Identification of Glycoprotein 330 as an Endocytic Receptor for Apolipoprotein J/Clusterin

doi:10.1074/jbc.270.22.13070

Identification of Glycoprotein 330 as an Endocytic Receptor for Apolipoprotein J/Clusterin (*)

From the (1) J. H. Holland Laboratory, Biochemistry Department American Red Cross, Rockville, Maryland 20855, the
(2) Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0575, and the
(3) Molecular Diseases Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892

** To whom correspondence should be addressed. Tel.: 301-738-0725; Fax: 301-738-0794; E-mail: argraves{at}hlsun.red-cross.org.

Abstract

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 (K= 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 I-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.

Footnotes

↵^§ Recipient of Individual National Research Service Award HL08744 from NHLBI, National Institutes of Health.
↵^¶ Recipient of a Merit Award from NHLBI, National Institutes of Health.
↵* This work was supported in part by National Institutes of Health Grants DK45598 (to W. S. A.) and GM42581 (to D. K. S.) and the Program of Excellence in Molecular Biology of the Heart and Lung HL41496 (to J. A. K. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

gp330

glycoprotein 330

LDLR

low density lipoprotein receptor

LRP

low density lipoprotein receptor-related protein

RAP

receptor-associated protein

apoJ

apolipoprotein J

apoE

apolipoprotein E

uPA

two chain urokinase

pro-uPA

prourokinase

PAI-1

plasminogen activator inhibitor-1

DBC

dibutyryl cAMP

TBS

Tris-buffered saline

tPA

tissue-type plasminogen activator

ELISA

enzyme-linked immunosorbent assay

RA

retinoic acid

PAGE

polyacrylamide gel electrophoresis.
↵²Apolipoprotein J is synonymous with clusterin, sulfated glycoprotein-2 (SPG-2), complement lysis inhibitor (CLI), testosterone-repressed prostate message-2 (TRPM-2), Gp80 and SP-40-40.
↵³S. Stefansson, D. A. Chappell, K. M. Argraves, and W. S. Argraves, manuscript in preparation.
↵⁴D. Swertfeger, D. Witte, and J. A. K. Harmony, manuscript in preparation.