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.
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.
Sirinian
Maria Isabella
MI
Department of Clinical and Applied Medical Therapy, University of Rome La Sapienza, Italy.
Belleudi
Francesca
F
Campagna
Filomena
F
Ceridono
Mara
M
Garofalo
Tina
T
Quagliarini
Fabiana
F
Verna
Roberto
R
Calandra
Sebastiano
S
Bertolini
Stefano
S
Sorice
Maurizio
M
Torrisi
Maria Rosaria
MR
Arca
Marcello
M
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
D048868:Adaptor Proteins, Signal Transducing; D002966:Clathrin; C469745:LDLRAP1 protein, human; D008077:Lipoproteins, LDL; D034741:RNA, Small Interfering; D011973:Receptors, LDL; D013395:Sucrose