Type I IL-1 receptor: ligand specific confirmation differences and the role of glycosylation in ligand binding.

The extracellular domain of the type I Interleukin-1 receptor (sIL-1R) was expressed in Drosophila S2 cells as a secreted 43 kDa glycoprotein, as evidenced by its binding to Concanavalin A and enzymatic deglycosylation. sIL-1R bound IL-1 beta with a K(D) of 2 nM as determined by competition ELISA. N-Glycanase treated sIL-1R had a C. 100 fold lower affinity than glycosylated sIL-1R for IL-1 beta, suggesting that glycosylation is a key component of the IL-1 beta/IL-1 receptor interaction. Crosslinking of sIL-1R to (125)I-IL-1 beta could be competed with unlabelled IL-1 alpha, IL-1 beta, IL-1 receptor antagonist (IL-1ra), and a mutant of IL-1 (Th9Gly) which has reduced bioactivity but wild type receptor binding affinity. Limited proteolysis of sIL-1R in the presence of IL-1 alpha, IL-1 beta, IL-1ra, and Thr9Gly IL-1 beta with several different proteases followed by analysis of sIL-1R by Western blot was used to assess the effect of binding on sIL-1R conformation. While some proteases showed no differences in cleavage patterns or sensitivity between free and bound sIL-1R, others showed differences in either cleavage sites or sensitivity with different ligands. This implies that upon ligand binding there is a conformational change in the receptor which is sensitive to the particular ligand bound, and hence has implications for the ability of different ligands to trigger responses after binding to receptor.