Xenopus Autosomal Recessive Hypercholesterolemia Protein Couples Lipoprotein Receptors with the AP-2 Complex in Oocytes and Embryos and Is Required for Vitellogenesis
2003; Elsevier BV; Volume: 278; Issue: 45 Linguagem: Inglês
10.1074/jbc.m308870200
ISSN1083-351X
AutoresYi Zhou, Jian Zhang, Mary Lou King,
Tópico(s)Cancer, Lipids, and Metabolism
ResumoARH 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 α- and β-adaptins, subunits of the AP-2 endocytic complex. Mutations within either of the two Dφ(F/W) motifs found in xARH abolished binding to α- and β-adaptins. Expression of a dominant negative mutant of xARH missing the clathrin box and one functional Dφ(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. 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 α- and β-adaptins, subunits of the AP-2 endocytic complex. Mutations within either of the two Dφ(F/W) motifs found in xARH abolished binding to α- and β-adaptins. Expression of a dominant negative mutant of xARH missing the clathrin box and one functional Dφ(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. In Xenopus, at least 8 months are required for stage I oocytes to develop into fully grown stage VI oocytes. One purpose of this lengthy process is to stockpile components required for early embryo development (1Dumont J.N. J. Morphol. 1972; 136: 153-179Crossref PubMed Scopus (1415) Google Scholar). Large amounts of macromolecules and nutrients in the form of serum-borne lipoproteins accumulate and the oocyte volume increases 10,000 times as a consequence. These lipoproteins are essential for the assembly of structures such as the plasma membrane in embryos. The major imported lipoproteins are very low density lipoproteins (VLDL) 1The abbreviations used are: VLDL, very low density lipoproteins; LDL, low density lipoprotein; ARH, autosomal recessive hypercholesterolemia; GST, glutathione S-transferase; HRP, horseradish peroxidase; LDLR, LDL receptor; LRP, LDL-receptor-related protein; VTG, vitellogenin; VTGR, VTG receptor; PBS, phosphate-buffered saline; PTB, phosphotyrosine binding; AP-2, adaptor protein complex 2; MOPS, 4-morpholinepropanesulfonic acid.1The abbreviations used are: VLDL, very low density lipoproteins; LDL, low density lipoprotein; ARH, autosomal recessive hypercholesterolemia; GST, glutathione S-transferase; HRP, horseradish peroxidase; LDLR, LDL receptor; LRP, LDL-receptor-related protein; VTG, vitellogenin; VTGR, VTG receptor; PBS, phosphate-buffered saline; PTB, phosphotyrosine binding; AP-2, adaptor protein complex 2; MOPS, 4-morpholinepropanesulfonic acid. and vitellogenin (VTG). Vitellogenin is a lipophosphoglycoprotein produced under hormonal control in the liver and transported to the female gonads in the blood (2Clemens M.J. Lofthouse R. Tata J.R. J. Biol. Chem. 1975; 250: 2213-2218Abstract Full Text PDF PubMed Google Scholar). Once internalized by receptor-mediated endocytosis, vitellogenin (∼250 kDa) is cleaved into multiple polypeptides and then stored as microcrystals within membrane-bound structures called yolk platelets (3Berridge M.V. Lane C.D. Cell. 1976; 8: 283-297Abstract Full Text PDF PubMed Scopus (48) Google Scholar, 4Opresko L.K. Methods Cell Biol. 1991; 36: 117-132Crossref PubMed Scopus (13) Google Scholar). Yolk platelets are thought to be the oocyte equivalent of late endosomes or lysosomes but with low proteolytic activity (5Dumont J.N. J. Exp. Zool. 1978; 204: 193-217Crossref PubMed Scopus (47) Google Scholar). During embryogenesis, yolk platelets become progressively more acidic (pH < 5), allowing yolk proteins to be degraded and utilized as nutrients for early development (6Fagotto F. Maxfield F.R. J. Cell Sci. 1994; 107: 3325-3337PubMed Google Scholar). VTG is transported into oocytes through a specific receptor (VTGR) that is internalized in clathrin-coated pits (7Richter H.P. Bauer A. Eur. J. Cell Biol. 1990; 51: 53-63PubMed Google Scholar). A mutant chicken strain carrying a single mutation in the VTGR gene cannot lay eggs (8Nimpf J. Radosavljevic M.J. Schneider W.J. J. Biol. Chem. 1989; 264: 1393-1398Abstract Full Text PDF PubMed Google Scholar). The oocytes of this mutant strain lose the ability to accumulate VTG and VLDL. In addition to VTG and VLDL, the VTG receptor also transports other molecules including riboflavin binding protein (9MacLachlan I. Nimpf J. Schneider W.J. J. Biol. Chem. 1994; 269: 24127-24132Abstract Full Text PDF PubMed Google Scholar), α2-macroglobulin (10Jacobsen L. Hermann M. Vieira P.M. Schneider W.J. Nimpf J. J. Biol. Chem. 1995; 270: 6468-6475Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), and activin (11Uchiyama H. Nakamura T. Komazaki S. Takio K. Asashima M. Sugino H. Biochem. Biophys. Res. Commun. 1994; 202: 484-489Crossref PubMed Scopus (27) Google Scholar), suggesting that this receptor is important not only in nutrient uptake during oogenesis but also in the development of early embryos. For example, the mammalian VLDL receptor, closely related to the VTG receptor, is involved in brain development in mouse embryos (12Trommsdorff M. Gotthardt M. Hiesberger T. Shelton J. Stockinger W. Nimpf J. Hammer R.E. Richardson J.A. Herz J. Cell. 1999; 97: 689-701Abstract Full Text Full Text PDF PubMed Scopus (1066) Google Scholar). To date, all molecularly characterized VTG receptors belong to the LDL receptor family (13Cheon H.M. Seo S.J. Sun J. Sappington T.W. Raikhel A.S. Insect Biochem. Mol. Biol. 2001; 31: 753-760Crossref PubMed Scopus (78) Google Scholar, 14Okabayashi K. Shoji H. Nakamura T. Hashimoto O. Asashima M. Sugino H. Biochem. Biophys. Res. Commun. 1996; 224: 406-413Crossref PubMed Scopus (50) Google Scholar, 15Schonbaum C.P. Lee S. Mahowald A.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1485-1489Crossref PubMed Scopus (155) Google Scholar). In mammals, the core members of this family include LDL receptor, LRP, VLDL receptor, ApoE receptor-2, LRP1b, megalin, and MEGF7. The cytoplasmic tails of these mammalian receptors contain one or more NPXY motifs, required for the endocytosis of the LDL receptor (16Chen W.J. Goldstein J.L. Brown M.S. J. Biol. Chem. 1990; 265: 3116-3123Abstract Full Text PDF PubMed Google Scholar). However, this motif is not used universally within the LDLR family, as LRP requires an YXXφ motif for endocytosis (17Li Y. Marzolo M.P. van Kerkhof P. Strous G.J. Bu G. J. Biol. Chem. 2000; 275: 17187-17194Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). Recently, genetic and biochemical studies have linked a gene to a disease in humans, autosomal recessive hypercholesterolemia (ARH) that clinically resembles familial hypercholesterolemia. The LDL receptors in ARH patients fail to efficiently clear LDL particles from the plasma, resulting in high blood cholesterol levels (18Garcia C.K. Wilund K. Arca M. Zuliani G. Fellin R. Maioli M. Calandra S. Bertolini S. Cossu F. Grishin N. Barnes R. Cohen J.C. Hobbs H.H. Science. 2001; 292: 1394-1398Crossref PubMed Scopus (457) Google Scholar) despite increased levels of LDL receptors on the cell surface (19Wilund K.R. Yi M. Campagna F. Arca M. Zuliani G. Fellin R. Ho Y.K. Garcia J.V. Hobbs H.H. Cohen J.C. Hum. Mol. Genet. 2002; 11: 3019-3030Crossref PubMed Scopus (90) Google Scholar). These findings strongly suggest that hARH is required for LDL receptor endocytosis. ARH contains a phosphotyrosine binding (PTB) domain in the N-terminal region that is present in many adaptor proteins such as SHC, IRS-1, Dab-1, and Numb (20Margolis B. Borg J.P. Straight S. Meyer D. Kidney Int. 1999; 56: 1230-1237Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). PTB domains can interact with NPXY motifs as well as non-NPXY motifs (20Margolis B. Borg J.P. Straight S. Meyer D. Kidney Int. 1999; 56: 1230-1237Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 21Li S.C. Zwahlen C. Vincent S.J. McGlade C.J. Kay L.E. Pawson T. Forman-Kay J.D. Nat. Struct. Biol. 1998; 5: 1075-1083Crossref PubMed Scopus (105) Google Scholar). hARH has been shown to be an adaptor protein coupling the LDLR and endocytic machinery (22He G. Gupta S. Yi M. Michaely P. Hobbs H.H. Cohen J.C. J. Biol. Chem. 2002; 277: 44044-44049Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 23Mishra S.K. Watkins S.C. Traub L.M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16099-16104Crossref PubMed Scopus (144) Google Scholar). However, the functions of ARH in other species or in other pathways have not been reported. In a screen for localized RNAs in frog oocytes, we unexpectedly isolated the Xenopus homologue of ARH (24Zhou, Y. (2003) Xenopus ARH, a Localized Maternal RNA, Encodes an Adaptor Protein Involved in Receptor Mediated Endocytosis. Ph.D. dissertation, University of Miami School of MedicineGoogle Scholar). We showed that maternal xARH, unlike hARH, is present as two transcripts, almost certainly the products of two genes, which differ in their 3′-untranslated regions. The longer transcript is found primarily in the vegetal cortex, whereas the shorter transcript is present almost exclusively in the oocyte animal half. Similar to hARH, xARH is found in the adult liver and spleen, but at low levels compared with oocytes. Other adaptor proteins mediating receptor endocytosis play important roles in development. A related PTB domain-containing protein, Drosophila Numb, links the Notch receptor with the endocytic machinery (25Santolini E. Puri C. Salcini A.E. Gagliani M.C. Pelicci P.G. Tacchetti C. Di Fiore P.P. J. Cell Biol. 2000; 151: 1345-1352Crossref PubMed Scopus (297) Google Scholar), which down-regulates the Notch signal (26Berdnik D. Torok T. Gonzalez-Gaitan M. Knoblich J. Dev. Cell. 2002; 3: 221Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar) and thus specifies neuron cell fate (27Hawkins N. Garriga G. Genes Dev. 1998; 12: 3625-3638Crossref PubMed Scopus (55) Google Scholar). Additional examples of the endocytic regulation of signaling pathways in development are likely to be found (28Seto E. Bellen H. Lloyd T. Genes Dev. 2002; 16: 1314-1336Crossref PubMed Scopus (182) Google Scholar). Here we show that xARH can interact with the LDL and VTG receptors through the PTB domain. Affinity purification of proteins binding to the C-terminal region of xARH identified α-adaptin and β-adaptin, subunits of the adaptor protein complex 2 (AP-2) endocytic complex. Dφ(F/W) motifs were required for the interaction between xARH and the AP-2 complex. Finally, we show that a dominant negative mutant of xARH containing a nonfunctional clathrin box and Dφ(F/W) motif inhibits endocytosis of VTG in cultured oocytes. Our results strongly suggest that maternal xARH is required for vitellogenesis. Our data are consistent with xARH acting as an adaptor protein linking LDL receptor family members and the endocytic machinery in oocytes and embryos. Our findings point to an evolutionarily conserved function for ARH in nutrient uptake. Materials, Oocytes, and Embryos—Frogs were purchased from Xenopus Express or Nasco. Individual oocytes were obtained by collagenase treatment as detailed in MacArthur et al. (29MacArthur H. Bubunenko M. Houston D. King M.L. Mech. Dev. 1999; 84: 75-88Crossref PubMed Scopus (69) Google Scholar). Oocytes were staged according to Dumont (1Dumont J.N. J. Morphol. 1972; 136: 153-179Crossref PubMed Scopus (1415) Google Scholar). Ovulated eggs from human chorionic gonadotropin-induced females were fertilized in vitro and placed in 0.1× MBS (1× MBS: 88 mm NaCl, 1 mm KCl, 1 mm MgSO4, 2.5 mm NaHCO3, 0.7 mm CaCl2, 5mm HEPES, pH 7.6). Embryos were subsequently dejellied with 2% cysteine and staged according to the normal table of Nieuwkoop and Faber (30Nieuwkoop P.D. Faber J. Normal Table of Xenopus laevis (Daudin). North-Holland Publishing Company, Amsterdam1975: 16-42Google Scholar). Anti-α-adaptin MA3-061 (1:100 dilution) was obtained from Affinity BioReagents, Inc. and anti-β–adaptin clone 74 (1:1000 dilution) was obtained from BD Bioscience. Glutathione beads were purchased from Amersham Biosciences. Expression and Purification of Fusion Proteins—The original xARH 2-kb cDNA clone (Xcat4) was obtained from a differential screen of a cDNA library enriched in localized RNAs (31Zhang J. King M.L. Methods Mol. Biol. 2000; 136: 309-314PubMed Google Scholar). A full-length clone was subsequently generated by 5′-RACE (rapid amplification of cDNA ends) and cloned into pSPORT1 (Invitrogen), and Xcat4 was identified as xARHα by sequence analysis (24Zhou, Y. (2003) Xenopus ARH, a Localized Maternal RNA, Encodes an Adaptor Protein Involved in Receptor Mediated Endocytosis. Ph.D. dissertation, University of Miami School of MedicineGoogle Scholar). The full-length (primers: HU, 5′-AGAATTCAGCGGGGAGATGGATGCACT-3′; HD, 5′-ACTCGAGCTGGTAGCTTCAGAAGTGT-3′) and C-terminal region (primers: CU, 5′-GGAATTCATGGAAGACTGTACCAAAGC-3′; HD) of xARHα were PCR-amplified and cloned into pGEX-5X-1 (Amersham Biosciences) to generate GST-tagged fusion proteins. Vector plasmid and the inserts were doubly digested with EcoRI and XhoI. Plasmids were transformed into Escherichia coli strains BL21 for prokaryotic expression. A large scale purification of GST-C protein was used to produce antigen for the generation of polyclonal antisera. PGEX-C was grown in a 50-ml overnight culture in LB medium containing ampicillin (LB-ampicillin) at room temperature. The culture was then diluted into 600 ml of fresh TB (Terrific Broth)-ampicillin and grown to an A 600 of ∼2.0. Expression was induced by the addition of isopropyl-1-thio-β-d-galactopyranoside to a final concentration of 1 mm. After 1.5 h of induction at room temperature, cells were collected by centrifugation. To purify GST-C protein, the pelleted cells were resuspended in 40 ml of B-PER reagent (Pierce) to which 800 μl of lysozyme (10 mg/ml) was added. After an incubation period of 20 min with shaking at room temperature, insoluble proteins were removed by centrifugation at 20,000 × g for 20 min. 2 ml of glutathione beads (Amersham Pharmacia Biotech) were added to the supernatant fluid and incubated for 1 h with mixing. After washing three times with 20 ml of PBS, the GST-C fusion protein was eluted with glutathione buffer (50 mm Tris, pH 8.0, 10 mm reduced glutathione). Approximately 6 mg of GST-C could be obtained (quantified with Bio-Rad Protein Assay reagent). The yield from one preparation was used for custom polyclonal antibody production in mice (Zymed Laboratories Inc.). To purify GST-xARH protein, pGEX-xARH was grown in 300 ml of LB medium with ampicillin to an A 600 of 0.8. Expression was induced by 0.3 mm isopropyl-1-thio-β-d-galactopyranoside for 2.5 h at 37 °C. The cells were pelleted and resuspended in 20 ml of B-PER reagent. After a 20-min incubation at room temperature, the inclusion bodies were collected by centrifugation at 20,000 × g for 20 min. The pellets were dissolved in 2 ml of denaturing buffer (50 mm Tris, pH 7.5, 6 m guanidine-HCl, 25 mm dithiothreitol) at 0 °C for 3 h. Insoluble material was removed by centrifugation at 14,000 × g for 30 min. The supernatant fraction was diluted to 20 ml with the denaturing solution. The protein solution was then dispensed slowly into 200 ml of refolding solution (50 mm Tris, pH 7.5, 0.2 m NaCl, 1 mm dithiothreitol, 1 m non-detergent sulphobetaines (NDSB 201)) at 0 °C. Aggregated protein was removed by centrifugation at 8000 × g for 30 min. 1 ml of glutathione beads was added to the supernatant fraction to purify the GST fusion protein at 4 °C overnight. The beads were washed three times with 10 ml of PBS, and 2 mg of GST-xARH fusion protein was eluted with glutathione buffer. Affinity Purification of Anti-xARH Antibodies and Immunoblotting— For affinity purification of xARH-specific antibodies, 2 ml of ascites to GST-xARH were thawed and diluted to 5 ml with PBS. The diluted ascites was passed over a column containing 1 mg of GST cross-linked to glutathione beads to remove GST-specific antibodies following a previously published method (32Koff A. Giordano A. Desai D. Yamashita K. Harper J.W. Elledge S. Nishimoto T. Morgan D.O. Franza B.R. Roberts J.M. Science. 1992; 257: 1689-1694Crossref PubMed Scopus (907) Google Scholar). The flow-through was collected as precleared ascites and then passed through a column containing 1 mg of GST-C cross-linked to glutathione beads. The column was then washed in PBS, 0.5 m NaCl and 10 mm Tris, pH 7.5, to remove the unbound antibodies. The bound antibodies were eluted with 2 ml of 100 mm glycine, pH 2.2, into tubes containing 0.2 ml of 1 m Tris, pH 8.0. The eluted antibody was concentrated with a Centricon YM-30 (Millipore) and stored at –20 °C in 50% glycerol. For immunoblot analysis, proteins were fractionated by 10% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked in 5% nonfat dry milk in PBS, 0.1% Tween 20 at 4 °C overnight. Primary antibody incubation was done at room temperature for 1 h. Following six washes, membranes were incubated with a 1:100,000 dilution of HRP-conjugated secondary antibody (Pierce). After six additional washes, detection was performed using the SuperSignal West Femto substrate (Pierce) as recommended by the supplier. Whole-mount Immunostaining—Embryos were fixed for 2 h at room temperature in freshly made MEMFA (100 mm MOPS, pH 7.4, 2 mm EGTA, 1 mm MgSO4, 3.7% formaldehyde). Samples were rehydrated to PBS and then incubated in PBT (PBS, 2 mg/ml bovine serum albumin, 0.1% Triton X-100) for 15 min with rocking. Nonspecific binding sites were blocked in PBT containing 10% goat serum for 1 h at room temperature. Primary antibody incubation (affinity-purified anti-xARH antibodies, 1:100 dilution) was done in blocking solution overnight at 4 °C. To demonstrate specificity for xARH, control samples contained excessive amounts of purified GST-C protein (mass ratio of GST-C to antibody, >100) to block the primary antibody. The following day, the embryos were washed four times at room temperature with PBT, 1 h each with rocking. This was followed by an overnight incubation at 4 °C in a 1:2000 dilution of goat anti-mouse HRP-conjugated secondary antibody (Pierce). Embryos were washed as described above. Detection was carried out using a diaminobenzidine colorimetric HRP substrate (Roche Applied Science). Affinity Purification of xARH Putative Binding Proteins from Embryo Extracts and Mass Spectrometry—Early blastula stage embryos were homogenized in buffer H (50 mm Tris, pH 7.5, 250 mm KCl, 5 mm EDTA, 1 mm dithiothreitol, 0.1% Triton X-100) with complete protease inhibitors (Roche Applied Science). Insoluble materials were removed through two 30-min cycles of centrifugation at 15,000 × g. The supernatant was cleared by preincubation with GST and glutathione-Sepharose beads overnight at 4 °C. After centrifugation at 15,000 × g for 30 min, the precleared supernatant was incubated overnight now with GST-C fusion protein and glutathione-Sepharose beads at 4 °C. The beads were washed three times with buffer H. The bound proteins were released by boiling in SDS loading buffer and resolved by 8% SDS-PAGE. Oocyte extracts routinely resulted in significantly higher background and more variable results and therefore were not used for these studies. For mass spectrometry, the gels were stained with Coomassie Blue. The band of interest at 108 kDa was excised from the gels. A total of 200 ng of p108 protein was obtained from two preparations and sent for mass spectrometry protein identification to the HHMI Biopolymer Laboratory and W. M. Keck Foundation Biotechnology Resource Laboratory at Yale University. Site-directed Mutagenesis—Mutagenesis was carried out using the QuikChange method (Stratagene) according to the manufacturer's protocol. Primers were designed to mutate the Dφ(F/W) motifs in GST-C fusion protein to AAA (primers are shown 5′ to 3′: DLFA5, AATCATATTGAAGCAGCTGCCAGGCAAAATGCA; DLFA3, TGCATTTTGCCTGGCAGCTGCTTCAATATGATT; DLFB5, GATGGACTGGATGCTGCCGCAGCAAGGCTTGCC; DLFB3, GGCAAGCCTTGCTGCGGCAGCATCCAGTCCATC; DLFC5, GCAGAAGCAGATGCCGCAGCTATGTTCTGACAC; DLFC3, GTGTCAGAACATAGCTGCGGCATCTGCTTCTGC). For the xARH-DM mutant, the clathrin box (LLDLE) was mutated to LLALA (primers: LLALAF, GCAAACCTTTTGGCCTTGGCCGACTGTACCAAA; LLALAR, TTTGGTACAGTCGGCCAAGGCCAAAAGGTTTGC), and the Dφ(F/W) motif 258 was mutated to AAA (primers: DLFB5 and DLFB3). All mutant constructs were sequenced to confirm the presence of the designed mutations. In Vitro Translation and GST Pull-down Assays—Oligonucleotides were synthesized to encode Xenopus LDLR-1 (nucleotides 2649–2804) and VTGR (nucleotides 2650–2811) cytoplasmic tails according to published sequence data (14Okabayashi K. Shoji H. Nakamura T. Hashimoto O. Asashima M. Sugino H. Biochem. Biophys. Res. Commun. 1996; 224: 406-413Crossref PubMed Scopus (50) Google Scholar, 33Mehta K.D. Chen W.J. Goldstein J.L. Brown M.S. J. Biol. Chem. 1991; 266: 10406-10414Abstract Full Text PDF PubMed Google Scholar). A T7 promoter sequence (TAATACGACTCACTATAGGGAG) and Kozak sequence (GCCACCATGG) were added to the 5′-ends. The synthesized DNA fragments were translated using the TnT Quick Transcription and Translation Coupled System from Promega. 5 μg of GST fusion proteins were dissolved in 500 μl of PBS + 0.1% Tween 20. 20 μl of glutathione beads (Amersham Biosciences) and 5 μl of an in vitro translation reaction (TnT Quick System) containing LDLR or VTGR 35S-labeled cytoplasmic tails were added. After incubation at room temperature for 3 h, the beads were washed three times with 500 μl of PBS + 0.1% Tween 20. The bound proteins were released by boiling in SDS loading buffer and resolved on a 10% SDS-PAG. GST fusion proteins were visualized by Coomassie Blue staining. The candidate partners were visualized by exposing the dried gel to a PhosphorImager screen. Purification and Labeling of Xenopus Vitellogenin—Vitellogenin purification was performed according to published procedures (4Opresko L.K. Methods Cell Biol. 1991; 36: 117-132Crossref PubMed Scopus (13) Google Scholar). Briefly, female frogs were injected with 17β-estradiol dissolved in propylene glycol (10 mg/ml) at a dose of 1.5 mg/100 g of frog on day 1 followed by a second injection on day 3. Plasma was collected on day 10 into 15-ml tubes containing 1 ml of 50% PBS and 70 mm sodium citrate. After centrifugation at 2500 × g for 15 min to remove the blood cells, the supernatant fraction was transferred to another tube. 10 ml of 20 mm EDTA, pH 7.7, for every 2.5 ml of plasma was added to the tube, and the contents were mixed gently. 0.8 ml of 0.5 m MgCl2 was added and mixed again. Vitellogenin was collected by centrifugation at 2500 × g for 15 min. The green pellet was dissolved in 1.5 ml of 1 m NaCl, 50 mm Tris, pH 7.5, and stored in aliquots at –20 °C (4Opresko L.K. Methods Cell Biol. 1991; 36: 117-132Crossref PubMed Scopus (13) Google Scholar). To biotinylate vitellogenin, the protein was transferred into PBS by ultrafiltration, which was repeated three times using Microcon YM-30 filter devices (Millipore). 10 mg of vitellogenin was dissolved in 1 ml of PBS. 0.5 mg of Sulfo-NHS-LC-Biotin (Pierce) powder was added directly into the protein solution, mixed well, and incubated on ice for 2 h. The unconjugated biotin was removed using a desalting column (Amersham Biosciences) (molecular weight cutoff, 5,000). The biotinylated vitellogenin was concentrated with Microcon YM-30 and stored at –20 °C in 50% glycerol. Vitellogenin Uptake Assay—Stage III/IV oocytes were obtained after collagenase treatment and incubated overnight in OCM (60% Liebovitz L-15 medium, 0.4 mg/ml bovine serum albumin, 1 mm glutamine, pH 7.6). Dominant negative mutant or control transcripts were injected into oocytes and the oocytes cultured overnight. Oocytes were then transferred into OR2 medium (82.5 mm NaCl, 2.5 mm KCl, 1 mm MgCl2, 1 mm CaCl2, 1 mm Na2HPO4, 5 mm HEPES, pH 7.8) containing 5 mg/ml bovine serum albumin and 0.25 mg/ml biotinylated vitellogenin. After incubation at room temperature for varying periods of time, the oocytes were washed with OR2. Any labeled vitellogenin was removed from the surface by washing with 25 mm EDTA, 0.5 m NaCl, 0.1 m glycine-NaOH, pH 9.5, for 40 min (4Opresko L.K. Methods Cell Biol. 1991; 36: 117-132Crossref PubMed Scopus (13) Google Scholar). Oocyte samples were boiled in SDS loading buffer and analyzed by Western blot using Streptavidin-HRP (Pierce) at 1:50,000 dilution. We had previously cloned two forms of xARH, xARHα and xARHβ, which appear to represent two genes that arose when the entire Xenopus genome duplicated an estimated 30 million years ago. Although xARHα RNA is the same length as hARH RNA, xARHβ has a much shorter 3′-untranslated regions (24Zhou, Y. (2003) Xenopus ARH, a Localized Maternal RNA, Encodes an Adaptor Protein Involved in Receptor Mediated Endocytosis. Ph.D. dissertation, University of Miami School of MedicineGoogle Scholar). The two isoforms are distributed differentially along the animal/vegetal axis in fully grown oocytes, with xARHα concentrated predominantly within the vegetal cortex and xARHβ restricted to the animal hemisphere. The ARH proteins are 97% identical by sequence analysis, with amino acid changes found dispersed throughout the ORF (24Zhou, Y. (2003) Xenopus ARH, a Localized Maternal RNA, Encodes an Adaptor Protein Involved in Receptor Mediated Endocytosis. Ph.D. dissertation, University of Miami School of MedicineGoogle Scholar). The xARHα clone was used in this study, and the results presented here are likely applicable to xARHβ as well, based on the virtual identity of their functional domains. To begin to address the function of xARH in oocytes and embryos, we first examined its developmental expression pattern using a polyclonal antibody raised against the C-terminal region of xARH (Fig. 1, residues 217–309). This region includes sequences immediately after the conserved clathrin box and thus avoids any cross-reactivity with the conserved PTB domain found in other proteins. Within this 92-amino acid region, xARHα and xARHβ differ by only four nonclustered residues (24Zhou, Y. (2003) Xenopus ARH, a Localized Maternal RNA, Encodes an Adaptor Protein Involved in Receptor Mediated Endocytosis. Ph.D. dissertation, University of Miami School of MedicineGoogle Scholar). xARH was detected at all stages of oogenesis, and in most oocytes, it appeared more concentrated at the cell periphery (Fig. 2A). The signal could be eliminated almost completely if GST-C fusion protein was added to block the primary antibody (Fig. 2, control), confirming the specificity of the signal. Immunostaining for xARH in bisected late stage V oocytes shows the accumulation of xARH protein around the nucleus. In the egg and after nuclear (germinal vesicle) breakdown, xARH was detected distributed throughout the animal hemisphere (Fig. 2B). In embryos, xARH was present in all cells and at all stages examined, from early cleavage stage 5 to tail bud (Fig. 2C). As nuclei were formed in the early embryo, histological sections revealed the protein concentrated within the perinuclear and cortical region (Fig. 2D, brown signal distinguished from the black granular melanosomes). In human liver, ARH has also been localized near the plasma membrane in hepatocytes (34Jones C. Hammer R.E. Li W.P. Cohen J.C. Hobbs H.H. Herz J. J. Biol. Chem. 2003; 278: 29024-29030Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar).Fig. 2xARH expression in oocytes and embryos. Affinity-purified mouse polyclonal anti-xARH
Referência(s)