XB-ART-4776J Biol Chem November 7, 2003; 278 (45): 44584-92.
Xenopus autosomal recessive hypercholesterolemia protein couples lipoprotein receptors with the AP-2 complex in oocytes and embryos and is required for vitellogenesis.
ARH is required for normal endocytosis of the low density lipoprotein (LDL) receptor in liver and mutations within this gene cause autosomal recessive hypercholesterolemia in humans. xARH is a localized maternal RNA in Xenopus with an unknown function in oogenesis and embryogenesis. Like ARH, xARH contains a highly conserved phosphotyrosine binding domain and a clathrin box. To address the function of xARH, we examined its expression pattern in development and used pull-down experiments to assess interactions between xARH, lipoprotein receptors and proteins in embryo extracts. xARH was detected concentrated at the cell periphery as well as in the perinuclear region of oocytes and embryos. In pull-down experiments, the xARH phosphotyrosine binding domain interacted with the LDL and vitellogenin receptors found in Xenopus oocytes and embryos. Mutations within the receptor internalization signal specifically abolished this interaction. The xARH C-terminal region pulled-down several proteins from embryo extracts including alpha- and beta-adaptins, subunits of the AP-2 endocytic complex. Mutations within either of the two Dvarphi(F/W) motifs found in xARH abolished binding to alpha- and beta-adaptins. Expression of a dominant negative mutant of xARH missing the clathrin box and one functional Dvarphi(F/W) motif severely inhibited endocytosis of vitellogenin in cultured oocytes. The data indicate that xARH acts as an adaptor protein linking LDL and vitellogenin receptors directly with the AP-2 complex. In oocytes, we propose that xARH mediates the uptake of lipoproteins from the blood for storage in endosomes and later use in the embryo. Our findings point to an evolutionarily conserved function for ARH in lipoprotein uptake.
PubMed ID: 12944396
Article link: J Biol Chem
Genes referenced: ap2a2 ap2b1 cltc ldlr ldlrap1 myc odc1 ptbp1 tfap2a
Antibodies: A2b1 Ab1 Ap2a2 Ab1 Ldlrap1 Ab1
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|FIG. 1. Sequence alignment of ARH homologues from different species. Conserved residues are shaded using the Boxshade program. The PTB domain is underlined, the DφF/W motifs are boxed, and the putative clathrin box is boxed with broken lines. The phenylalanine residue required for the interaction between hARH and LDLR is marked with an arrow. The complete ascidian sequence is not available. The GenBank™ accession numbers for the ARH homologues are as follows: zebrafish, BC045926; ascidian, AV384534; mosquito, AJ281467.|
|FIG. 2. xARH expression in oocytes and embryos. Affinity-purified mouse polyclonal anti-xARH-C antibody was used for whole-mount immunostaining of oocytes and embryos. No signal was detected in oocytes or embryos stained with antibody blocked with the purified antigen GST-C. A, in oocytes, ARH appears to be concentrated peripherally. B, in bisected stage V oocytes, ARH is perinuclear (arrow), and in bisected eggs after germinal vesicle breakdown, the signal is mostly in the animal hemisphere (arrow). N, nucleus. C, xARH protein is found broadly distributed in early stage embryos. D, in sectioned blastula stage embryos, ARH is found in the perinuclear (arrows) as well as the cortical region (arrows). Black pigment granules (open arrow) are distinct from the brown ARH-positive staining. Bar = 1 mm.|
|FIG. 3. xARH interacts with the LDL and VTG receptors. A, total RNA was isolated from embryos at different stages and xARH expression analyzed by reverse transcriptase-PCR. Ornithine decarboxylase (ODC) served as the loading control. Reaction without reverse transcriptase added (–RT) was used as the negative control. B, GST pull-down assays showing interaction between xARH and the LDL and VTG receptors. In vitro translated and 35S-Met labeled xARH-N terminus (residues 1–212) or VTGR was mixed with purified GST-LDLR or GST-xARH. The GST fusion proteins and bound proteins were pulled down with glutathione-Sepharose beads and analyzed by SDS-PAGE. The GST fusion proteins were visualized by Coomassie Blue staining. The in vitro translated proteins were detected by exposure to a PhosphorImager screen. C, the cytoplasmic tails of the LDLR and VTGR were translated in vitro and labeled by 35S-Met incorporation. In mutants (mt), the NPVY motifs were changed to NPVA. Purified GST-xARH fusion protein from E. coli was mixed with the cytoplasmic tails and glutathione-Sepharose beads. The beads were spun down and boiled in SDS loading buffer (pellet). Both the supernatant (S) and pellet (P) fractions were resolved by SDS-PAGE. The cytoplasmic tails were visualized by exposure to a PhosphorImager screen. The GST-xARH protein was detected by Coomassie Blue staining.|
|FIG. 4. Identification of proteins binding to xARH-α C terminus. A, diagram showing the arrangement of known functional regions within xARH. The clathrin box is striped, and the DφF/W motifs are shown as three solid bars in the C-terminal region. Proteins from embryo extracts that bound to the C-terminal region of xARH were affinity-purified with GST-C and resolved by SDS-PAGE and silver staining. Five proteins with molecular masses of 119, 108, 53, 48, and 35 kDa were pulled down by GST-C but not by GST alone. B, the 108-kDa band was excised from the gel and identified by quadrupole time-of-flight mass spectrometry. One spectrum matched a peptide sequence in α-adaptin. The only nonmatching residue of 13 is underlined.|
|FIG. 5. xARH interacts with both α- and β-adaptin. XARH-binding proteins were pulled down with GST-C. Western blot analysis was performed using either monoclonal anti-α or -β adaptin antibody (Affinity BioReagents, Inc. and BD Bioscience). Both α- and β-adaptin (arrowheads) can be pulled down by GST-C but not by GST alone. The lower molecular weight mass are most likely β-adaptin degradation products.|
|FIG. 6. Dφ(F/W) motifs are required for the interaction between xARH and the AP-2 complex. A, diagram of xARH mutants fused to GST used in the analysis of the Dφ(F/W) motifs. The Dφ(F/W) motifs at positions 235, 258, and 305, respectively, were individually mutated to AAA. B, fusion proteins were used in pull-down assays to test for the binding of β2-adaptin to ARH and were analyzed subsequently by Western blotting. The nitrocellulose membrane was stained with Ponceau S to visualize the GST fusion proteins and to confirm equal loading. GST and GST-C served as the negative and positive controls, respectively.|
|FIG. 7. Expression of xARH dominant negative mutant (xARH-DM) in cultured oocytes inhibited vitellogenin endocytosis. A, schematic diagram of xARH-DM showing mutations in the clathrin box (LLDLE) and the 258 Dφ(F/W) motif. B, stage III oocytes were incubated in culture medium containing biotinylated VTG for various periods of time. Surface-bound VTG was removed by washing with a high pH buffer. The internalized biotinylated VTG was detected using streptavidin-HRP conjugate. The nitrocellulose membrane was stained with Ponceau S to visualize the lipovitellin band used as a loading control. Biotin-VTG was progressively internalized over the culture period. C and D, after the injection of 5 ng of xARH-DM or control galactosidase (Gal) transcripts, oocytes were cultured overnight. The expression of Myc-tagged xARH-DM protein was confirmed by Western blot analysis (C), and VTG uptake was measured (D) as described in B. xARH-DM severely inhibited VTG uptake compared with the galactosidase control.|
|FIG. 8. Models favored for xARH function in LDL receptor family mediated endocytosis in oocytes and embryos. Lipoprotein receptor and ligand are internalized through clathrin-mediated endocytosis. The receptor and ligand dissociate in the endosome, and yolk protein is stored there (Yolk Platelet). The receptor is recycled back to the plasma membrane. Later in development, the ligand (vitellogenin, lipoprotein) is degraded and used as a source of nutrients by the embryos (not shown). The xARH-N-terminal PTB domain interacts with the internalization motif of the receptor directly or indirectly through interaction with unknown protein (X). For the LDL receptor, this motif is NPVY and is likely YXXφ for the VTG receptor. The C-terminal region of xARH has multiple motifs to associate with the endocytic machinery, AP-2 complex, and clathrin.|