out this apparent conversion appeared to be associated both with plasma membranes prepared from these cells and also with solubilized receptors. Moreover, when p140, crosslinked with both dithiobis succinimidylpropionate and glutaraldehyde, was purified and reelectrophoresed under reducing conditions, p70 could be generated. N-glycanase digestion of p140 and p70 revealed a similar level of N-linked carbohydrate, which upon closer study appeared to consist of two chains, a 3-kd and an 8-kd moiety. Consistent with this data, we propose that the receptor is a 140-kd glycoprotein that is cleaved to a 70-kd surface protein upon mIL-3 binding and chemical crosslinking.
Terry Fox Laboratory, B.C. Cancer Research Center, Vancouver, Canada.
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Murthy
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Mui
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Krystal
D000818:Animals; D002462:Cell Membrane / Q000032:analysis; D003432:Cross-Linking Reagents; D006031:Glycosylation; D007377:Interleukin-3 / Q000378:metabolism*; D007457:Iodine Radioisotopes; D051379:Mice; D008961:Models, Structural; D011971:Receptors, Immunologic / Q000032:analysis*; D016185:Receptors, Interleukin-3
D003432:Cross-Linking Reagents; D007377:Interleukin-3; D007457:Iodine Radioisotopes; D011971:Receptors, Immunologic; D016185:Receptors, Interleukin-3