Ramanujam K S, Seetharam S, Dahms N M, Seetharam B
Department of Medicine Division of Gastroenterology, Medical College of Wisconsin, Milwaukee 53226.
J Biol Chem. 1991 Jul 15;266(20):13135-40.
Previous studies from our laboratory (Seetharam, B., Levine, J. S., Ramasamy, M., and Alpers, D. H. (1988) J. Biol. Chem. 263, 4443-4449; Fyfe, J. C., Ramanujam, K. S., Ramaswamy, K., Patterson, D. F., and Seetharam, B. (1991) J. Biol. Chem. 266, 4489-4494) have identified and isolated a 230-kDa receptor from rat and canine kidney which binds with high affinity [57Co]cyanocobalamin (Cbl) complexed to gastric intrinsic factor (IF). Although these studies have identified a renal receptor which binds intrinsic factor-cobalamin (IFCR), it is not known whether the binding is specific for IF-Cbl and whether renal cells internalize [57Co]Cbl bound to IF and transport [57Co]Cbl across the cell. Using a variety of renal cells, our results show that IF-[57Co]Cbl binding activity is detected in proximal tubular-derived epithelial cells from opossum (OK) and porcine kidney (LLC-PK1) but not in distal tubular-derived cells from canine kidney cells (MDCK). Metabolic labeling studies with Tran 35S-label confirmed the presence of a 230-kDa IFCR in OK and LLC-PK1 cells. Cell surface labeling and binding studies demonstrated that IFCR is targeted to the apical membrane. This apical expression of IFCR in OK cells is inhibited by the microtubule-disruptive drugs, colchicine and nocodazole. Opossum kidney cells when grown on culture inserts are polarized and transport [57Co]Cbl only when bound to IF and not to other Cbl binders. Furthermore, the transport of [57Co]Cbl occurred unidirectionally from the apical to the basolateral surface. Treatment of cells with colchicine or nocodazole inhibited the surface binding of IF-[57Co]Cbl as well as the transcytosis of [57Co]Cbl by 70-75%. IFCR retained intracellualarly by incubation of cells with colchicine or nocodazole is degraded by leupeptin-sensitive proteases. Based on these results, we suggest that proximal tubular-derived epithelial cells transport [57Co]Cbl bound to IF in a saturable way via receptor-mediated endocytosis.
我们实验室之前的研究（Seetharam, B., Levine, J. S., Ramasamy, M., and Alpers, D. H. (1988) J. Biol. Chem. 263, 4443 - 4449; Fyfe, J. C., Ramanujam, K. S., Ramaswamy, K., Patterson, D. F., and Seetharam, B. (1991) J. Biol. Chem. 266, 4489 - 4494）已经从大鼠和犬肾中鉴定并分离出一种230 kDa的受体，该受体与与胃内因子（IF）复合的[57Co]氰钴胺素（Cbl）具有高亲和力结合。尽管这些研究已经鉴定出一种结合内因子 - 钴胺素的肾受体（IFCR），但尚不清楚这种结合是否对IF - Cbl具有特异性，以及肾细胞是否内化与IF结合的[57Co]Cbl并使[57Co]Cbl跨细胞运输。使用多种肾细胞，我们的结果表明，在负鼠（OK）和猪肾（LLC - PK1）的近端小管来源的上皮细胞中检测到IF - [57Co]Cbl结合活性，但在犬肾细胞（MDCK）的远端小管来源的细胞中未检测到。用Tran 35S - 标记进行的代谢标记研究证实了OK和LLC - PK1细胞中存在230 kDa的IFCR。细胞表面标记和结合研究表明，IFCR定位于顶端膜。OK细胞中IFCR的这种顶端表达受到微管破坏药物秋水仙碱和诺考达唑的抑制。负鼠肾细胞在培养插入物上生长时是极化的，并且仅在与IF结合而非与其他Cbl结合剂结合时才转运[57Co]Cbl。此外，[57Co]Cbl的运输从顶端表面单向发生到底侧表面。用秋水仙碱或诺考达唑处理细胞可使IF - [57Co]Cbl 的表面结合以及[57Co]Cbl 的转胞吞作用受到70 - 75% 的抑制。通过用秋水仙碱或诺考达唑孵育细胞而保留在细胞内的IFCR被亮抑酶肽敏感的蛋白酶降解。基于这些结果,我们认为近端小管来源的上皮细胞通过受体介导的内吞作用以饱和方式转运与IF结合的[57Co]Cbl。
