Functional expression of intrinsic factor-cobalamin receptor by renal proximal tubular epithelial cells.
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.
Ramanujam
K S
KS
Department of Medicine Division of Gastroenterology, Medical College of Wisconsin, Milwaukee 53226.
Seetharam
S
S
Dahms
N M
NM
Seetharam
B
B
D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002460:Cell Line; D003078:Colchicine / Q000494:pharmacology; D004285:Dogs; D004848:Epithelium / Q000378:metabolism; D007437:Intrinsic Factor / Q000378:metabolism*; D007687:Kidney Tubules, Proximal / Q000378:metabolism*; D007700:Kinetics; D008871:Microvilli / Q000378:metabolism; D015739:Nocodazole / Q000494:pharmacology; D009893:Opossums; D051381:Rats; D011956:Receptors, Cell Surface / Q000096:biosynthesis / Q000187:drug effects / Q000378:metabolism*; D014805:Vitamin B 12 / Q000378:metabolism*
D011956:Receptors, Cell Surface; C029486:intrinsic factor-cobalamin receptor; D007437:Intrinsic Factor; D014805:Vitamin B 12; D015739:Nocodazole; D003078:Colchicine