DIFFERENTIAL FUNCTIONS OF TRIPLICATED REPEATS SUGGEST 2 INDEPENDENT ROLES FOR THE RECEPTOR-ASSOCIATED PROTEIN AS A MOLECULAR CHAPERONE

The 39-kDa receptor-associated protein (RAP) is a molecular chaperone for the low density lipoprotein receptor-related protein (LRP), a larg e endocytic receptor that binds multiple ligands, The primary function of RAP has been defined as promotion of the correct folding of LRP, a nd prevention of premature interaction of ligands with LRP within the early secretory pathway, Previous examination of the RAP sequence reve aled an internal triplication, However, the functional implication of the triplicated repeats was unknown, In the current study using variou s RAP and LRP domain constructs, we found that the carboxyl-terminal r epeat of RAP possesses high affinities to each of the three ligand-bin ding domains on LRP, whereas the amino-terminal and central repeats of RGP exhibit only low affinity to the second and the fourth ligand-bin ding domains of LRP, respectively, Using truncated soluble minirecepto rs of LRP, we identified five independent RAP-binding sites, two on ea ch of the second and fourth, and one on the third ligand-binding domai n of LRP. By coexpressing soluble LRP minireceptors and RAP repeat con structs, we found that only the carboxyl-terminal repeat of RAP was ab le to promote the folding and subsequent secretion of the soluble LRP minireceptors However, when the ability of each RAP repeat to inhibit Ligand interactions with LRP was examined, differential effects were o bserved for individual LRP ligands, Most striking, both the amino-term inal and central repeats, but not the carboxyl-terminal repeat, of RAP inhibited the interaction of alpha(2)-macroglobulin with LRP. These d ifferential functions of the RAP repeats suggest that the roles of RAP in the folding of LRP and in the prevention of premature interaction of ligand with the receptor are independent.