Characterization of recombinant extracellular domain of human interleukin-10 receptor.
The extracellular region of the human interleukin-10 (hIL-10) receptor was expressed using a myeloma cell line and was purified to homogeneity by ligand-affinity chromatography. SDS-polyacrylamide gel electrophoresis analysis indicated that the soluble receptor is glycosylated and has an apparent molecular mass of 35,000-45,000. Under native conditions, soluble hIL-10 receptor was determined by gel filtration to be a monomeric protein. Soluble hIL-10 receptor was able to inhibit the binding of 125I-hIL-10 to the full-length receptor and was able to antagonize the effect of human IL-10 in cell proliferation and cytokine synthesis inhibition. The apparent dissociation constant (Kd) of soluble hIL-10 receptor was determined to be 563 +/- 59 pM, approximately 2- to 10-fold higher than that found on intact cells (Tan, J. C., Indelicato, S. R., Narula, S. K., Zavodny, P. J., and Chou, C.-C. (1993) J. Biol. Chem. 268, 21053-21059; Liu, Y., Wei, S. H.-Y., Ho, A. S.-Y., de Waal Malefyt, R., and Moore, K. W. (1994) J. Immunol. 152, 1821-1829). When hIL-10 binds soluble hIL-10 receptor in solution, a single complex was detected by gel filtration, and the complex was found to consist of two hIL-10 dimers and four soluble receptor monomers, suggesting that hIL-10 may induce a novel mode of oligomerization of the receptor upon binding.
Tan
J C
JC
Schering-Plough Research Institute, Kenilworth, New Jersey 07033-0530, USA.
Braun
S
S
Rong
H
H
DiGiacomo
R
R
Dolphin
E
E
Baldwin
S
S
Narula
S K
SK
Zavodny
P J
PJ
Chou
C C
CC
D000595:Amino Acid Sequence; D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism; D016764:Cell Polarity; D003432:Cross-Linking Reagents; D016207:Cytokines / Q000096:biosynthesis; D006801:Humans; D016753:Interleukin-10 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D011485:Protein Binding; D018123:Receptors, Interleukin / Q000235:genetics / Q000378:metabolism*; D053701:Receptors, Interleukin-10; D011994:Recombinant Proteins / Q000378:metabolism; D017421:Sequence Analysis; D012995:Solubility
D003432:Cross-Linking Reagents; D016207:Cytokines; D010446:Peptide Fragments; D018123:Receptors, Interleukin; D053701:Receptors, Interleukin-10; D011994:Recombinant Proteins; D016753:Interleukin-10