LDL receptor related protein (LRP) is a ubiquitously expressed cell surface receptor that binds, at least in vitro, a plethora of ligands among them c~2-macroglobulin and lactoferrin (Lf). The function of LRP in internalisation and distribution of ligands within cellular metabolism is still unclear. We here investigated by combined ligand-and immunoblotting the participation of LRP/a2MR and its associated protein (RAP) in receptor mediated endocytosis of Lf into rat liver. We found LRP highly enriched in sucrose density gradient fractions around density I.I0 g/ml, previously characterised as endosomal fractions. RAP was concentrated in distinct fractions around density 1.14 g/ml. This separation of RAP from LRPIa2MR is physiologically meaningful as RAP avidly binds to LRPIa2MR and by that shuts off aH ligand binding function. In endosomal fractions we found one single binding protein for ~2Sl-labelled Lf. With a specific anti LRPla2MR antibody and ligand blotting with 125I-labelled RAP this endosomal Lf binding site was verified to be LRP/azMR. Endosomes did not bind labelled Lf when prepared from rats that received an intravenous injection of Lf (20 mg per animal) 20 rain prior to preparation. Surprisingly we immunodetected Lf in these endosomes at a position around 600 kDa, comigrating with LRP/ c~2MR. We determined Lf binding to be optimal at pH 5.8, what led us to suggest the existence of a very stable LF-LRP/c~2MR complex in endosomes. These data support the idea of effective binding of Lf at pH as found in inflamed tissue environment where Lf is reported to be involved in leukocyte mediated inflammation regulation.

Receptor-associated protein (RAP) was originally described as a 39-kDa intracellular protein copurifying with mammalian low density lipoprotein (LDL) receptor-related protein/alpha 2-macroglobulin receptor (LRP/alpha 2MR). RAP has a high affinity for LRP/alpha 2MR and interferes with the receptor's ability to bind a variety of ligands. The laying hen expresses, in a tissue-specific manner, at least four different proteins which belong to the same family of receptors as LRP/alpha 2MR. Here we show that the chicken also produces RAP, so far thought to be expressed only in mammals. Studies on the interaction of recombinant human RAP with the LDL receptor family in the chicken revealed that RAP binds with high affinity to the abundant oocyte receptor for yolk precursors (OVR) as well as to the somatic cell-specific LRP/alpha 2MR. Significantly, RAP interacts with a lower affinity with the LDL receptor, but does not bind to the oocyte-specific form of LRP. Binding of RAP to OVR inhibits the interaction of the receptor with all known physiological ligands, i.e. the yolk precursors very low density lipoprotein, vitellogenin, and alpha 2-macroglobulin. In COS cells transfected with OVR, RAP is internalized and degraded in a concentration-dependent and saturable manner. Lactoferrin, another protein with a high affinity for mammalian LRP/alpha 2MR, also binds to OVR and abolishes its interaction with yolk precursors. Cross-competition experiments show that RAP and lactoferrin recognize sites different from those involved in yolk precursor binding. The availability of pure OVR and LDLR enable us to determine kinetic parameters for the binding of RAP and lactoferrin to these receptors by surface plasmon resonance. Taken together, our results strongly suggest that chicken OVR, which is easily accessible and highly abundant in growing oocytes, represents a superior system for studying mechanistic and structural aspects of the interaction of ligands and modulating proteins with members of the LDL receptor gene family.

Complement component 3 (C3) and ␣ 2 -macroglobulin evolved from a common, evolutionarily old, ancestor gene. Low density lipoprotein-receptor-related protein/ ␣ 2 -macroglobulin receptor (LRP/␣ 2 MR), a member of the low density lipoprotein receptor family, is responsible for the clearance of ␣ 2 -macroglobulin-protease complexes. In this study, we examined whether C3 has conserved affinity for LRP/␣ 2 MR. Ligand blot experiments with human 125 I-C3 on endosomal proteins show binding to a 600-kDa protein, indistinguishable from LRP/␣ 2 MR by the following criteria: it is competed by receptorassociated protein (the 39-kDa receptor-associated protein that impairs binding of all ligands to LRP/␣ 2 MR) and by lactoferrin and Pseudomonas exotoxin, other well known ligands of the multifunctional receptor. Binding of C3 is sensitive to reduction of the receptor and is Ca 2؉ -dependent. All these features are typical for cysteine-rich binding repeats of the low density lipoprotein receptor family. In LRP/␣ 2 MR, they are found in four cassettes (2, 8, 10, and 11 repeats). Ligand blotting to chicken LR8 demonstrates that a single 8-fold repeat is sufficient for binding. Confocal microscopy visualizes initial surface labeling of human fibroblasts incubated with fluorescent labeled C3, which changes after 5 min to an intracellular vesicular staining pattern that is abolished in the presence of receptor-associated protein. Cell uptake is abolished in mouse fibroblasts deficient in LRP/␣ 2 MR. Native plasma C3 is not internalized. We demonstrate that the capacity to internalize C3 is saturable and exhibits a K D value of 17 nM. After intravenous injection, rat hepatocytes accumulate C3 in sedimentable vesicles with a density typical for endosomes. In conclusion, our ligand blot and uptake studies demonstrate the competence of the LRP/␣ 2 MR to bind and endocytose C3 and provide evidence for an LRP/␣ 2 MRmediated system participating in C3 metabolism. LRP/␣ 2 MR1 is a member of the low density lipoprotein (LDL) receptor family, which includes the LDL receptor, LRP/␣ 2 MR, megalin (gp330), the very low density lipoprotein receptor, apolipoprotein E receptor 2, and LR8B (1). A gene closely related to that of LRP/␣ 2 MR was identified in Caenorhabditis elegans, showing that LRP/␣ 2 MR is an evolutionarily old molecule (2). LRP/␣ 2 MR was discovered by its homology to structures of complement components (3), and it was suggested that it might function together with the LDL receptor as receptor for apoE-containing lipoproteins executing the mass transport of chylomicron remnants (4). Later, LRP/␣ 2 MR was shown to be identical with the ␣ 2 MR (5, 6). In recent years, a plethora of new ligands were reported, and LRP/␣ 2 MR was classified as a multifunctional receptor (1, 7). Members of the LDL receptor family all consist of the same basic structural components: (i) a class of cysteine-rich repeats of approximately 40 amino acids, which are also present in the terminal complement components and are therefore referred to as complemen...

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