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 7 in human-mouse cell hybrids (hormone/ 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 based on Analyses of isozyme markers and 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 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 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). At 370C the binding of EGF 1 (EC 2.7.4.3; AK1), adenine phosphoribosyltransferase (EC usually decreases after reaching a maximum; at 0C there is 2.4.2.7; APRT), esterase D (EC 3.1.1.1; ESD), galactokinase (EC no net loss of cell-bound EGF (1, 3, 20). A typical binding 2.7.1.6; GALK), glucose-6-phosphate dehydrogenase (EC profile for human HeLa cells at 230C shows no decrease of 1.1.1.49; G6PD), glutamic-oxaloacetic transaminase (soluble) (EC 2.6.1.1; GOTS), glutathione reductase (EC 1.6.4.2; GSR), Table 1. Binding of 125I-EGF to human and mouse hexosaminidase B (EC 3.2.1.30; HEXB), hypoxanthine phos- cultured cell lines phoribosyltransferase (EC 2.4.2.8; HPRT), isocitrate dehy- EGF binding drogenase (soluble) (EC 1.1.1.42; IDHS), superoxide dismutase ability, % input/ (soluble) (EC 1.15.1.1; SODS), lactate dehydrogenases A and Cell line Cell type 106 cells B (EC 1.1.1.27; LDHA and LDHB), malic enzyme (EC WI-38 Human female diploid 1.9 1.1.1.40; MES), mannosephosphate isomerase (EC 5.3.1.8; MPI), (CCL75)* embryonic lung fibroblasts nucleoside phosphorylase (EC 2.4.2.1; NP), peptidases A, B, and GM1696t Human female fibroblasts 2.9 C (EC 3.4.11-; PEPA, PEPB, and PEPC), phosphoglucomutase carrying an X/7 chromosome 1 (EC 2.7.5.1; PGM1), phosphoglycerate kinase (EC 2.7.2.3; translocation PGK), and triosephosphate isomerase (EC 5.3.1.1; TPI). HeLa S3t Human cervical epithelioid 1.5 All of these enzymes except ADK, APRT, HPRT, and GALK carcinoma were detected by conventional histochemical staining (15). HT1080-lb§ Human fibrosarcoma-derived 1.5 Activities of ADK, APRT, and HPRT were detected by auto- epithelial cells, 8-azaguanine radiography of DE81-ion-exchange paper to which radioactive resistant reaction products had been transferred from the cellulose ac- Swiss/3T3 etate plate. To assay GALK, the lanthanum chloride method, (CCL 92)* Mouse embryonic fibroblasts 3.6 followed by autoradiography, was used. The detailed proce- BALBc/3T3T Mouse embryonic fibroblasts 2.1 dures will be published elsewhere. Double immunodiffusion RAGW Mouse BALB/cd renal 0.42 was carried out for mitochondrial NAD-linked malate dehy- adenocarcinoma, HPRT drogenase (EC 1.1.1.37; MDHM) and for mitochondrial deficient NADP-linked isocitrate dehydrogenase (EC 1.1.1.42; IDHM) A9W Mouse L-cell-derived HPRT 0.00 with rabbit antisera specific for the relevant human enzymes deficient mutant, from normal (16, 17). subcutaneous areolar and Rocket immunoelectrophoresis was carried out for glucose- adipose tissue of C3H/An phosphate isomerase (EC 5.3.1.9; GPI) with rabbit antisera mouse specific for human GPI (18). Briefly, 60 ul of rabbit anti-human * American Type Culture Collection, Rockville, MD. t Human Genetic Mutant Cell Repository, Camden, NJ. GPI antiserum was added to 12 ml of 1% agarose in 40mM Na From F. H. Ruddle and B. P. Dorman, Yale University, New Haven, barbital buffer (pH 8.6) kept at 50°C. The antibody-containing CT. agarose was cast on a precleaned glass plate (10 X 12.5 cm) on § From R. S. Kucherlapati, Princeton University, Princeton, NJ. a leveled platform. After the agarose solidified, 10 sample wells I From J. D. Hall, University of Arizona, Tucson, AZ- Downloaded by guest on September 26, 2021 3602 Genetics: Shimizu et al. Proc. Natl. Acad. Sci. USA 77 (1980)

Table 3. Concordant segregation of EGF-binding ability and Q A B c human MDHM in four subclones of TA-4 hybrid line 0 EGF ^-40 1.5 0 binding, A .5 Cell line cpm/dish MDHM LDHB GALK GPI * 1.0 I TA-4 229 + + + + c TA-4a 0 - + +++ 0.5 .0 TA-4b 79 + + + +++ TA-4c 583 +++ + - ++ 0 0 TA-4d 220 ++ + + + 0 10 30 60 0 10 30 60 0 10 30 60 The presence ofhuman MDHM and GPI was classified into three Incubation time, min subclasses (+, ++, +++) on the basis of intensity ofthe histochem- FIG. 1. Time course of 125I-EGF bindingto variousculturedcell ically stained enzyme-antibody complex shown in Fig. 2. lines. Incubation temperature was 23°C. (A) Human HeLa S3; (B) human WI-38 (0) and mouse A9 (@); (C) human-mouse hybrids virtually no activity (see also Fig. 1B). Two other mouse cell TA-4a (0) and TA-4d (0). lines, BALBc/3T3 and RAG, had significantly high binding cell-bound radioactive EGF during 60 min of incubation (Fig. activity. When a similar binding assay was performed for 1A). These conditions were used throughout this study to ex- 125I-labeled insulin, all the cell lines listed in Table 1, including amine steady state EGF-binding capacity. Table 1 summarizes mouse A9, had substantial binding ability (data not shown). the results for EGF binding to a number of established cell lines. Although decreased EGF-binding ability is incidental to sar- Mouse Swiss/3T3 had the highest binding ability; mouse A9 had coma virus-induced transformation (21), we suspected that the A9 cells might have mutated in a gene(s) for the EGF receptor or certain functions related to EGF-binding ability. To test these Table 2. Correlation of EGF binding ability to the presence of possibilities we undertook cell hybridization analysis. human chromosome 7 or 19 and the corresponding marker The A9 cells were fused with human diploid fibroblasts enzymes in four human-mouse cell hybrid lines (WI-38) which possess EGF-binding ability (Table 1; Fig. 1B), Presence or absence of human and the fusion progenies were cultured in HAT medium con- chromosomest and markerst taining ouabain. Twenty-three colonies were isolated from two Human Corresponding in each human-mouse cell independent fusions and passaged more than 20 times in non- chromo- phenotypic hybrid line selective medium to allow chromosome segregation. Isozyme some marker* TA-1 TA-2 TA-3 TA-4 analysis revealed that all these cell lines had the nature of true 1 PEPC, PGM1 +(++) +(++) -(--) -(--) human-mouse cell hybrids. Four representative lines of these 2 IDHS +(+) +(+) +(+) -(-) hybrids (TA series) were extensively characterized for EGF 3 ND -(ND) -(ND) +(ND) -(ND) binding and the presence or absence of human chromosomes 4 ND +(ND) +(ND) +(ND) -(ND) and their corresponding marker enzymes (Table 2). Lines TA-I 5 HEXB +(+) -(-) -(-) -(-) and TA-4 exhibited EGF-binding ability and the other two did 6 MES -(- () -() -- not. The ability of A9 cells to bind EGF was restored when the 7 MDHM +(+) -(-) -(-) +(+) hybrid retained human chromosome 7 or 19 or both. No other 8 GSR +(+) -(-) +(+) -(-) 9 AK1 -(- -(- -(- -(- 10 ADK, GOTS +(++) -(--) -(--) -(--) A I B 11 LDHA -(-) -(-) +(+) -(-) (+) 12 LDHB,.PEPB, TPI +(+++) -(---) +(+++) +(+++) 13 ESD +(+) +(+) +(+) -(-) 14 NP -(-) -(-) +(+) -(-) 3 15 MPI, IDHM +(++) -(--) -(--) -(--) -o 16 APRT +(+) +(+) +(+) -(-) 17 GALK -(-) +(+) -(-) +(+) A 5 18 PEPA +(+) +(+) -(-) -(-) 19 GPI +(+) -(-) -(-) +(+) 20 ADA +(+) -(-) +(+) -(-) 6 (-) 21 SODS +(+) -(-) -(-) -(-) 1 2 3 4 a 6 22 ND +(ND) -(ND) -(ND) -(ND) X HPRT, G6PD, PGK +(+++) +(+++) +(+++)-(---) FIG. 2. (A) Double immunodiffusion analysis of human mito- Y§ ND - - - - chondrial NAD-linked MDH (MDHM). Antibody-enzyme complex 71 EGF-binding was visualized by histochemical staining. The central well contained activity + - - + 8 ,l of the absorbed anti-human MDHM antiserum. Test samples: 1, human WI-38; 2, human-mouse hybrid TA-4d; 3, human-mouse * For details concerning these markers, see McKusick and Ruddle hybrid TA-4b; 4, human-mouse hybrid TA-4a; 5, human WI-38; 6, (22). ND, Not tested. mouse A9; 7, human HeLa; 8, human-mouse hybrid TA-4c. (B) t Frequency of individual human chromosomes, if present, in each Rocket immunoelectrophoresis analysis of human GPI. Test samples: of these hybrids ranged from 12% to 72% and these were scored as 1, mouse A9; 2, human-mouse hybrid TA-4d; 3, human-mouse hybrid positive (+) for that chromosome. TA-4b; 4, human-mouse hybrid TA-4c; 5, human-mouse hybrid Noted in the parentheses. TA-4a; 6, human WI-38. 0, origin; (+) and (-) direction of electric § Human parental line WI-38 is from a female. current. Size ofthe stained area (rocket) is proportional to the amount I This work. of human GPI. Downloaded by guest on September 26, 2021 Genetics: Shimizu et al. Proc. Natl. Acad. Sci. USA 77 (1980) 3603

7 XIC Table 4. Syntenic relationship of EGF-binding ability with G6PD human MDHM in 27 AUG series human-mouse cell hybrid lines iiHPRT Phenotypic Pattern of expression* markers +/+ +/- -/+ -I- EGF binding/MDHM 20 0 1 6 MDHMg SHPRT HMDHM G6PD EGF binding/GPI 18 2 2 5 * FIG. 3. Diagram of the translocation in human Each number represents the number of clones that exhibited con- X/7 fibroblast, cordant (+/+, -/-) or discordant (+/-,-/+) segregation of two line GM1696. phenotypes. human chromosomes were correlated with the EGF-binding ability. The expression pattern of phenotypic markers (noted Twenty-seven AUG hybrid lines were chosen, including 18 in the parentheses) is consistent with this conclusion. lines that were kept in HAT medium and 9 lines that were In order to test if EGF-binding ability further segregates with grown in nonselective medium. Table 4 summarizes the results either one of these human chromosomes we isolated four sub- of analysis of them. Twenty lines expressed EGF-binding ability clones from the EGF-binding-positive hybrid line TA-4, which together with human MDHM; six lines expressed neither EGF retains human chromosomes 7, 12, 17, and 19, and examined binding nor human MDHM. One clone, A5B7, expressed the corresponding marker enzymes (Table 3). There was one human MDHM but did not express EGF-binding ability. EGF-binding-negative subclone, TA-4a, in which human Nevertheless, nearly perfect correlation between human MDHM was no longer found (Fig. 2A). All three other sub- MDHM and EGF-binding ability expressions was evident. clones retained EGF-binding ability to different extents in Again, no correlation was found with any other markers parallel with the intensity of MDHM activity. Human GPI was tested. expressed in all four subclones although activity varied (Fig. Two representative AUG hybrid lines, C2B5 and C3B4, were 2B). Human LDHB and GALK segregated discordantly with passaged three times in nonselective medium and processed for EGF-binding as from the data in Table 2. Thus, the EGF- 8-azaguanine (AG) back-selection (16). In the cell populations binding ability is correlated solely with the expression of human that survived AG selection, human MDHM and the two human MDHM. These results strongly suggest that human chromo- X-linked markers, HPRT and G6PD, were lost (Table 5). These some 7 carries a gene(s) that complements the lesion of A9 cells AG back-selectants had lost EGF-binding ability, as expected. in EGF-binding activity. The presence or absence of other markers such as GPI and We produced another series of cell hybrids (AUG series) to LDHA was unaltered by this selection. When chromosomes confirm the above provisional assignment by fusing mouse A9 were examined, the loss of translocation chromosome 7p+ cells with a human fibroblast GM1696 line that was derived coincided with the loss of EGF-binding ability. It is noteworthy from a patient carrying an X/7 chromosome translocation (14). that the line C2B5 retained only the 7p+ as the The break-points in this translocation occur at p22 on the short and still maintained EGF-binding ability and that this chro- arm of chromosome 7 and q21 on the long arm of the X chro- mosome was absent in the AG back-selectant C2B5-AG (un- mosome, resulting in two rearranged products, 7p+ and Xq- published results). Taking all these data together, we conclude (14) (Fig. 3). Thus, the human GM1696 line possesses one intact that a human gene(s) which is located in the p22-qter region X chromosome and one intact chromosome 7 besides these of chromosome 7 is responsible for reversing the lesion of A9 translocation products. In human-mouse cell hybrids involving cells in EGF-binding ability. an X/autosome translocation, the intact X chromosome is late-replicating and often is lost in an early stage of hybrid DISCUSSION development (23). The translocation chromosome 7p+ is re- The data presented in this paper provide strong evidence that tained because it carries active HPRT which is necessary for binding of '25I-labeled EGF to the surface of hybrid cells, which hybrids to survive HAT selection. In fact, all the AUG hybrid were produced between mouse A9 cells and human fibroblasts lines that were maintained in HAT medium for more than 20 WI-38 or GM1696, is dependent on the presence of a single passages expressed human MDHM together with two X-linked human chromosome, chromosome 7. human markers, HPRT and G6PD, that are located on the long EGFs isolated from both humans and mice have a similar arm of the X chromosome (Fig. 3). Twenty-five additional molecular weight, 6000, share the same cell surface receptor human marker enzymes (Table 2) which represent 19 different sites, and exhibit identical biological functions, although there human chromosomes were also tested and found to segregate are differences in the amino acid sequences at 16 positions (1, independently of human MDHM (data not shown). Therefore, 3). Receptors for EGF are thought to be glycoproteins because coexpression of MDHM, HPRT, and G6PD can be attributed a number of lectins block binding of labeled EGF to the target to the translocation chromosome 7p+. cells (7). Purification of EGF receptors has been hampered by

Table 5. Concordant loss of EGF-binding ability, three human marker enzymes, and translocation chromosome 7p+ in two AUG hybrid lines after AG back-selection EGF- Translocation binding Human marker enzymes chromosome Cell line ability MDHM HPRT G6PD GPI LDHA 7p+* C2B5 + + + + + C2B5-AGt ------C3B4 + + + + + + + C3B4-AG7t - - - - + + * See Fig. 3. t Cells isolated in the presence of AG (20 ,g/ml). Downloaded by guest on September 26, 2021 3604 Genetics: Shimizu et al. Proc. Natl. Acad. Sci. USA 77 (1980) the fact that binding activity is lost after the plasma membrane the cell-bound EGF is internalized (unpublished results). By is solubilized (8). EGF receptors, however, were detected as a using a number of mutants with different lesions one would be chemically crosslinked complex with 125I-labeled EGF and their able to dissect the cascade of EGF/receptor-mediated hormonal subunit molecular weights were estimated to be in the range signal transfer mechanisms. Considering the complexity as 180,000-195,000 (6, 8, 24). By definition, the specific cell sur- discussed above, we use a gene symbol EGFS to represent the face components to which radioactive polypeptide hormones human gene(s) that complements the lesion of mouse A9 cells bind are hormone receptors (25). Therefore, the lack of EGF in EGF binding and that is involved in determining sensitivity binding in mouse A9 cells must be correlated with a deficiency to the EGF interaction. EGFS is located on the p22-qter region in receptors or components involved in synthesizing functional of human chromosome 7. receptors. The simplest and most favorable interpretation of We thank Drs. H. V. Aposhian and F. H. Ruddle for their continuous our results is that human chromosome 7 carries a gene(s) for the interest and encouragement throughout this work. This work was receptor structural protein. supported by National Institutes of Health Grant GM 24375. N.S. is It has been postulated that membrane proteins embedded a recipient of American Cancer Society Junior Faculty Research Award in the lipid bilayer are synthesized on membrane-bound ribo- JFRA-9. somes (26). During polypeptide synthesis the ribosomes are directed to specific sites in the endoplasmic reticulum and the 1. Carpenter, G. & Cohen, S. (1978) in Biochemical Actions of growing polypeptide chain is extruded through the membrane. Hormones, ed. Litwack, G. (Academic, New York), Vol. 5, pp. Carbohydrate is then added to the proteins, and the completed 203-247. glycoproteins finally appear in the plasma membrane. It is 2. Gospodarowicz, D. & Moran, J. S. (1976) Annu. Rev. Biochem. therefore possible that the A9 cells are capable of producing 45,531-558. EGF receptors but they are either not exposed at the cell surface 3. Carpenter, G. & Cohen, S. (1979) Annu. Rev. Biochem. 48, 193-216. or they are exposed but the carbohydrate moiety, presumably 4. Gospodarowicz, D., Greenburg, G., Bialecki, H. & Zetter, B. R. necessary for EGF binding, is lacking. We have tested possible (1978) In Vitro 14,85-118. accumulation of premature receptor proteins with negative 5. Schlessinger, J., Shecter, Y., Willingham, M. C. & Pastan, I. (1978) results-no binding of 125I-labeled EGF was found in A9 cells Proc. Natl. Acad. Sci. USA 75,2659-2663. fixed with ethanol and treated with acetone or dilute Triton 6. Das, M. & Fox, F. (1978) Proc. Natl. Acad. Sci. USA 75,2644- X-100 (unpublished results). 2648. Not only a defect in glycosylation of the receptor proteins 7. Carpenter, G. & Cohen, S. (1977) Biochem. Biophys. Res. but also a defect in the biosynthesis of amino sugars could lead Commun. 79,545-552. to incomplete glycosylation of receptor proteins and to de- 8. Hock, R. A., Nexo, E. & Hollenberg, M. D. (1979) Nature creased exposure to the cell surface (27). Such defects would (London) 277, 403-405. 9. Shimizu, N., Behzadian, M. A. & Shimizu, Y. (1979) Cytogenet. probably result in decreased formation of complex glycolipids Cell Genet., 25,201-202. and this may indirectly contribute to diminished EGF-binding 10. Hayflick, L. (1965) Exp. Cell Res. 37,614-636. ability. Alternatively, the full function of the EGF receptors 11. Littlefield, J. W. (1966) Exp. Cell Res. 41, 190-196. may require the presence of normal surface components in the 12. Littlefield, J. W. (1964) Science 145,709-710. vicinity of the receptor that facilitate EGF binding in a ste- 13. Siminovitch, L. (1976) Cell 7, 1-11. reospecific manner. If this is the case for the deficiency in A9, 14. Sauer, F., Greenstein, R. M., Reardon, P. & Riddick, D. (1977) these components should be encoded by the gene(s) on human Obstet. Gynecol. 49, 101-104. chromosome 7. 15. Nichols, E. A. & Ruddle, F. H. (1973) J. Histochem. Cytochem. Another possibility relates to the findings that sarcoma 21,1066-1081. 16. Shimizu, N., Giles, R. E., Kucherlapati, R. S., Shimizu, Y. & virus-transformed cells release a family of EGF-like growth Ruddle, F. H. (1977) Somat. Cell Genet. 3,47-60. factors into culture fluid and that they compete for EGF re- 17. Shimizu, N., Shimizu, Y. & Ruddle, F. H. (1978) Cytogenet. Cell ceptors (21,28). A9 cells are mutants derived from mouse L cells Genet. 22,441-445. which are known to secrete nerve growth factor-like poly- 18. Shimizu, N., Shimizu, Y., Kucherlapati, R. S. & Ruddle, F. H. peptides (29). A9 cells are not known for the presence of murine (1976) Cell 7, 123-130. sarcoma virus although they possess many features that are 19. Kozak, C. A., Lawrence, J. B. & Ruddle, F. H. (1977) Exp. Cell commonly associated with transformed cells (11). Thus, the Res. 105, 109-117. apparent loss of EGF-binding ability could be attributed to the 20. Carpenter, G. & Cohen, S. (1976) J. Cell Biol. 71, 159-171. blocking or masking of EGF receptors by closely related 21. Todaro, G. J., De Larco, J. E. & Cohen, S. (1976) Nature (Lon- polypeptides. Then, restoring don) 264,26-31. of binding ability in hybrid cells 22. McKusick, V. A. & Ruddle, F. H. (1977) Science 196, 390- could be considered to be the result of suppression of poly- 405. peptide synthesis by human on chromosome 7. This in- 23. Kahan, B. & DeMars, R. (1975) Proc. Natl. Acad. Sci. USA 72, teresting possibility must be extensively examined although our 1510-1514. preliminary experiments indicated that culture medium of A9 24. Das, M., Miyakawa, T., Fox, C. F., Pruss, R. M., Aharonov, A. & cells does not inhibit the binding of 125I-labeled EGF to human Herschman, H. R. (1977) Proc. Natl. Acad. Sci. USA 74, HeLa cells (unpublished results). 2790-2794. In summary, we have demonstrated the expression and 25. Roth, J. (1975) Methods Enzymol. 37B, 66. segregation of a polypeptide hormone-binding ability on the 26. Blobel, G. & Dobberstein, B. (1975) J. Cell Biol. 67,852-862. cell surface of human-mouse cell hybrids. 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