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J. Biol. Chem., Vol. 280, Issue 46, 38416-38423, November 18, 2005

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Adaptor Protein ARH Is Recruited to the Plasma Membrane by Low Density Lipoprotein (LDL) Binding and Modulates Endocytosis of the LDL/LDL Receptor Complex in Hepatocytes^*

Maria Isabella Sirinian, Francesca Belleudi¶, Filomena Campagna, Mara Ceridono, Tina Garofalo, Fabiana Quagliarini, Roberto Verna, Sebastiano Calandra||, Stefano Bertolini**, Maurizio Sorice, Maria Rosaria Torrisi¶, and Marcello Arca1

From the Departments of Clinical and Applied Medical Therapy and Experimental Medicine and Pathology and ^¶Azienda Ospedaliera Sant'Andrea, University of Rome "La Sapienza," 00161 Rome, Italy, ^||Department of Biomedical Sciences, University of Modena and Reggio Emilia, 41100 Modena, Italy, and ^**Department of Internal Medicine, University of Genoa, 16132 Genoa, Italy

ARH is a newly discovered adaptor protein required for the efficient activity of low density lipoprotein receptor (LDLR) in selected tissues. Individuals lacking ARH have severe hypercholesterolemia due to an impaired hepatic clearance of LDL. It has been demonstrated that ARH is required for the efficient internalization of the LDL-LDLR complex and to stabilize the association of the receptor with LDL in Epstein-Barr virus-immortalized B lymphocytes. However, little information is available on the role of ARH in liver cells. Here we provide evidence that ARH is codistributed with LDLR on the basolateral area in confluent HepG2-polarized cells. This distribution is not modified by the overexpression of LDLR. Conversely, the activation of the LDLR-mediated endocytosis, but not the binding of LDL to LDLR, promotes a significant colocalization of ARH with LDL-LDLR complex that peaked at 2 min at 37 °C. To further assess the role of ARH in LDL-LDLR complex internalization, we depleted ARH protein using the RNA interference technique. Twenty-four hours after transfection with ARH-specific RNA interference, ARH protein was depleted in HepG2 cells by more than 70%. Quantitative immunofluorescence analysis revealed that the depletion of ARH caused about 80% reduction in LDL internalization. Moreover, our findings indicate that ARH is associated with other proteins of the endocytic machinery. We suggest that ARH is an endocytic sorting adaptor that actively participates in the internalization of the LDL-LDLR complex, possibly enhancing the efficiency of its packaging into the endocytic vesicles.

Received for publication, April 20, 2005 , and in revised form, August 19, 2005.

^* This work was supported by Telethon Grant GGP02149 and an Ateneo 2004 grant (to M. A.) and by grants from Ministero dell' Instruzione, Università e Ricera, from the Ministero della Salute, and from the Associazione Italiana per la Ricerca sul Cancro, Italy (to M. R. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dipartimento di Clinica e Terapia Medica Applicata, Università di Roma "La Sapienza" Policlinico Umberto I, Viale del Policlinico 155, 00161 Rome, Italy. Tel.: 39-06-4450074; Fax: 39-06-4440290; E-mail: marcelloarca{at}libero.it.

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