Jesaitis A J, Naemura J R, Sklar L A, Cochrane C G, Painter R G
J Cell Biol. 1984 Apr;98(4):1378-87. doi: 10.1083/jcb.98.4.1378.
When human granulocytes were exposed to 50 nM N-formyl-Met-Leu-[3H]Phe at 37 degrees C they rapidly formed ligand-receptor complexes that dissociated 50-100 times more slowly than those on cells initially exposed to the peptide at 4 degrees C. These complexes of apparent higher affinity were stable after detergent solubilization of the cells with Triton X-100. The complexes co-isolated with the detergent insoluble cytoskeletal residues and were free of the cytosolic and Golgi markers, lactate dehydrogenase and galactosyl transferase, respectively. After 5 s of exposure to f-Met-Leu-Phe, 2,000-3,000 molecules of ligand per cell were trapped in such complexes. Continued exposure resulted in capture of a maximum of 14,000 molecules per cell by 5 min. Exposure at 15 degrees C, a temperature at which endocytosis of the receptor is prevented, resulted in complex formation at a linear rate for at least 20 min to levels twice those measured at 37 degrees C. At 4 degrees C, complex formation was approximately 10% of the maximum amount formed at 37 degrees C. Pulse-chase experiments revealed that the complex was in transient association with the cytoskeleton with a half life ranging between 30 s to 4 min depending on the length of the original incubation. Electron microscopic autoradiography indicated that after 1 min of incubation at 37 degrees C, the majority of the specific autoradiographic grains were localized to the outer circumference of the cellular cytoskeleton. After 4 min of incubation, the grains were less frequent at the cytoskeleton periphery but still threefold enriched over a random cellular distribution. We conclude that a metabolically controlled modulation of the state of the N-formyl chemotactic peptide receptor occurs in the plasma membrane which may be the result of transient association of ligand-receptor complex and the cell cytoskeleton.
当人粒细胞在37℃下暴露于50 nM的N-甲酰甲硫氨酰-亮氨酰-[3H]苯丙氨酸时，它们迅速形成配体-受体复合物，其解离速度比最初在4℃下暴露于该肽的细胞上的复合物慢50-100倍。这些表观亲和力更高的复合物在用Triton X-100对细胞进行去污剂溶解后仍保持稳定。这些复合物与去污剂不溶性细胞骨架残余物共同分离，并且分别没有胞质和高尔基体标志物乳酸脱氢酶和半乳糖基转移酶。暴露于甲硫氨酰-亮氨酰-苯丙氨酸5秒后，每个细胞有2000-3000个配体分子被困在这种复合物中。持续暴露导致到5分钟时每个细胞最多捕获14000个分子。在15℃下暴露，这是一个阻止受体胞吞作用的温度，导致复合物以线性速率形成至少20分钟，达到37℃下测量水平的两倍。在4℃下，复合物形成约为37℃下形成的最大量的10%。脉冲追踪实验表明，该复合物与细胞骨架短暂结合，半衰期在30秒到4分钟之间，具体取决于最初孵育的时间长度。电子显微镜放射自显影表明，在37℃孵育1分钟后，大多数特异性放射自显影颗粒定位于细胞骨架的外周。孵育4分钟后，颗粒在细胞骨架周边的出现频率降低，但仍比随机细胞分布富集三倍。我们得出结论，在质膜中发生了N-甲酰趋化肽受体状态的代谢控制调节，这可能是配体-受体复合物与细胞骨架短暂结合的结果。
