title: 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.
abstract: 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 degrees 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.
authors.affiliation: Department of Clinical and Applied Medical Therapy, University of Rome La Sapienza, Italy.
authors.forename: Maria Isabella
authors.initials: MI
authors.lastname: Sirinian
authors.forename: Francesca
authors.initials: F
authors.lastname: Belleudi
authors.forename: Filomena
authors.initials: F
authors.lastname: Campagna
authors.forename: Mara
authors.initials: M
authors.lastname: Ceridono
authors.forename: Tina
authors.initials: T
authors.lastname: Garofalo
authors.forename: Fabiana
authors.initials: F
authors.lastname: Quagliarini
authors.forename: Roberto
authors.initials: R
authors.lastname: Verna
authors.forename: Sebastiano
authors.initials: S
authors.lastname: Calandra
authors.forename: Stefano
authors.initials: S
authors.lastname: Bertolini
authors.forename: Maurizio
authors.initials: M
authors.lastname: Sorice
authors.forename: Maria Rosaria
authors.initials: MR
authors.lastname: Torrisi
authors.forename: Marcello
authors.initials: M
authors.lastname: Arca
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism / Q000502:physiology*; D001402:B-Lymphocytes / Q000821:virology; D001483:Base Sequence; D001671:Biochemistry / Q000379:methods; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002499:Centrifugation, Density Gradient; D002966:Clathrin / Q000737:chemistry / Q000378:metabolism; D004705:Endocytosis; D020868:Gene Silencing; D022781:Hepatocytes / Q000378:metabolism* / Q000821:virology; D004854:Herpesvirus 4, Human / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D008077:Lipoproteins, LDL / Q000737:chemistry / Q000378:metabolism*; D008099:Liver / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D011485:Protein Binding; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D011973:Receptors, LDL / Q000378:metabolism; D013395:Sucrose / Q000494:pharmacology; D013696:Temperature; D013997:Time Factors
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D002966:Clathrin; C469745:LDLRAP1 protein, human; D008077:Lipoproteins, LDL; D034741:RNA, Small Interfering; D011973:Receptors, LDL; D013395:Sucrose