The adaptor protein beta-arrestin2 enhances endocytosis of the low density lipoprotein receptor.
Endocytosis of the low density lipoprotein (LDL) receptor (LDLR) in coated pits employs the clathrin adaptor protein ARH. Similarly, agonist-dependent endocytosis of heptahelical receptors in coated pits employs the clathrin adaptor beta-arrestin proteins. In mice fed a high fat diet, we found that homozygous deficiency of beta-arrestin2 increased total and LDL plus intermediate-density lipoprotein cholesterol levels by 23 and 53%, respectively (p < 0.05), but had no effect on high density lipoprotein cholesterol levels. We therefore tested whether beta-arrestins could affect the constitutive endocytosis of the LDLR. When overexpressed in cells, beta-arrestin1 and beta-arrestin2 each associated with the LDLR, as judged by co-immunoprecipitation, and augmented LDLR endocytosis by approximately 70%, as judged by uptake of fluorescent LDL. However, physiologic expression levels of only beta-arrestin2, and not beta-arrestin1, enhanced endogenous LDLR endocytosis (by 65%) in stably transfected beta-arrestin1/beta-arrestin2 double-knockout mouse embryonic fibroblasts (MEFs). Concordantly, when RNA interference was used to suppress expression of beta-arrestin2, but not beta-arrestin1, LDLR endocytosis was reduced. Moreover, beta-arrestin2-/- MEFs demonstrated LDLR endocytosis that was 50% less than cognate wild type MEFs. In fusion protein pull-down assays, beta-arrestin2 bound to the LDLR cytoplasmic tail stoichiometrically, and binding was abolished by mutation of LDLR Tyr807 to Ala. Mutation of LDLR cytoplasmic tail Ser833 to Asp enhanced both the affinity of LDLR fusion protein binding to beta-arrestin2, and the efficiency of LDLR endocytosis in cells expressing beta-arrestin2 physiologically. We conclude that beta-arrestin2 can bind to and enhance endocytosis of the LDLR, both in vitro and in vivo, and may thereby influence lipoprotein metabolism.
Wu
Jiao-Hui
JH
Department of Medicine (Cardiology), Duke University Medical Center, Durham, North Carolina 27710, USA.
Peppel
Karsten
K
Nelson
Christopher D
CD
Lin
Fang-Tsyr
FT
Kohout
Trudy A
TA
Miller
William E
WE
Exum
Sabrina T
ST
Freedman
Neil J
NJ
D000818:Animals; D019390:Arrestins / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D016466:CHO Cells; D002460:Cell Line; D002784:Cholesterol / Q000097:blood; D008076:Cholesterol, HDL / Q000097:blood; D008078:Cholesterol, LDL / Q000097:blood; D003001:Cloning, Molecular; D006224:Cricetinae; D004041:Dietary Fats / Q000008:administration & dosage; D004622:Embryo, Mammalian; D004705:Endocytosis; D005347:Fibroblasts; D015870:Gene Expression; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D007163:Immunosorbent Techniques; D007668:Kidney; D008074:Lipoproteins / Q000097:blood; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D016296:Mutagenesis; D011973:Receptors, LDL / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D014162:Transfection; D000071557:beta-Arrestins
D019390:Arrestins; D008076:Cholesterol, HDL; D008078:Cholesterol, LDL; D004041:Dietary Fats; D008074:Lipoproteins; D011973:Receptors, LDL; D011993:Recombinant Fusion Proteins; D000071557:beta-Arrestins; C030104:lipoprotein cholesterol; D002784:Cholesterol; D005982:Glutathione Transferase