Quehenberger O, Prossnitz E R, Cochrane C G, Ye R D
Department of Immunology, Scripps Research Institute, La Jolla, California 92037.
J Biol Chem. 1992 Oct 5;267(28):19757-60.
We investigated the interaction of the N-formyl peptide receptor (NFPR) with G proteins in infected Sf9 insect cells expressing the recombinant NFPR. Recombinant receptor expression of up to 27 pmol/mg protein was achieved in these cells. The receptor was recognized by an antiserum raised against an NFPR carboxyl-terminal peptide, and displayed specific and saturable binding of the formyl peptide ligand fMet-Leu-[3H]Phe. Scatchard analysis of the binding data yielded a dissociation constant of approximately 62 nM, a binding affinity of 60- to 120-fold lower than that of the high affinity sites in neutrophils and in transfected mammalian cell lines expressing the NFPR. That this low binding affinity was due to a lack of receptor coupling to G protein was suggested by the failure of guanine nucleotides to regulate receptor affinity and by the lack of formyl peptide-stimulated GTPase activity in these cells. Furthermore, immunoblotting with an anti-G(i) antibody and ADP-ribosylation experiments indicated that the approximately 40-kDa G(i) alpha subunit, which couples to the NFPR in neutrophils, is not present in Sf9 cell membranes. Thus, the current study provides for the first time evidence that a major G protein is absent in the Sf9 insect cells. Potential applications of the Sf9 system for in vitro reconstitution of the NFPR-G protein interaction are discussed.
我们研究了N-甲酰肽受体（NFPR）与G蛋白在表达重组NFPR的感染Sf9昆虫细胞中的相互作用。在这些细胞中实现了高达27 pmol/mg蛋白质的重组受体表达。该受体可被针对NFPR羧基末端肽产生的抗血清识别，并表现出甲酰肽配体fMet-Leu-[³H]Phe的特异性和饱和性结合。对结合数据的Scatchard分析得出解离常数约为62 nM，结合亲和力比中性粒细胞和表达NFPR的转染哺乳动物细胞系中的高亲和力位点低60至120倍。鸟嘌呤核苷酸未能调节受体亲和力以及这些细胞中缺乏甲酰肽刺激的GTP酶活性，表明这种低结合亲和力是由于受体与G蛋白缺乏偶联所致。此外，用抗G(i)抗体进行的免疫印迹和ADP-核糖基化实验表明，在中性粒细胞中与NFPR偶联的约40 kDa的G(i)α亚基不存在于Sf9细胞膜中。因此，本研究首次提供证据表明Sf9昆虫细胞中不存在主要的G蛋白。讨论了Sf9系统在体外重建NFPR-G蛋白相互作用方面的潜在应用。
