Characterization of mouse interleukin-12 p40 homodimer binding to the interleukin-12 receptor subunits.
Interleukin-12 (IL-12) is a heterodimeric cytokine composed of two disulfide-bonded subunits, p35 and p40, which has important regulatory effects on T cells and natural killer (NK) cells. In contrast to heterodimeric IL-12, a homodimer of the p40 subunit, designated (p40)2, has been shown to be a potent IL-12 antagonist. To study the interaction between (p40)2 and the known IL-12 receptor (IL-12R) subunits, IL-12Rbeta1 and IL-12Rbeta2, we directly measured the binding activity of mouse (p40)2 to ConA-activated lymphoblasts and purified B cells from splenocytes of C57BL/6J mice. These results demonstrated the presence of both high (Kd about 5 pM) and low affinity (Kd about 15 nM) binding sites for mouse 125I-labeled (p40)2. To elucidate which of the IL-12R subunits binds mouse (p40)2, binding studies of mouse 125I-labeled (p40)2 to Ba/F3 cells expressing recombinant mouse IL-12Rbeta1 and/or mouse IL-12Rbeta2 were carried out. Mouse IL-12Rbeta1 bound mouse 125I-labeled (p40)2 with high and low affinities, comparable to that observed on Con A blasts and B cells. In contrast, mouse IL-12Rbeta2 bound mouse 125I-labeled (p40)2 very poorly. These data demonstrate that similar to IL-12, mouse (p40)2 binds with both high and low affinity to Con A blasts and B cells, and that IL-12Rbeta1 is responsible for mediating the specific binding of mouse (p40)2.
Wang
X
X
Department of Inflammation/Autoimmune Diseases, Hoffmann-La Roche Inc., Nutley, USA.
Wilkinson
V L
VL
Podlaski
F J
FJ
Wu
C
C
Stern
A S
AS
Presky
D H
DH
Magram
J
J
D000818:Animals; D001402:B-Lymphocytes / Q000276:immunology; D001665:Binding Sites; D001667:Binding, Competitive; D002460:Cell Line; D003208:Concanavalin A / Q000494:pharmacology; D019281:Dimerization; D066298:In Vitro Techniques; D018664:Interleukin-12 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008213:Lymphocyte Activation; D051379:Mice; D008810:Mice, Inbred C57BL; D011487:Protein Conformation; D018123:Receptors, Interleukin / Q000737:chemistry / Q000378:metabolism*; D053707:Receptors, Interleukin-12; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D018123:Receptors, Interleukin; D053707:Receptors, Interleukin-12; D011994:Recombinant Proteins; D003208:Concanavalin A; D018664:Interleukin-12