Rathinavelu A, Isom G E
Department of Pharmacology and Toxicology, School of Pharmacy and Pharmacal Sciences, Purdue University, West Lafayette, IN 47907.
Biochem J. 1991 Jun 1;276 ( Pt 2)(Pt 2):493-7. doi: 10.1042/bj2760493.
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.
PC12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）。通过在C-ANF（一种C受体选择性类似物）存在下的饱和结合研究确定，B受体亚型占细胞表面受体群体的65%，其余35%为C受体。能与B和C受体亚型都结合的ANF-(99 - 126)-肽[ANF(99 - 126)]，在37℃孵育后迅速内化进入细胞。125I-ANF(99 - 126)的内化被用作受体介导的内吞作用指标，并用于量化受体内化。在C-ANF饱和浓度存在下，125I-ANF(99 - 126)的受体介导内化减少了24%，表明B受体介导76%的配体内化。在37℃用10μM-ANF孵育细胞会下调两种受体亚型，这通过表面结合减少得以体现。时间依赖性研究表明B和C受体亚型经历不同的下调过程。在无ANF培养基中孵育下调的细胞120分钟后，细胞表面结合恢复到原始水平的35%。重新孵育（上调）细胞后，125I-ANF与质膜上受体的亲和交联显示主要表达B受体亚型。莫能菌素阻断72%的受体上调，而环己酰亚胺抑制43%，这表明存在一种活跃的再循环机制参与介导B受体的上调。本研究证明了PC12细胞中B受体存在快速内化和细胞内再循环机制。C受体也会发生内化和下调，但与B受体相比，这种受体亚型再循环到质膜的速率较低且程度较小。
