Identification of a critical Ig-like domain in IL-18 receptor alpha and characterization of a functional IL-18 receptor complex.
Steady state mRNA levels in various human tissues reveal that the proinflammatory cytokine IL-18 is constitutively and ubiquitously expressed. However, limited IL-18R alpha-chain (IL-18Ralpha) expression in tissues may restrict ligand-acting sites and contribute to a specific response for IL-18. To study the IL-18R complex, [(125)I]IL-18 was studied for binding to the cell surface receptors of IL-18-responsive NK and macrophagic KG-1 cells. After cross-linking, [(125)I]IL-18 formed three IL-18R complexes with sizes of approximately 93, 160, and 220 kDa. In KG-1 cells, Scatchard analysis revealed the presence of 135 binding sites/cell, with an apparent dissociation constant (K(d)) of 250 pM; in NK cells, there were 350 binding sites per cell with an apparent K(d) of 146 pM. Each domain of extracellular IL-18Ralpha was cloned and individually expressed in Escherichia coli. An mAb specifically recognized the membrane-proximal third domain; this mAb blocked IL-18-induced IFN-gamma production in NK cells. Furthermore, deletion of the membrane-proximal third domain of IL-18Ralpha prevented the formation of IL-18R ternary complex with IL-18R beta-chain. The present studies demonstrate that the biologically active IL-18R complex requires the membrane-proximal third Ig-like domain in IL-18Ralpha for the formation of IL-18R ternary complex as well as for signal transduction involved in IL-18-induced IFN-gamma in NK cells.
Azam
Tania
T
University of Colorado Health Sciences Center, Denver, CO 80262, USA.
Novick
Daniela
D
Bufler
Philip
P
Yoon
Do-Young
DY
Rubinstein
Menachem
M
Dinarello
Charles A
CA
Kim
Soo Hyun
SH
D002460:Cell Line; D045744:Cell Line, Tumor; D006801:Humans; D007136:Immunoglobulins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D020382:Interleukin-18 / Q000096:biosynthesis / Q000378:metabolism*; D053727:Interleukin-18 Receptor alpha Subunit; D007457:Iodine Radioisotopes / Q000378:metabolism; D007694:Killer Cells, Natural / Q000276:immunology / Q000378:metabolism; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D009928:Organ Specificity / Q000235:genetics / Q000276:immunology; D011485:Protein Binding / Q000276:immunology; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology; D021122:Protein Subunits / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D012333:RNA, Messenger / Q000096:biosynthesis; D018123:Receptors, Interleukin / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D053724:Receptors, Interleukin-18; D017384:Sequence Deletion; D013329:Structure-Activity Relationship
C508572:IL18R1 protein, human; D007136:Immunoglobulins; D020382:Interleukin-18; D053727:Interleukin-18 Receptor alpha Subunit; D007457:Iodine Radioisotopes; D046911:Macromolecular Substances; D008565:Membrane Proteins; D021122:Protein Subunits; D012333:RNA, Messenger; D018123:Receptors, Interleukin; D053724:Receptors, Interleukin-18