Partial functional mapping of the human interleukin-8 type A receptor. Identification of a major ligand binding domain.
We have previously demonstrated that a basic amino acid residue of interleukin (IL)-8, namely Arg-6, is critical for the binding of IL-8 to its receptor. We reasoned that this residue is likely to be poised to directly interact with a counterpart acidic residue on the receptor. To identify this key residue, we systematically mutated to Ala all acidic residues present on the ligand accessible surface of IL-8 receptor type A. Using this strategy, we demonstrate that two residues which are present in extracellular loop 3 of the receptor, namely Glu-275 and Arg-280, are critical for ligand binding. In addition, we show that although Asp-11 is critical for ligand binding, a conservative mutation of Asp-11 to Glu or a substitution of Asp-11 with Lys (the residue found at position 11 in IL-8 receptor type B) does not affect the Kd of the receptor/ligand interaction. These data suggest that Lys-11 recruits a new and favorable interaction with IL-8 (analogous to that of IL-8 receptor type B with IL-8) or that the cavity created by mutating Asp-11 to Ala is particularly deleterious. Finally, we discuss fluorescence-activated cell sorter staining data which support the hypothesis that the N-terminal region and the extracellular loop 3 of the receptor may lie in close proximity of one another and constitute a major binding domain for IL-8.
Hébert
C A
CA
Department of Immunology, Genentech Inc., South San Francisco, California 94080.
Chuntharapai
A
A
Smith
M
M
Colby
T
T
Kim
J
J
Horuk
R
R
D000409:Alanine; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001120:Arginine; D001665:Binding Sites; D002460:Cell Line; D017124:Conserved Sequence; D004926:Escherichia coli / Q000235:genetics; D005434:Flow Cytometry; D005971:Glutamates; D018698:Glutamic Acid; D006801:Humans; D016209:Interleukin-8 / Q000378:metabolism; D007668:Kidney; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D017433:Protein Structure, Secondary; D011971:Receptors, Immunologic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D023062:Receptors, Interleukin-8A; D011994:Recombinant Proteins; D014162:Transfection
D000911:Antibodies, Monoclonal; D005971:Glutamates; D016209:Interleukin-8; D011971:Receptors, Immunologic; D023062:Receptors, Interleukin-8A; D011994:Recombinant Proteins; D018698:Glutamic Acid; D001120:Arginine; D000409:Alanine