Jesaitis A J, Naemura J R, Painter R G, Schmitt M, Sklar L A, Cochrane C G
J Cell Biochem. 1982;20(2):177-91. doi: 10.1002/jcb.240200209.
Experiments were performed to examine how human granulocytes, stimulated by N-formyl-chemotactic peptides, process the N-formyl peptide receptor. One percent of the surface N-formyl-chemotactic peptide receptors of purified human granulocytes were covalently, specifically, and radioactively labeled at 4 degrees C using the photochemically reactive N-formyl-chemotactic hexapeptide CHO-Nle-Leu-Phe-Nle-[125I] Tyr-N epsilon (6-(4'-azido-2'-nitrophenyl-amino)hexanoyl)-Lys. After incubation in the presence of 500 nM of N-formyl-Met-Leu-Phe at 37 degrees C, the cells were lysed and fractionated by isopycnic surcrose density gradient sedimentation. Receptor-associated radioactivity cosedimented with plasma membrane in fractions from cells kept at 4 degrees C or incubated at 37 degrees C for 2 min or less. Fractionation of cells incubated at 37 degrees C for longer times revealed that the radioactivity sedimented to lower densities coincident with Golgi markers and the site of noncovalently bound and internalized formyl-chemotactic peptide. To follow the redistribution of unoccupied receptors, human granulocytes were stimulated with 500 nM N-formyl-Met-Leu-Phe at 37 degrees C for 5 min, washed, lysed by N2 cavitation, and fractionated by rate zonal sucrose density gradient sedimentation. Compared to unstimulated controls the specific binding of N-formyl-Met-Leu-[3H]Phe decreased 76% +/- 9% in plasma membrane fractions. N-formyl-Met-Leu-[3H]Phe-binding activity associated with an intracellular pool cosedimenting with specific granules remained unchanged. Approximately 20% of the activity lost in the plasma membrane could be accounted for by a redistribution of specific N-formyl-Met-Leu-Phe binding to fractions enriched in azurophil granules. We conclude that the receptor is the carrier in the internalization of the N-formyl-chemotactic peptides to a Golgi-enriched fraction and hypothesize that after a short residency in this fraction, the receptor may dissociate from the ligand and pass onto a fraction cosedimenting with dense granules.
进行了实验以研究人粒细胞在N-甲酰基趋化肽刺激下如何处理N-甲酰基肽受体。使用光化学反应性N-甲酰基趋化六肽CHO-Nle-Leu-Phe-Nle-[125I]Tyr-Nε(6-(4'-叠氮基-2'-硝基苯基-氨基)己酰基)-Lys，在4℃下对纯化的人粒细胞表面1%的N-甲酰基趋化肽受体进行共价、特异性和放射性标记。在37℃下于500 nM N-甲酰基-Met-Leu-Phe存在下孵育后，将细胞裂解并通过等密度蔗糖密度梯度沉降进行分级分离。在4℃保存或在37℃孵育2分钟或更短时间的细胞分级分离物中，与受体相关的放射性与质膜一起沉降。对在37℃孵育更长时间的细胞进行分级分离发现，放射性沉降到较低密度，与高尔基体标志物以及非共价结合和内化的甲酰基趋化肽的部位一致。为了追踪未占据受体的重新分布，在37℃下用500 nM N-甲酰基-Met-Leu-Phe刺激人粒细胞5分钟，洗涤，通过N2空化裂解，并通过速率区带蔗糖密度梯度沉降进行分级分离。与未刺激的对照相比，质膜分级分离物中N-甲酰基-Met-Leu-[3H]Phe的特异性结合降低了76%±9%。与特异性颗粒一起沉降的细胞内池相关的N-甲酰基-Met-Leu-[3H]Phe结合活性保持不变。质膜中丧失的活性约20%可由特异性N-甲酰基-Met-Leu-Phe结合重新分布到富含嗜天青颗粒的分级分离物中来解释。我们得出结论，该受体是N-甲酰基趋化肽内化至富含高尔基体的分级分离物中的载体，并推测在该分级分离物中短暂停留后，受体可能与配体解离并转移至与致密颗粒一起沉降的分级分离物中。
