Quehenberger O, Prossnitz E R, Cavanagh S L, Cochrane C G, Ye R D
Department of Immunology, Scripps Research Institute, La Jolla, California 92037.
J Biol Chem. 1993 Aug 25;268(24):18167-75.
Binding of the chemotactic tripeptide fMet-Leu-Phe (fMLP) to its receptor on phagocytes activates these cells through a G protein-coupled pathway. To delineate the structural requirement of the N-formyl peptide receptor (FPR) for ligand binding and signaling, we constructed chimeric receptors between FPR and a recently identified granulocyte receptor, FPR2 (Ye, R. D., Cavanagh, S. L., Quehenberger, O., Prossnitz, E. R., and Cochrane, C. G. (1992) Biochem. Biophys. Res. Commun. 184, 582-589). FPR2 shares 69% sequence homology with the FPR; yet it binds fMLP with a low affinity (Kd = 430 nM), as compared with the high affinity (Kd = 1 nM) displayed by the FPR. This property of the FPR2 was utilized for mapping the FPR ligand binding domains. Seven chimeric FPR/FPR2 receptors were generated by sequential replacement of the FPR segments with the corresponding regions from FPR2. Three reciprocal FPR2/FPR chimeric receptors were also constructed by selective substitution of the FPR segments into FPR2. These chimeric receptors were stably expressed in transfected fibroblasts and analyzed for their ligand binding and transmembrane signaling properties. Replacement of the FPR domains, including the first and the third extracellular loops, resulted in 275- and 85-fold decrease in ligand binding affinity, respectively. Introduction of both domains into the FPR2 significantly increased ligand binding affinity (Kd = 18 nM), whereas substitution of the domains containing the first or third extracellular loop alone improved ligand binding to a lesser degree (Kd = 90 and 372 nM, respectively). In contrast, substitution of either the amino or the carboxyl-terminal regions with those of the FPR2 had little effect on ligand binding affinity. An analysis of the sequences of the two receptors revealed several key residues in the first and the third extracellular loops of the FPR and their adjacent transmembrane domains that may be essential for binding of fMLP. We propose that multiple domains of the FPR are required for high-affinity ligand binding, with a major determinant located in the first extracellular loop and its adjacent transmembrane domains.
趋化性三肽甲酰甲硫氨酰 - 亮氨酰 - 苯丙氨酸（fMet-Leu-Phe，fMLP）与其在吞噬细胞上的受体结合，通过G蛋白偶联途径激活这些细胞。为了阐明N - 甲酰肽受体（FPR）在配体结合和信号传导方面的结构要求，我们构建了FPR与最近鉴定的粒细胞受体FPR2之间的嵌合受体（Ye, R. D., Cavanagh, S. L., Quehenberger, O., Prossnitz, E. R., and Cochrane, C. G. (1992) Biochem. Biophys. Res. Commun. 184, 582 - 589）。FPR2与FPR有69%的序列同源性；然而，与FPR显示的高亲和力（Kd = 1 nM）相比，它与fMLP的结合亲和力较低（Kd = 430 nM）。FPR2的这一特性被用于绘制FPR配体结合结构域。通过用FPR2的相应区域依次替换FPR片段，产生了七个嵌合FPR/FPR2受体。还通过将FPR片段选择性地替换到FPR2中构建了三个反向FPR2/FPR嵌合受体。这些嵌合受体在转染的成纤维细胞中稳定表达，并分析它们的配体结合和跨膜信号传导特性。替换FPR结构域，包括第一个和第三个细胞外环，分别导致配体结合亲和力下降275倍和85倍。将这两个结构域引入FPR2显著增加了配体结合亲和力（Kd = 18 nM），而单独替换包含第一个或第三个细胞外环的结构域对配体结合的改善程度较小（Kd分别为90和372 nM）。相比之下，用FPR2的氨基或羧基末端区域替换对配体结合亲和力影响很小。对这两个受体序列的分析揭示了FPR的第一个和第三个细胞外环及其相邻跨膜结构域中的几个关键残基，这些残基可能对fMLP的结合至关重要。我们提出，FPR的多个结构域是高亲和力配体结合所必需的，主要决定因素位于第一个细胞外环及其相邻跨膜结构域中。
