ISOLATED RAT HEPATOCYTES BIND LACTOFERRINS BY THE RHL-1 SUBUNIT OF THE ASIALOGLYCOPROTEIN RECEPTOR IN A GALACTOSE-INDEPENDENT MANNER

Isolated rat hepatocytes bind and internalize the iron-binding protein lactoferrin (Lf) by a set of high-affinity, recycling, Ca2+-dependent binding sites. We have purified a 45-kDa membrane protein (p45) from rat hepatocytes that exhibits Ca2+-dependent receptor activity. In thi s study, we found p45 to be identical to the major subunit (RHL-1) of the rat asialoglycoprotein receptor. Two tryptic fragments of p45 show ed 100% identity with RHL-1 internal sequences (Leu(121) --> Lys(126) and Phe(198) --> Lys(220)), and monospecific antisera against p45 and RHL-1 cross-reacted equally well with each protein. Molar excesses of anti-p45 IgG, anti-RHL-1 IgG, asialoorosomucoid, and asialofetuin comp etitively blocked the binding of I-125-Lf to isolated rat hepatocytes at 4 degrees C. Similarly, either excess anti-p45 or Lf blocked the bi nding of I-125-asialoorosomucoid to cells at 4 degrees C. We did not d etect the minor subunits of the rat asialoglycoprotein receptor (RHL-2 /3) in p45 preparations from Triton X-100 extracts of hepatocytes and I-125-Lf bound to purified RHL-1 but not to RHL-2/3 immobilized on nit rocellulose, Nonetheless, anti-RHL-2/3 IgG reduced the binding of I-12 5-Lf to hepatocytes at 4 degrees C. Exoglycosidases were used to remov e terminally-exposed N-acetylneuraminyl, alpha- and beta-galactosyl, a nd N-acetylhexosaminyl sugars from human and bovine Lf glycans, and le ctin blotting confirmed that glycosidase-treated Lfs lacked detectable terminal galactosyl sugars, Unexpectedly, these deglycosylated Lfs ex hibited no loss in their ability to compete with unmodified Lfs for bi nding to isolated hepatocytes. In addition, molar excess of beta-lacto se but not sucrose competitively blocked the binding of I-125-Lf to ce lls, indicating that Lf bound at or very near the carbohydrate-recogni tion domain of RHL-1. We conclude that RHL-1 is the Ca2+-dependent Lf receptor on hepatocytes and that it binds Lf at its carbohydrate-recog nition domain yet in a galactose-independent manner.