IDENTIFICATION OF GLYCOPROTEIN-330 AS AN ENDOCYTIC RECEPTOR FOR APOLIPOPROTEIN-J CLUSTERIN/

Glycoprotein 330 (gp330) is a member of a family of endocytic receptor s related to the low density lipoprotein receptor. gp330 has previousl y been shown to bind a number of ligands in common with its family mem ber, the low density Lipoprotein receptor-related protein (LRP). To id entify ligands specific for gp330 and relevant to its localization on epithelia such as in the mammary gland, gp330-Sepharose affinity chrom atography was performed. As a result, a 70-kDa protein was selected fr om human milk and identified by protein sequencing to be apolipoprotei n J/clusterin (apoJ). Solid-phase binding assays confirmed that gp330 bound to aporJ with high affinity (K-d = 14.2 nM). Similarly, gp330 bo und to apoJ transferred to nitrocellulose after SDS-polyacrylamide gel electrophoresis. LRP, however, showed no binding to apoJ in either ty pe of assay. The binding of gp330 to apoJ could be competitively inhib ited with excess apoJ as well as with the gp330 ligands apolipoprotein E, lipoprotein Lipase, and the receptor-associated protein, a 39-kDa protein that acts to antagonize binding of all known ligands for gp330 and LRP. Several cultured cell Lines that express gp330 and ones that do not express the receptor were examined for their ability to bind a nd internalize I-125-apoJ. Only cells that expressed gp330 endocytosed and degraded radiolabeled apoJ. Furthermore, F9 cells treated with re tinoic acid and dibutyryl cyclic AMP to increase expression levels of gp330 displayed an increased capacity to internalize and degrade apoJ. Cellular internalization and degradation of radiolabeled apoJ could b e inhibited with unlabeled apoJ, receptor-associated protein, and gp33 0 antibodies. The results indicate that gp330 but not LRP can bind to apoJ in vitro and that gp330 expressed by cells can mediate apoJ endoc ytosis leading to lysosomal degradation.