Identification of glycoprotein 330 as an endocytic receptor for apolipoprotein J/clusterin.
Glycoprotein 330 (gp330) is a member of a family of endocytic receptors related to the low density lipoprotein receptor. gp330 has previously been shown to bind a number of ligands in common with its family member, the low density lipoprotein receptor-related protein (LRP). To identify ligands specific for gp330 and relevant to its localization on epithelia such as in the mammary gland, gp330-Sepharose affinity chromatography was performed. As a result, a 70-kDa protein was selected from human milk and identified by protein sequencing to be apolipoprotein J/clusterin (apoJ). Solid-phase binding assays confirmed that gp330 bound to apoJ with high affinity (Kd = 14.2 nM). Similarly, gp330 bound to apoJ transferred to nitrocellulose after SDS-polyacrylamide gel electrophoresis. LRP, however, showed no binding to apoJ in either type of assay. The binding of gp330 to apoJ could be competitively inhibited 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 and internalize 125I-apoJ. Only cells that expressed gp330 endocytosed and degraded radiolabeled apoJ. Furthermore, F9 cells treated with retinoic 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 be inhibited with unlabeled apoJ, receptor-associated protein, and gp330 antibodies. The results indicate that gp330 but not LRP can bind to apoJ in vitro and that gp330 expressed by cells can mediate apoJ endocytosis leading to lysosomal degradation.
Kounnas
M Z
MZ
J. H. Holland Laboratory, Biochemistry Department American Red Cross, Rockville, Maryland 20855, USA.
Loukinova
E B
EB
Stefansson
S
S
Harmony
J A
JA
Brewer
B H
BH
Strickland
D K
DK
Argraves
W S
WS
D000818:Animals; D053318:Apolipoprotein E3; D001057:Apolipoproteins E / Q000378:metabolism; D002846:Chromatography, Affinity; D051152:Clusterin; D004705:Endocytosis; D006023:Glycoproteins / Q000378:metabolism*; D026541:Heymann Nephritis Antigenic Complex; D006801:Humans; D008071:Lipoprotein Lipase / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000302:isolation & purification / Q000378:metabolism*; D008895:Milk, Human / Q000378:metabolism; D018832:Molecular Chaperones; D011485:Protein Binding; D011973:Receptors, LDL / Q000378:metabolism*; D013552:Swine
D053318:Apolipoprotein E3; D001057:Apolipoproteins E; C495242:CLU protein, human; D051152:Clusterin; D006023:Glycoproteins; D026541:Heymann Nephritis Antigenic Complex; D008562:Membrane Glycoproteins; D018832:Molecular Chaperones; D011973:Receptors, LDL; D008071:Lipoprotein Lipase