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 lip oproteins, lipoprotein lipase, alpha(2)-macroglobulin-protease complex es, plasminogen activator-inhibitor complexes, and the active protease tissue-type plasminogen activator. Ligand binding and competition exp eriments suggest that most LRP ligands bind to specific, independent s ites on the large 515-kDa subunit of the receptor. In a previous study (Moestrup, S. K., Holtet, T. L., Etzerodt, M., Thogersen, H. C., Nykj aer, A., Andreasen, P. A., Rasmussen, H. H., Sottrup-Jensen, L., and G liemann, J. (1993) J. Biol. Chem. 268, 13691-13696), ligand blotting w as used to localize the binding sites for urokinase-type plasminogen a ctivator-plasminogen activator inhibitor-1 (PAI-1) complexes and for a lpha(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 ch imeric ''LRP-minireceptors'' containing two different regions of the e xtracellular domain of the receptor in cultured cells. Receptor-associ ated protein, a negative modulator of LRP activity, is bound and inter nalized by cells transfected with either construct. A minireceptor con taining the cluster of eight complement-type cysteine-rich repeats fol lowed by four epidermal growth factor precursor homologous domains bin ds and internalizes I-125-labeled plasminogen activator-PAI-1 complexe s. It also mediates the cellular uptake of the uncomplexed protease ti ssue-type plasminogen activator (tPA), suggesting that the tPA and PAI -1 binding sites on LRP are in close vicinity and might promote cooper ative binding of tPA PAI-1 complexes. However, alpha(2)-macroglobulin is not internalized by this minireceptor suggesting that this ligand r equires the presence of a single epidermal growth factor-repeat which is contained in the previously studied proteolytic fragment but is abs ent from the minireceptor.