Complete mutagenesis of the extracellular domain of interleukin-8 (IL-8) type A receptor identifies charged residues mediating IL-8 binding and signal transduction.
We systematically converted each of the amino acids in the extracellular domain of the interleukin-8 (IL-8) type A receptor to alanine for the purpose of identifying amino acids contributing to IL-8 binding and IL-8-mediated signal transduction. We identified 20 mutations which cause a decrease in receptor affinity from a Kd of 2 nM to a Kd > or = 25 nM. We then analyzed these receptor mutants for their ability to mobilize intracellular calcium upon stimulation with 10 nM IL-8. The majority of the mutants were able to produce calcium fluxes at levels approximating that of wild-type IL-8 receptor A, with the exception of six mutants (R199A, R203A, C30A, C110A, C187A, and C277A) which showed no significant response. In addition, we performed calcium mobilization experiments to further characterize a series of previously constructed mutants which had only been characterized by their binding affinities in our previous report and found that mutant D265A showed no response upon stimulation with 10 nM IL-8. Our study shows that, besides the extracellular domain cysteines which may be critical for the overall folding of the receptor, three residues, Arg-199, Arg-203, and Asp-265, are important for IL-8 binding and IL-8-mediated signal transduction.
Department of Immunology, Genentech, Inc., South San Francisco, California 94080.
S R
SR
Leong
R C
RC
Kabakoff
C A
CA
Hébert
D000409:Alanine / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001120:Arginine / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D006801:Humans; D016209:Interleukin-8 / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D018123:Receptors, Interleukin / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D023062:Receptors, Interleukin-8A; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D014162:Transfection
D016209:Interleukin-8; D018123:Receptors, Interleukin; D023062:Receptors, Interleukin-8A; D011994:Recombinant Proteins; D001120:Arginine; D000409:Alanine