Differential internalization and processing of atrial-natriuretic-factor B and C receptor in PC12 cells.
Differential internalization and processing of atrial-natriuretic-factor B and C receptor in PC12 cells.
复制标题
PC12 细胞中心房钠尿因子 B 和 C 受体的差异内化和加工。
作者：
A. Rathinavelu;G. Isom
PC 12细胞表达两种心房利钠因子（ANF）受体亚型，分子量分别为130，000（B受体）和70，000（C受体）。B受体亚型占细胞表面受体群体的65%，其余35%为C受体，如在C-ANF（一种C受体选择性类似物）存在下通过饱和结合研究所确定。ANF-（99-126）-肽[ANF（99-126）]可与B-和C-受体亚型结合，在37 ℃孵育后迅速内化到细胞中。125 I-ANF（99-126）的内化被用作受体介导的内吞作用的指标并定量受体内化。在饱和浓度的C-ANF存在下，受体介导的125 I-ANF（99-126）的内化减少了24%，表明B受体介导了76%的配体内化。在37 ℃下用10 μ M的ANF孵育细胞下调了两种受体亚型，这反映在表面结合减少。时间依赖性研究表明，B-和C-受体亚型进行差异下调。在无ANF的培养基中孵育下调的细胞120分钟产生了35%的原始细胞表面结合的恢复。亲和交联的125 I-ANF的质膜上的受体在重新孵育（上调）的细胞表现出主要的B-受体亚型的表达。莫能菌素抑制72%的受体上调，而环己酰亚胺抑制43%，这表明参与介导B受体上调的主动再循环机制。本研究证实了PC 12细胞中B受体的快速内化和细胞内循环机制。C受体也经历内化和下调，但这种受体亚型再循环进入质膜的速率和程度低于B受体。
PC12 cells express two atrial-natriuretic-factor-(ANF)-receptor subtypes with molecular masses of 130,000 (B receptor) and 70,000 (C receptor). The B-receptor subtype constitutes 65% of the cell-surface receptor population, and the remaining 35% are C receptors as determined by saturation binding studies in the presence of C-ANF, a C-receptor-selective analogue. ANF-(99-126)-peptide [ANF(99-126)], which can bind to both B- and C-receptor subtypes, was rapidly internalized into the cells after incubation at 37 degrees C. Internalization of 125I-ANF(99-126) was used as an index of the receptor-mediated endocytosis and to quantify receptor internalization. In the presence of a saturating concentration of C-ANF, receptor-mediated internalization of 125I-ANF(99-126) was reduced by 24%, indicating B receptor mediate 76% of ligand internalization. Incubation of cells with 10 microM-ANF at 37 degrees C down-regulated both receptor subtypes as reflected by decreased surface binding. Time-dependent studies suggest that B- and C-receptor subtypes undergo differential down-regulation. Incubation of down-regulated cells for 120 min in ANF-free medium produced a recovery of 35% of the original cell-surface binding. Affinity cross-linking of 125I-ANF to the receptors on the plasma membrane in re-incubated (up-regulated) cells demonstrated expression of predominantly the B-receptor subtype. Monensin blocked 72% of receptor up-regulation, whereas cycloheximide inhibited 43%, suggesting an active recycling mechanism involved in mediating up-regulation of the B receptors. The present study demonstrates a rapid internalization and intracellular recycling mechanism for B receptors in PC12 cells. C receptors also undergo internalization and down-regulation, but recycling of this receptor subtype into the plasma membrane occurs at a lower rate and to a lesser extent than is the case for the B receptor.