Enhanced degradation of EGF receptors by a sorting nexin, SNX1

Science

Volumn 272, Issue 5264, 1996, Pages 1008-1010

  Kurten, Richard C ^a   Cadena, Deborah L ^a   Gill, Gordon N ^a  

^a UNIVERSITY OF CALIFORNIA   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

EPIDERMAL GROWTH FACTOR RECEPTOR; PROTEASE NEXIN I;

AMINO ACID SEQUENCE; ARTICLE; HUMAN; MOLECULAR RECOGNITION; NONHUMAN; PRIORITY JOURNAL; PROTEIN DEGRADATION; PROTEIN TARGETING; PROTEIN TRANSPORT; SEQUENCE HOMOLOGY; TRANSCRIPTION REGULATION; TRANSPORT KINETICS;

EID: 0029762883     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.272.5264.1008     Document Type: Article

References (19)

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5. 35S-labeled deoxyadenosine triphosphate and a primer (5′-GATGTTAACGATACCAGCCTCTTGCT-GAGT-3′) that bound upstream of the vector-cDNA fusion junction. The sequence was used to search the nucleotide sequence databases of the National Center for Biotechnology Information using the BLAST algorithm (15).
6. 32P-labeled oligonucleotide complementary to the 5′ end of the SNX1 library plasmid insert (5′-CTTTCTCAAACCTCACT-TCT-3′). Six phages were plaque-purified, the cDNAs subcloned into the Eco RI site of pMOBII (Gold Biotechnology, St. Louis, MO), and transposon insertions used to determine the complete sequence of two of the inserts on both strands. Areas of ambiguity were clarified with gene-specific primers. The sequence of SNX1 is deposited in GenBank (accession number U53225).
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8. R. C. Kurten, D. L. Cadena, G. N. Gill, data not shown.
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11. L. K. Opresko et al., ibid., p. 4325.
12. 6 cells) for 4 to 6 hours. For transient assays, lysates from human embryonic kidney 293 cells were prepared 48 hours later for protein immunoblotting. Stable African green monkey kidney CV-1 cells were prepared by expanding the cells 48 hours after transfection onto 10-cm-diameter plates in the presence of hygromycin (320 μg/ml) and selected for 14 to 21 days. Clonal lines were prepared from isolated colonies with cloning rings.
13. 35S-methionine (0.65 mCi/ml) for 1 hour. Cells were incubated in complete medium, and radio immunoprecipitation assay extracts [1 % sodium deoxycholate, 1% NP-40, 0.1% SDS, 10 mM Hepes (pH 7.6), and 150 mM NaCl] were prepared after incubation for various times and immunoprecipitated with antibodies 528 and 13A9 to EGFR. After they were washed, the immunoprecipitates were solubilized in SDS sample buffer, electrophoresed, and visualized by fluorography, and the radioactivity was quantified by liquid scintillation counting.
14. F. M. Ausubel et al., Eds., Current Protocols in Molecular Biology (Greene, Brookyln, NY, 1995).
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16. The detergent extraction buffer was 1% Triton X-100,50 mM Hepes (pH 7.4), 10% glycerol, and 75 mM NaCl. Whole-cell lysate buffer was 1% SDS and 10 mM Hepes (pH 7.4). Buffers were supplemented with the following phosphatase and protease inhibitors: 10 mM NaF, 1 mM sodium vanadate, 1 mM EDTA, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 10 mM benzamidine, leupeptin (10 μg/ml), antipain (10 μg/ml), and aprotinin (10 μg/ml). Products of the coupled rabbit reticulocyte lysate transcription and translation reaction (Promega, Madison, WI) were separated by gel electrophoresis and visualized by fluorography.
17. Cells were fixed in paraformaldehyde; permeabilized with saponin; stained with antibody to SNX1 (anti-SNX1) and a mixture of antibodies to EGFR [immunoglobulin G's (IgGs) 528, 13A9, and 225], followed by Texas Red-conjugated goat anti-rabbit IgG and fluorescein isothiocynate-conjugated goat anti-mouse IgG; and visualized by epifluorescence illumination.
18. - broth containing 2% dextrose or 2% galactose (to induce the library plasmid insert) and grown at 30°C for 18 to 22 hours (14).
19. We thank R. Brent for providing the components of the yeast two-hybrid system and A. Nesterov and H. S. Wiley for helpful discussions. Supported by NIH grants F32DK08666 (to R.C.K.) and CA58689 (to G.N.G.) and by funds provided by the Breast Cancer Fund of the State of California through the Breast Cancer Research Program of the University of California, grant numbers 1FB-0314 (to R.C.K.) and 1KB-0140(toD.LC.).