Leong S R, Kabakoff R C, Hébert C A
Department of Immunology, Genentech, Inc., South San Francisco, California 94080.
J Biol Chem. 1994 Jul 29;269(30):19343-8.
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.
为了确定参与白细胞介素-8（IL-8）结合及IL-8介导的信号转导的氨基酸，我们系统地将IL-8 A型受体胞外域中的每个氨基酸替换为丙氨酸。我们鉴定出20个突变，这些突变导致受体亲和力从2 nM的解离常数（Kd）降至Kd≥25 nM。然后，我们分析了这些受体突变体在用10 nM IL-8刺激后动员细胞内钙的能力。除了六个无明显反应的突变体（R199A、R203A、C30A、C110A、C187A和C277A）外，大多数突变体能够产生与野生型IL-8受体A水平相近的钙通量。此外，我们进行了钙动员实验，以进一步表征一系列先前构建的突变体，这些突变体在我们之前的报告中仅通过其结合亲和力进行了表征，结果发现突变体D265A在用10 nM IL-8刺激时无反应。我们的研究表明，除了可能对受体整体折叠至关重要的胞外域半胱氨酸外，三个残基Arg-199、Arg-203和Asp-265对IL-8结合及IL-8介导的信号转导很重要。
