Ye R D, Quehenberger O, Thomas K M, Navarro J, Cavanagh S L, Prossnitz E R, Cochrane C G
Department of Immunology, Scripps Research Institute, La Jolla, CA 92037.
J Immunol. 1993 Feb 15;150(4):1383-94.
The rabbit neutrophil N-formyl peptide receptor (FPR) has been well studied for its ligand binding properties. Recent gene cloning experiments have established the existence of a subfamily of G protein-coupled receptors that share extensive sequence homology with the FPR, yet lack the capability of high affinity binding to FMLP. These findings prompted us to identify the structural requirement for formyl peptide ligand binding by delineation of the primary structure of the rabbit FPR. A rabbit neutrophil cDNA library was screened with a cloned human FPR cDNA probe and the insert of one positive isolate (B6) was sequenced. The 1268-bp cDNA insert encodes a peptide of 352 amino acids. Stably transfected L cell fibroblasts expressing the rabbit cDNA displayed specific binding of the ligand fMet-Leu-[3H]Phe with two affinities (Kd = 0.31 and 7.5 nM). Addition of the nonhydrolyzable guanosine triphosphate analogue, GTP gamma S, converted > or = 85% of the high affinity sites to the low affinity sites. FMLP induced mobilization of intracellular calcium in the transfected cells (EC50 = 0.5 nM), a response sensitive to pertussis toxin. FMLP stimulation desensitized the receptor such that subsequent stimulation with the same ligand produced a significantly reduced signal. These results indicate that the cloned rabbit receptor represents a high affinity FPR, and that FPR-mediated early signal transduction events can be fully reconstituted in transfected mammalian cells. The rabbit FPR sequence is 78% identical to that of the human FPR, and 68% identical to FPR2, a homologue of FPR with a low binding affinity (Kd > or = 400 nM) for FMLP. Analysis of the aligned sequences of these three proteins revealed that: 1) the amino termini and the second extracellular loops have the lowest sequence homology; 2) sequence in the intracellular domains that couple to G protein are highly conserved; and 3) the first and the third extracellular loops and their adjacent transmembrane domains of the FPR may contain residues essential for the high affinity binding of FMLP.
兔中性粒细胞N-甲酰基肽受体（FPR）的配体结合特性已得到充分研究。最近的基因克隆实验证实存在一个G蛋白偶联受体亚家族，它们与FPR具有广泛的序列同源性，但缺乏与FMLP高亲和力结合的能力。这些发现促使我们通过描绘兔FPR的一级结构来确定甲酰基肽配体结合的结构要求。用克隆的人FPR cDNA探针筛选兔中性粒细胞cDNA文库，并对一个阳性分离株（B6）的插入片段进行测序。1268 bp的cDNA插入片段编码一个352个氨基酸的肽。稳定转染表达兔cDNA的L细胞成纤维细胞显示配体fMet-Leu-[3H]Phe具有两种亲和力的特异性结合（Kd = 0.31和7.5 nM）。加入不可水解的鸟苷三磷酸类似物GTPγS可使≥85%的高亲和力位点转变为低亲和力位点。FMLP诱导转染细胞内钙动员（EC50 = 0.5 nM），该反应对百日咳毒素敏感。FMLP刺激使受体脱敏，以至于随后用相同配体刺激产生的信号明显减弱。这些结果表明克隆的兔受体代表高亲和力FPR，并且FPR介导的早期信号转导事件可以在转染的哺乳动物细胞中完全重建。兔FPR序列与人FPR序列的同源性为78%，与FPR2的同源性为68%，FPR2是FPR的一个同源物，对FMLP的结合亲和力较低（Kd≥400 nM）。对这三种蛋白质的比对序列分析表明：1）氨基末端和第二个细胞外环的序列同源性最低；2）与G蛋白偶联的细胞内结构域序列高度保守；3）FPR的第一个和第三个细胞外环及其相邻的跨膜结构域可能包含FMLP高亲和力结合所必需的残基。
