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Genetics of Cell Surface Receptors for Bioactive Polypeptides

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Genetics of Cell Surface Receptors for Bioactive Polypeptides Proc. Nati. Acad. Sci. USA Vol. 77, No. 6, pp. 3600-3604, June 1980 Genetics Genetics of cell surface receptors for bioactive polypeptides: Binding of epidermal growth factor is associated with the presence of human chromosome 7 in human-mouse cell hybrids (hormone/gene mapping/gene regulation) NOBUYOSHI SHIMIZU, M. ALI BEHZADIAN, AND YOSHIKO SHIMIZU Department of Cellular and Developmental Biology, University of Arizona, Tucson, Arizona 85721 Communicated by Frank H. Ruddle, March 24, 1980 ABSTRACT Mouse A9 cells, L-cell-derived mutants defi- Although recent work has identified the EGF receptor as a cient in hypoxanthine phosphoribosyltransferase (HPRT; glycoprotein with subunit structure (6-8), little is known about IMP:pyrophosphate Ihosphoribosyltransferase, EC 2.4.2.8) were its genetics and biosynthesis. By an application of somatic cell foun to e incapable of binding 25I-labeled epidermal growth factor (EGF) to the cell surface. The A9 cells were fused with genetics we have been studying the genetic and molecular basis human diploid fibroblasts (WI-38) possessing EGF-binding of receptor-mediated mitogenic action of EGF (9). In this report ability, and human-mouse cell hybrids (TA series) were isolated we present the evidence for the dominance of EGF-binding after hypoxanthine/aminopterin/thymidine/ouabain selection. ability and its linkage with human chromosome 7 based on Analyses of isozyme markers and chromosomes of four repre- analysis of human-mouse cell hybrids. sentative clones of TA hybrids indicated that the expression of EGF-binding ability is correlated with the presence of human chromosome 7 or 19. Four subclones were isolated from an MATERIALS AND METHODS EGF-binding-positive line, TA4, and segregation of EGF- Cell Lines. TA hybrid lines were progenies of fusion between binding was found to be concordant with the expression of human diploid fibroblasts WI-38 (10) and mouse L-cell-derived human mitochondrial malate dehydrogenase (MDHM; L-mal- ate:NAD+ oxidoreductase, EC 1.1.1.37), a marker for chromo- A9 cells deficient in hypoxanthine phosphoribosyltransferase some 7, but not with glucosephosphate isomerase (GPI; Dglu- (HPRT; IMP:pyrophosphate phosphoribosyltransferase, EC cose-phosphate ketol-isomerase, EC 5.3.1.9), a marker for 2.4.2.8) (11). Fusion was mediated by f3-propiolactone-inacti- chromosome 19. Furthermore, evidence from 27 clones of AUG vated Sendai virus (Connaught) and selection was performed hybrids that were produced between A9 and another human in hypoxanthine/aminopterin/thymidine (HAT) medium (12) fibroblast line, GM1696, carrying an X/7 chromosome trans- containing 1 ,uM ouabain (13). Four representative lines of TA location indicated that EGF-binding ability segregates together with human MDHM and two X-linked markers, HPRT and hybrids (TA-1, -2, -3, and -4) were chosen from 23 isolated glucose-6phosphate dehydrogenase (G6PD; i.-glucose-6phos- clones that were maintained in nonselective medium for more phate:NADP+ 1-oxidoreductase, EC 1.1.1.49), that are located than 20 passages and used for the present study after extensive on the translocation chromosome 7p+. These results permit as- characterization. These hybrids were maintained in Dulbecco's signment of the gene, designated EGFS, which is associated modified Eagle's medium (GIBCO) supplemented with 10% with the expression of EGF-binding ability, to human chromo- fetal calf serum (GIBCO), penicillin (100 units/ml), and some 7 and its localization to the p22-qter region. Because the streptomycin (100 (GIBCO) in culture dishes EGF receptor is reported to be a glycoprotein the EGFS could ,ug/ml) plastic be either a structural gene(s) for receptor protein or a gene(s) for or flasks (Falcon or Corning) under a humidified atmosphere modifying the receptor protein through glycosylation. of 5% C02/95% air at 370C. AUG hybrid lines were progenies of fusion between human fibroblasts GM1696 carrying an X/7 Epidermal growth factor (EGF), a single-chain bioactive chromosome translocation (14) and mouse A9 cells. The isola- polypeptide of molecular weight approximately 6000, is isolated tion procedures were similar to those for TA hybrids except that from the submaxillary glands of adult male mice and from the AUG hybrids were maintained in Dulbecco's modified human urine (1, 2). EGF has a unique stimulatory effect in Eagle's medium containing HAT. Twenty-seven AUG hybrids epidermal and epithelial tissue development in newborn ani- were chosen from 45 primary clones and 15 secondary clones mals, and in various cultured cells it remarkably stimulates that were isolated from three independent fusions and used for DNA synthesis and cell division (3, 4). Evidence has been ac- the present study. These include D5B1-3, D5B1-16, B4A1, cumulating that EGF specifically recognizes its receptor on the A4B3, B4A1-6, C2B5, C2BS-AG, C2B5-30, C3A1, B5B2, C3B4, plasma membrane and that, subsequent to the initial binding, C3B4-AG1, C3B4-AG3, C3B4-AG4, C3B4-AG6, C3B4-AG7, the bound EGF or EGF-receptor complex is compartmental- C3B4-AG8, C3B4-AG9, B4B4, B2B4, B4A1-17a, C3B1, B4B2, ized and internalized into endocytotic vesicles (1, 3, 5, 6). They C2B3, C2B3-AG, A5B7, and B2B1. Other cell lines used are are eventually degraded in lysosomes (1, 3, 5, 6). It has been listed in Table 1 together with source and cell type informa- assumed that a number of biochemical and biological alterations tion. induced by EGF result from the amplification and propagation Assay for EGF-Binding. Confluent cell cultures grown in of a series of signals (1, 3, 5, 6). Yet, it has not been possible to 60-mm-diameter plastic dishes were placed on ice and washed determine these signals in relation to the metabolic fate of the twice with 2 ml of ice-cold EBSS buffer [Earl's balanced salt receptor-bound EGF or to correlate them with the potent mi- solution (5.4 mM KCI/116 mM NaCl/1 mM NaH2PO4/1.4 mM togenic action. CaC12/0.8 mM MgSO4/5.5 mM glucose), 5 mM Hepes (pH 7.4), and 0.1% bovine serum albumin (Sigma, RIA grade)]. (These The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "ad- Abbreviations: EGF, epidermal growth factor; HAT, hypoxanthine/ vertisement" in accordance with 18 U. S. C. §1734 solely to indicate aminopterin/thymidine mixture; HPRT, hypoxanthine phosphori- this fact. bosyltransferase; AG, 8-azaguanine. 3600 Downloaded by guest on September 26, 2021 Genetics: Shimizu et al. Proc. Natl. Acad. Sci. USA 77 (1980) 3601 stock solutions were kept frozen until use.) The washed cells (3 mm diameter) were punched with a metal gel cutter (gel were then covered with 1 ml of EBSS buffer, and 10 ,ul of pieces were removed by aspiration). The gel plate was placed 1251-labeled EGF (150,uCi/,gg; 10,tCi/ml; 1 Ci = 3.7 X 1010 across the bridge gap on the electrophoresis chamber and becquerels; Collaborative Research) was added at a final con- bridged with Whatman no. 1 filter paper. An 8-pl sample of centration of 0.11 nM. After incubation at 230C for various each cell extract was deposited in a sample well by means of a times (60 min for standard assay), the plates were washed three Hamilton microsyringe. The electrophoresis was performed times with 3 ml of ice-cold EBSS buffer and dissolved in 1 ml with 40 mM Na barbital buffer (pH 8.6) at room temperature of 0.5 M NaOH. Aliquots (0.9 ml) were mixed with 7 ml of for 10 hr at 10 V/cm. After electrophoresis, soluble proteins Riafluor (New England Nuclear) and acidified with 75 ,l of were removed from the gel plate by extensive washing and the 6 M HCI. Radioactivity was assayed in a Packard liquid scin- GPI-antibody complex was stained as described (18). tillation spectrometer. To measure nonspecific binding, 1 ,p1 Chromosome Analysis. Chromosome preparations were of unlabeled EGF (100,gg/ml) was added at a final concen- made from hybrid cells that had been arrested at metaphase tration of 16 nM 10 min prior to the addition of radioactive by vinblastine sulfate (16). Specimens for karyotyping were EGF. Nonspecific binding thus measured was 10-15% and was obtained concurrently with the collection of cells for enzyme subtracted from the total binding to give specific binding. analysis or binding assay. The human chromosomes were Cell Extracts. Logarithmic phase cells in plastic petri dishes identified by sequential staining with Giemsa and Hoechst (15 cm diameter) were harvested by scraping with a Teflon- 33258 (19). Observations and microphotography were made taped razor blade. The cells were collected by centrifugation, with a Nikon microscope Fluophot. More than 25 chromosome washed with isotonic saline, and stored as frozen pellets, at spreads from each cell line were examined for the presence of -70'C. The frozen cells (1-3 X 107 cells) were thawed by human chromosomes. adding 0.27 ml of extraction buffer [10 mM Tris-HCI, pH 7.5/1 Cell Counting. Cells were removed from the culture dishes mM MgCl2/20 mM KCI/0.1 mM dithiothreitol/10% (vol/vol) by a standard trypsinization method and counted by either glycerol] and the cell suspension was mixed with 30,gl of 5% hemocytometer or Electrozone/Celloscope (Particle Data). Triton X-100 (Bio-Rad) in the same buffer. Cells were lysed in Counting variations were 5-10% on duplicate dishes, and fre- an ice bath for 30 min with intermittent agitation. The lysate quency of live cells was usually 95-99% as determined by the was clarified by centrifugation at 30,000 X g for 60 min and trypan blue exclusion test. stored at -70°C in small aliquots. Isozyme Analysis. Electrophoretic separation of the fol- RESULTS lowing marker enzymes were carried out on a cellulose acetate The binding of l25-Ilabeled EGF to cultured cells is known to plate (Titan III, Helena): adenosine deaminase (EC 3.5.4.4; take place at a wide range of temperatures and with time- ADA), adenosine kinase (EC 2.7.1.20; ADK), adenylate kinase dependent saturable kinetics (1, 3).
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