Two polypeptides identified by interleukin 3 cross-linking represent distinct components of the interleukin 3 receptor.
Two receptor polypeptides have been identified in several studies by using cross-linking with interleukin 3 (IL-3). It has been suggested that proteolytic degradation of the larger polypeptide yields the lower molecular weight fragment, but there is little proof that this is the case. We have used several different approaches to characterize the polypeptides cross-linked in R6X or FDC-P1 cells. Several bifunctional cross-linkers of various sizes were tested to determine their effectiveness in cross-linking IL-3 to its receptor. The longer cross-linker gave the highest proportion of labeling of the low molecular weight band. Incubation in the absence of protease inhibitors caused a decrease in labeling of both cross-linked polypeptides, but no indication of a significant increase in the lower molecular weight band. Direct comparison of the two cross-linked polypeptides by V8 protease mapping showed no common peptides that might be expected if they were related molecules, except those derived from the iodinated IL-3. Digestion with N-glycanase resulted in a decrease in apparent molecular weight of 5000 in the larger polypeptide but a decrease of 15,000 in the smaller polypeptide. These results suggest that the 70-kd polypeptide identified by cross-linking of IL-3 represents a second binding chain of the receptor. By analogy with some of the other hemopoietin receptors, the 70- and 125-kd polypeptides may form a complex necessary for high affinity binding.
Duronio
V
V
Biomedical Research Centre, University of British Columbia, Vancouver, Canada.
Clark-Lewis
I
I
Schrader
J W
JW
D000581:Amidohydrolases / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D003432:Cross-Linking Reagents; D066298:In Vitro Techniques; D007377:Interleukin-3 / Q000378:metabolism*; D051379:Mice; D008970:Molecular Weight; D010449:Peptide Mapping; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011480:Protease Inhibitors / Q000494:pharmacology; D011485:Protein Binding; D016185:Receptors, Interleukin-3 / Q000737:chemistry* / Q000378:metabolism; D013696:Temperature
D003432:Cross-Linking Reagents; D007377:Interleukin-3; D011480:Protease Inhibitors; D016185:Receptors, Interleukin-3; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase