Wenzel-Seifert K, Hurt C M, Seifert R
Howard Hughes Medical Institute, Stanford University Medical School, Stanford, California 94305-5428, USA.
J Biol Chem. 1998 Sep 11;273(37):24181-9. doi: 10.1074/jbc.273.37.24181.
The formyl peptide receptor (FPR) couples to pertussis toxin (PTX)-sensitive Gi-proteins to activate chemotaxis and exocytosis in neutrophils. PTX reduces not only formyl peptide-stimulated but also agonist-independent ("basal") Gi-protein activity, suggesting that the FPR is constitutively active. We aimed at identifying an inverse FPR agonist, i.e. a compound that suppresses constitutive FPR activity. In Sf9 insect cell membranes, the G-protein heterotrimer Gialpha2beta1gamma2 reconstituted N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP)-stimulated guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) binding and GTPgammaS-sensitive high affinity [3H]FMLP binding. The FPR "antagonist" cyclosporin H (CsH) potently and efficiently reduced basal GTPgammaS binding in Sf9 membranes. Another FPR antagonist, N-t-butoxycarbonyl-L-phenylalanyl-L-leucyl-L-phenylalanyl-L-leucyl-L- phenylalanine did not inhibit basal GTPgammaS binding but blocked the inhibitory effect of CsH on GTPgammaS binding. Na+ reduced basal GTPgammaS binding and eliminated the inhibitory effect of CsH. Similar effects of FMLP, CsH, and Na+ as in Sf9 membranes were observed with FPR expressed in the mammalian cell line HEK293. Our data show that the human FPR possesses high constitutive activity. CsH is an inverse FPR agonist and stabilizes the FPR in an inactive state. Na+ also stabilizes the FPR in an inactive state and, thereby, diminishes inverse agonist efficacy.
甲酰肽受体（FPR）与百日咳毒素（PTX）敏感的Gi蛋白偶联，以激活中性粒细胞的趋化性和胞吐作用。PTX不仅降低甲酰肽刺激的Gi蛋白活性，还降低激动剂非依赖性（“基础”）Gi蛋白活性，这表明FPR具有组成性活性。我们旨在鉴定一种FPR反向激动剂，即一种抑制FPR组成性活性的化合物。在Sf9昆虫细胞膜中，G蛋白异三聚体Gialpha2beta1gamma2重组了N-甲酰-L-蛋氨酰-L-亮氨酰-L-苯丙氨酸（FMLP）刺激的鸟苷5'-O-（3-硫代三磷酸）（GTPγS）结合和GTPγS敏感的高亲和力[3H]FMLP结合。FPR“拮抗剂”环孢菌素H（CsH）有效且显著地降低了Sf9膜中的基础GTPγS结合。另一种FPR拮抗剂N-叔丁氧羰基-L-苯丙氨酰-L-亮氨酰-L-苯丙氨酰-L-亮氨酰-L-苯丙氨酸不抑制基础GTPγS结合，但阻断了CsH对GTPγS结合的抑制作用。Na+降低基础GTPγS结合并消除CsH的抑制作用。在哺乳动物细胞系HEK293中表达的FPR也观察到与Sf9膜中FMLP、CsH和Na+类似的作用。我们的数据表明，人FPR具有高组成性活性。CsH是一种FPR反向激动剂，可使FPR稳定在无活性状态。Na+也使FPR稳定在无活性状态，从而降低反向激动剂的效力。
