The orphan mouse receptor interleukin (IL)-8R beta binds N51. Structure-function analysis using N51/IL-8 chimeric molecules.
We have demonstrated that the orphan receptor representing the putative mouse (mu) homolog of the human (hu) interleukin-8 receptor beta (IL-8R beta) binds the mouse N51 cytokine, also known as KC. The muIL-8R beta gene was constitutively expressed in NIH 3T3 cells (NIH-muIL-8R beta). Cells and plasma membranes from the NIH-muIL-8R beta clone showed binding of 125I-N51 that was displaced by unlabeled N51. Other related cytokines were assayed for their ability to displace 125I-N51. MIP-2 and GRO alpha/MGSA competed as well as N51 for the receptor, but huIL-8 and NAP-2 did not compete at all. Chimeric molecules between IL-8 and N51 were used to extend the binding analysis. The segment between the conserved cysteines 2 and 3, named domain I; cysteines 3 and 4, domain II; and cysteine 4 and the C terminus, domain III of IL-8 were replaced by the corresponding domains of N51 and vice versa. When studying the binding of 125I-N51 and the hybrid molecules to the receptor, we observed that chimeras of N51 containing either domain I, II, or III of IL-8 were agonists of N51, and chimeras of IL-8 containing domain II or III of N51 were partial agonists of N51. These results demonstrate that domain I of N51 does not confer binding specificity and suggest that the region from the third cysteine to the C terminus of the N51 molecule is more important for binding to muIL-8R beta.
Heinrich
J N
JN
Department of Molecular Biology, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543-4000.
Bravo
R
R
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D054360:Chemokine CXCL1; D019743:Chemokines, CXC; D002630:Chemotactic Factors / Q000378:metabolism*; D003001:Cloning, Molecular; D017124:Conserved Sequence; D003545:Cysteine; D017931:DNA Primers; D004220:Disulfides / Q000032:analysis; D006133:Growth Substances / Q000378:metabolism*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D007457:Iodine Radioisotopes; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000378:metabolism; D016133:Polymerase Chain Reaction; D018123:Receptors, Interleukin / Q000096:biosynthesis / Q000378:metabolism*; D023062:Receptors, Interleukin-8A; D023063:Receptors, Interleukin-8B; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014162:Transfection
C475829:CXCL1 protein, human; D054360:Chemokine CXCL1; D019743:Chemokines, CXC; D002630:Chemotactic Factors; C475895:Cxcl1 protein, mouse; D017931:DNA Primers; D004220:Disulfides; D006133:Growth Substances; D036341:Intercellular Signaling Peptides and Proteins; D007457:Iodine Radioisotopes; D009363:Neoplasm Proteins; D018123:Receptors, Interleukin; D023062:Receptors, Interleukin-8A; D023063:Receptors, Interleukin-8B; D011994:Recombinant Proteins; D003545:Cysteine