Molecular dissection of ligand binding sites on the low density lipoprotein receptor-related protein.

The low density lipoprotein receptor-related protein (LRP) is a large multifunctional receptor that is involved in the cellular uptake of a number of functionally diverse ligands including apoE-rich remnant lipoproteins, lipoprotein lipase, alpha 2-macroglobulin-protease complexes, plasminogen activator-inhibitor complexes, and the active protease tissue-type plasminogen activator. Ligand binding and competition experiments suggest that most LRP ligands bind to specific, independent sites on the large 515-kDa subunit of the receptor. In a previous study (Moestrup, S.K., Holtet, T.L., Etzerodt, M., Thøgersen, H.C., Nykjaer, A., Andreasen, P.A., Rasmussen, H.H., Sottrup-Jensen, L., and Gliemann, J. (1993) J. Biol. Chem. 268, 13691-13696), ligand blotting was used to localize the binding sites for urokinase-type plasminogen activator-plasminogen activator inhibitor-1 (PAI-1) complexes and for alpha 1-macroglobulin to a proteolytic fragment of LRP containing the second cluster of complement-type cysteine-rich repeats. Here, we have used a recombinant DNA approach to express functionally restricted chimeric "LRP-minireceptors" containing two different regions of the extracellular domain of the receptor in cultured cells. Receptor-associated protein, a negative modulator of LRP activity, is bound and internalized by cells transfected with either construct. A minireceptor containing the cluster of eight complement-type cysteine-rich repeats followed by four epidermal growth factor precursor homologous domains binds and internalizes 125I-labeled plasminogen activator-PAI-1 complexes. It also mediates the cellular uptake of the uncomplexed protease tissue-type plasminogen activator (tPA), suggesting that the tPA and PAI-1 binding sites on LRP are in close vicinity and might promote cooperative binding of tPA-PAI-1 complexes. However, alpha 2-macroglobulin is not internalized by this minireceptor suggesting that this ligand requires the presence of a single epidermal growth factor-repeat which is contained in the previously studied proteolytic fragment but is absent from the minireceptor.