Identification and isolation of a 45-kDa calcium-dependent lactoferrin receptor from rat hepatocytes

Isolated rat hepatocyes bind, internalize, and degrade bovine lactoferrin (Lf) via high-affinity Ca2+-dependent sites [<10(6) sites/cell; McAbee et al., (1993) Biochemistry 32, 13749-13760]. In this study, we identified a 45-kDa Ca2+-dependent Lf binding protein on rat hepatocytes by three independent approaches. First, dithiobis(sulfosuccimidylproprionate) (DTSSP) cross-linked I-125-Lf to a 45-kDa adduct in a Ca2+-dependent manner on intact cells. The I-125-labeled cross-linked complexes were absent when either surface-bound I-125-Lf was stripped prior to cross-linking or an excess of unlabeled Lf was included in the DTSSP reaction. Second, I-125-Lf bound to a 45-kDa hepatocyte polypeptide in a Ca2+-dependent fashion following incubation with SDS-PAGE fractioned hepatocyte membrane proteins absorbed on nitrocellulose. Third, when Triton X-100 extracts of hepatocyte membrane ghosts were chromatographed on Lf-agarose, a 45-kDa polypeptide (p45) was eluted by EGTA. Column fractions enriched in p45-but not those depleted of p45-possessed soluble Lf receptor activity as determined by competition binding assay. Monospecific polyclonal anti-p45 IgG detected p45 in crude hepatocyte ghost homogenates and blocked vigorously I-125-Lf binding and endocytosis to intact rat hepatocytes. We conclude, therefore, that p45 constitutes the Ca2+-dependent Lf receptor on isolated rat hepatocytes.