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.
Einstein
R
R
Department of Molecular Immunology, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania, USA.
Jackson
J R
JR
D'Alessio
K
K
Lillquist
J S
JS
Sathe
G
G
Porter
T
T
Young
P R
PR
D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents; D004331:Drosophila melanogaster; D010450:Endopeptidases; D006031:Glycosylation; D006801:Humans; D007375:Interleukin-1 / Q000378:metabolism*; D008024:Ligands; D008099:Liver / Q000378:metabolism; D011487:Protein Conformation; D017472:Receptors, Interleukin-1 / Q000737:chemistry* / Q000378:metabolism*; D053573:Receptors, Interleukin-1 Type I; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D014162:Transfection
D003432:Cross-Linking Reagents; D007375:Interleukin-1; D008024:Ligands; D017472:Receptors, Interleukin-1; D053573:Receptors, Interleukin-1 Type I; D011993:Recombinant Fusion Proteins; D010450:Endopeptidases