The IL-4 receptor: biochemical characterization of IL-4-binding molecules in a T cell line expressing large numbers of receptors.
Cross-linking of 125I-IL-4 to the surface of cells expressing IL-4R yields as the major IL-4-binding molecules, polypeptide chains with inferred m.w. of approximately 70,000 (p70) and approximately 120,000 to 140,000 (p120-p140). The demonstration that the functional product of the IL-4R cDNA clone has m.w. of approximately 140,000 and that no p70 product is detected in transfected COS-7 cells has led to an uncertainty regarding the nature of p70. To study this issue, we examined the relationship of the IL-4-binding molecules p120 and p70 and, in parallel, attempted to immunoprecipitate p70 from surface and internally labeled cells using IL-4 and two anti-IL-4R antibodies (M1 and M2), bound to Affigel 10, as ligands. Cross-linked complexes containing 125I-IL-4 and p70 or p120 were isolated and digested with chymotrypsin or with V8 protease. Three distinct IL-4-binding peptides could be compared; these were indistinguishable for cross-linked p70 and p120, strongly implying that p70 and p120 were structurally related. Furthermore, immunoprecipitates made with IL-4 or anti-IL-4R-Affigel did not contain p70. This led us to conclude that p70 is a breakdown product of p120. A second IL-4-binding molecule of 40,000 Da (p40) expressing the M1 and M2 epitopes of the IL-4R was detected and appears to be the product of an mRNA coding for the soluble form of the receptor. mRNA for p40 was detected in both the T cell line CT.4R and the mast cell line CFTL.12 using polymerase chain reaction primers unique to this species of message. Pulse-chase studies of IL-4R in [35S] methionine-labeled cells indicates that p40 is derived from a 42,000-Da precursor that is detectable at the end of the pulse period, and thus, further argue that p40 is an independently translated molecule and not a degradation product of p120. Although p40 has been previously shown to be a soluble, truncated form of the receptor, we failed to observe secretion of p40 into the medium by internally labeled CT.4R cells.
Keegan
A D
AD
Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892.
Beckmann
M P
MP
Park
L S
LS
Paul
W E
WE
D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D002460:Cell Line; D003432:Cross-Linking Reagents; D015847:Interleukin-4 / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009841:Oligonucleotides / Q000737:chemistry; D010449:Peptide Mapping; D016133:Polymerase Chain Reaction; D011233:Precipitin Tests; D012333:RNA, Messenger / Q000235:genetics; D019948:Receptors, Interleukin-4; D011975:Receptors, Mitogen / Q000737:chemistry / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D009841:Oligonucleotides; D012333:RNA, Messenger; D019948:Receptors, Interleukin-4; D011975:Receptors, Mitogen; D015847:Interleukin-4