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 homogenei ty by Ligand-affinity chromatography. SDS-polyacrylamide gel electroph oresis analysis indicated that the soluble receptor is glycosylated an d has an apparent molecular mass of 35,000-45,000. Under native condit ions, soluble hIL-10 receptor was determined by gel filtration to be a monomeric protein. Soluble hIL-10 receptor was able to inhibit the bi nding of I-125-hTL-10 to the full-length receptor and was able to anta gonize the effect of human IL-10 in cell proliferation and cytokine sy nthesis inhibition. The apparent dissociation constant (K-d) of solubl e 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., Idelic ato, S. R., Narula, S. IL, Zavodny, P. J., and Chou, C.-C. (1993) J. B iol. Chem. 268, 21053-21059; Liu, Y., Wei, S. H.-Y., Ho, A S.-Y., de W aal Malefyt, R., and Moore, IL m. (1994) J. Immunol. 152, 1821-1829). When hIL-10 binds soluble hIL-10 receptor in solution, a single comple x was detected by gel filtration, and the complex was found to consist of two hIL-10 dimers and four soluble receptor monomers, suggesting t hat hIL-10 may induce a novel mode of oligomerization of the receptor upon binding.