c-erbA: PROTOONCOGENE OR GROWTH SUPPRESSOR GENE? Klaus Damm Gene Expression Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037, and Department of Neuroendocrinology, Max-Planck-Institute for Psychiatry, 8000 Munich 40, Germany

I. Introduction Avian Erythroblastosis Virus as a Model for Cooperativity of Oncogenes T h e Protooncogene c-ei-bA Encodes a Thyroid Hormone Receptor Multiple c-erbA Loci Structural Differences between v-erbA and c-erbA Functional Properties of the ErbA Proteins A. Hormone Binding B. DN.4 Binding C. Oncogene (v-erbA) and Protooncogene (c-erbA) Act as Transcription Factors D. Cotransfection and Competition VII. Mutations Affecting the Biological Activity of v-erbA VIII. c-ErbA Regulation of Erythroid Differentiation and Gene Expression IX. c-erbA: Protooncogene o r Growth Suppressor Gene? X. Mutations Affecting c-erbA Function XI. Current Concepts and Open Questions References 11. I1 I. IV. V. VI.

I. Introduction

Cellular growth and differentiation are mutually exclusive and cancer has been commonly linked to aberrant differentiation and a failure of the tumor cells to differentiate. Tumor cells usually show multiple genetic lesions, including gene amplification, chromosomal translocations, and point mutations. In a number of cases cooperativity between oncogenes has been observed and a multistep transformation process may involve a primary oncogene providing a continuous proliferation signal with a second oncogene blocking the ability of these cells to differentiate. In a distinct mechanism cellular transformation may also be achieved by the inactivation of genes whose normal function is to constrain cell growth by either suppressing proliferation or inducing differentiation, thereby acting normally in a fashion opposite to the oncogenes, and it is the loss or inactivation of these genes that would trigger cancer development. The efficient cellular transformation induced by retroviruses carrying a pair of cooperating oncogenes might be achieved by a combination of these 89 ADVANCES IN CANCER RESEARCH, VOL. 59

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two mechanisms. One well-studied example is the v-erbA and v-erbB oncogenes of the avian erythroblastosis virus (AEV), where v-mbA function is needed to provide a complete block in differentiation of erythroid precursor cells transformed by v-erbB. In this article, the molecular mechanisms by which v-erbA potentiates transformation and influences the growth requirements of cells will be discussed in the context of normal c-erbAlthyroid hormone receptor function.

II. Avian Erythroblastosis Virus as a Model for Cooperativity of Oncogenes The avian erythroblastosis virus (AEV) represents an acute chicken retrovirus capable of inducing lethal erythroleukemia and sarcomas in vivo, properties paralleled by its transformation of hematopoietic cells and fibroblasts in vitro (for review, see Graf and Beug, 1978, 1983). Erythroblastosis induced by AEV is characterized by the appearance of large, blastlike cells in the peripheral blood as early as 5 days after intravenous injection. Infection of chicken bone marrow cells with AEV in uitro leads to the appearance of rapidly proliferating blastlike cells. In contrast to normal erythroid progenitor cells, AEV-transformed erythroblasts in culture are capable of extensive self-renewal in the absence of terminal differentiation throughout their life span of 10-40 generations. This suggests that the virus transforms its target cells either by arresting their differentiation or by inducing their proliferation. The cells that are susceptible to transformation by AEV have been characterized by using a series of antisera specific for cells of various hematopoietic cell lineages. These studies indicate that most of the target cells are at the burst forming unit-erythroid (BFU-E) stage. The molecular cloning of AEV revealed that the viral genome contains two oncogenes derived from the chicken genome (Fig. 1; Vennstrom et al., 1980; Vennstrom and Bishop, 1982). The first oncogene, v-erbB, is transduced from the gene that encodes the cell surface receptor for epidermal growth factor (Downward et al., 1984; Ullrich et al., 1984),whereas the second oncogene, v-erbA, exhibits extensive structural similarities to genes encoding intracellular receptors for steroid hormones and was subsequently shown to be a mutated derivative of the chicken thyroid hormone receptor a gene (Sap et al., 1986; Weinberger et al., 1986). An analysis of the transforming capacities of viral deletion mutants in both the v-erbA and the v-erbB genes led to the following conclusions (Fig. 2). Neither of the two mutant types is capable of inducing a typical AEV

-08kb+-

LTR

Agag

1 z k b __c

- -

erbA

lSkb

erbB

LTR

FIG. 1. Structure and expression of the proviral genome of AEV. In AEV the retroviral pol and e m genes have been replaced by two cell-derived oncogenes, erbA and erbB. The genome is transcribed into two RNAs, a genomic length RNA coding for the 75-kDa GagErbA fusion protein and a subgenomic RNA encoding the 68-kDa ErbB glycoprotein.

v-erbA + v-erbB

ftc

c) v-erbB

v-erbA

-0

ftc

FIG. 2. Contribution of v-erbA and v-erbB to the leukemic phenotype. Normal erythroblasts exhibit only a limited self-renewal capacity and differentiate to mature erythrocytes. v-erbE induces infected erythroid progenitors to self-renew and abrogates the requirements of these cells for erythroid growth factors, e.g., erythropoietin. However, v-erbB does not completely block erythroid differentiation and causes only a weak erythroleukemia in chicken. v-erbA cooperates with v-erbB by completely arresting their spontaneous differentiation and bypassing the complex growth requirements of v-erbBtransformed cells. Acting by itself, v-erbA is sufficient to arrest differentiation of normal erythroid progenitors but is unable to induce sustained self-renewal.

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erythroblastosis in uiuo or in uitro. However, v-erbB is the primary transforming gene of AEV since it is sufficient for transformation of fibroblasts and erythroid progenitor cells, the two types of target cells that AEV interacts with in uiuo and in uitro (Frykberg et al., 1983; Fung et al., 1983; Sealy et al., 1983; Yamamoto et al., 1983). The described effects of v-erbB in chicken cells include its ability to abrogate the requirement for mitogens such as epidermal growth factor (EGF), transforming growth factor a (TGFa), or the erythroid growth factor erythropoietin, the induction of an abnormal self-renewal in erythroid precursor cells, and phenotypical changes typical for transformed fibroblasts (Beug et al., 1982, 1985; Royer-Pokora et al., 1978; Khazaie et al., 1988; Pain et al., 1991). However, leukemia cells induced by v-erbB alone resemble normal CFU-E in that they exhibit a significant potential to differentiate spontaneously. Furthermore, they grow only under culture conditions similar to those that promote the differentiation of normal erythroid cells in uitro (Beug et al., 1982, 1985). T h e v-erbA oncogene, on the other hand, does not exhibit a strong transforming capacity by itself. In chicken fibroblasts v-erbA elicits only modest biological effects such as enhancement of agar colony formation, in vitro life span, and production of extracellular matrix proteins (Frykberg rt al., 1983; Jansson ct ul., 1987; Grandrillon et al., 1987). In hematopoietic cells, v-erbA by itself induces an aberrant, largely immature phenotype, characterized by the coexpression of erythroblast arid erythrocyte differentiation antigens and indicative of its ability to arrest differentiation (Frykberg et a[., 1983; Grandrillon et al., 1989; Schroeder et al., 1990). Important clues as to the respective roles of v-erbA in erythroblast transformation came from the analysis of two AEV derivatives, AEVtdSSY,a transformation defective mutant of AEV unable to transform erythroblasts (Royer-Pokora et al., 1979), and its revertant AEV' I Z , obtained after in uzuo passage of the AEVtclS.iSmutant (Damn1 et al., 1987). Nucleotide sequence analysis of the cloned viral genomes revealed that the v-ErbA protein of AEVtdS5gcontained among other substitutions one amino acid change at position 144 of c-ErbA, a Pro Arg change (Damm et al., 1987). The v-ErbA protein of AEV'12 had reverted to a proline in this position. T h e v-ErbB genes of both the mutant and the revertant carry identical deletions in their 3' ends: a 306nucleotide deletion removed most of the region located after the domain of v-ErbB homologous to tyrosine kinases. To analyze the role of these mutations in v-ErbA and v-ErbB for erythroblast transformation, recombinant viruses containing all possible combinations of AEV"', AEVtd35g, and AEVrl* v-erbA and v-erbB genes were constructed. T h e results of subsequent bone marrow transformation experiments with the resulting

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viruses showed that the mutant v-erbB genes were unable to transform erythroblasts in the absence of an active v-erbA gene: v-erbA of AEV'" or AEVwtbut not AEVtd359was able to restore the erythroblast-transforming activity to the mutant v-erbB genes, confirming that the reversion in the AEVr12 genome had occurred in its v-erbA oncogene (Damm et al., 1987). These and other studies showed that v-ErbA contributes to the transformed phenotype in two ways. First, v-ErbA enhances the transforming activity of v-ErbB, and restores the erythroblast-transforming potential of transformation-defective v-ErbB mutants (Damm et al., 1987; Jansson et al., 1987) by blocking the residual, spontaneous differentiation program in erythroid precursors. In addition, v-ErbA enables a variety of tyrosine kinase-encoding oncogenes (e.g., v-src, v-fps, v-sea, and v-fms) as well as v-Ha-ras to transform erythroblasts, all of which by themselves are unable to do so (Kahn et al., 1986). Second, v-ErbA abrogates the complex growth requirements of transformed erythroblasts and enables these cells to proliferate under a wide range of pH and HC0,-/Na+ concentration (Beug et al., 1985; Damm et al., 1987; Kahn et al., 1986). III. The Protooncogene c-erbA Encodes a Thyroid Hormone Receptor

The cloning of the human glucocorticoid receptor (hGR) cDNA represented a major breakthrough in our understanding of the mechanisms by which v-ErbA exerts its oncogenic activity. A comparison of the amino acid sequences revealed a remarkable homology between the carboxyterminal half of hGR and the v-ErbA oncoprotein (Hollenberg et al., 1985; Weinberger et al., 1985). The relationship of hormone receptors to v-ErbA was independently confirmed by the cloning of receptors for all major classes of steroid hormones as well as retinoic acid (vitamin A) and vitamin D, receptors (for review, see Evans, 1988; Green and Chambon, 1988; Beato, 1989). T h e region with maximal homology between hGR and v-ErbA corresponds to a cysteine-rich region; 9 out of 10 cysteine residues of hGR are conserved in this high-identity region with v-ErbA (Weinberger et al., 1985; Green et al., 1986). The presence of this structure, which embodies the DNA-binding domain in the hormone receptors, suggests that the v-ErbA oncoprotein itself might bind to DNA and may function in oncogenesis by inappropriately modulating transcription of specific target genes in the host cell. Significant homology was also found in the ligand-binding domain, which suggests that the cellular homolog of v-erbA, the protooncogene c-erbA, encodes a receptor for a steroid-related ligand. Two groups succeeded in the molecular cloning

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and characterization of the c-erbA protooncogene product: Sap et al. (1986) cloned the authentical c-erbA protooncogene from chicken embryo and Weinberger et al. (1986) identified a c-erbA gene from a human placenta library. To the surprise of most of the “receptorologists,” both groups could demonstrate the c-erbA protooncogene products do not bind steroids but bind thyroid hormones [3,5,3’-triiodo-~-thyronine (T3), 3,5,3’,5’-tetraiodo-~-thyronine (TJ, 3,5,3’-triiodo-~-thyroacetic acid (TRIAC)] with high affinity and specificity and may therefore be the chicken and human receptors for these hormones. The identity of c-erbA as a thyroid hormone receptor (TR) was further supported by the nuclear localization of the chicken c-erbA product (Sap et al., 1986). IV. Multiple c-erbA Loci

A surprising observation in the comparison of the chicken and human c-ErbA amino acid sequences was the relatively low (91%) homology in the DNA-binding domain, which is in contrast to human and chick estrogen receptors or human and rat glucocorticoid receptors, where the DNA-binding domains are entirely conserved. The two c-erbA products may thus correspond to similar but not identical TRs, suggesting the possibility of multiple TRs and corresponding genes in a given species. Indeed, previous studies localized a v-erbA related sequence to human chromosome 17 (17q11.2-17q21) Jansson et al., 1983; Dayton et al., 1984; Spurr et al., 1984) whereas the cloned human c-erbA is localized to chromosome 3 ( 3 ~ 2 2 - 3 ~ 2 4 . 1(Weinberger ) et al., 1986; Drabkin et al., 1988). Based on amino acid homologies and chromosomal localizations, thyroid hormone receptors from human, rat, mouse, chicken, and Xenopus laevis have been classified into two groups, a and (Fig. 3; Sap et al., 1986; Weinberger et al., 1986; Thompson et al., 1987; Benbrook and Pfahl, 1987; Nakai et al., 1988a,b; Lazar et al., 1988, 1989a; Murray et al., 1988; Koenig et al., 1988; Izumo and Mahdavi, 1988; Prost et al., 1988; Hodin et al., 1989; Forrest et al., 1990a; Yaoita et al., 1990). The c-erbAITRa gene locus produces different mRNAs that give rise to multiple divergent receptor proteins (Thompson et al., 1987; Benbrook and Pfahl, 1987; Nakai et al., 1988a; Lazar et al., 1989b; Murray et al., 1988; Mitsuhashi et al., 1988; Izumo and Mahdavi, 1988). The proteins encoded are identical for the first 370 amino acids and then diverge completely. This digression of TRa-2 from the sequence in rat and human T R a spans the ligand-binding domain, probably accounting for the fact that the protein encoded by TRa represents an authentic receptor whereas the protein encoded by TRa-2 does not bind hor-

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hormone binding

DNA binding

transactivation

+

TRa2

TRa

+

+

+

TRP

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FIG.3. Comparison of c-ErbAIthyroid hormone receptor isoforms. Homology of different domains of the TRs are compared; numbers refer to the percentage similarity at the amino acid level and domains with little or no homology are indicated with different shading. Functional properties such as hormone binding, DNA binding, and trans-activation, are listed.

mone. Both proteins, however, contain the DNA-binding domain and can interact with putative TREs (Koenig et al., 1988; Izumo and Mahdavi, 1988; Lazar et al., 1988). Furthermore, in rat and human there seem to be multiple splicing variants of the TRa-2 (Izumo and Mahdavi, 1988); at least one of these forms differs in its amino acid sequence by a deletion of 39 amino acids at the beginning of the divergent carboxyterminal region (Mitsuhashi et al., 1988; C . Thompson, K. Damm, and R. M. Evans, unpublished results). In the chicken, no TRa-2 has been found and the 4.5-kb mRNA is apparently expressed ubiquitously, with elevated levels during the late stages of erythrocyte differentiation (Sap et al., 1986; Hentzen et al., 1987; Forrest et al., 1990b).In rat and human, Rev-ErbAa, a non-T,-binding variant of the hormone receptor family, is also encoded at this genomic locus (Miyajima et al., 1989; Lazar et al., 1989a). The DNA strand coding for Rev-ErbAa is opposite of that encoding T R a proteins. As a result, the mRNAs encoding Rev-ErbA and TRa-2 are complementary for 269 nucleotides whereas T R a and RevErbA mRNAs are derived from nonoverlapping regions (Miyajima et al., 1989; Lazar et al., 1990). The c-erbA/TRP gene locus encodes at least two receptors, TRP, the human c-erbA clone described by Weinberger et al. (1986), and TRP-2, which differs completely at the N terminus but represents a bona fide T R by the criteria of T, and DNA binding (Hodin et al., 1989). TRP is expressed in most tissues of rat and humans, with somewhat elevated levels in liver, kidney, and hypothalamus. An intriguing aspect of the rat TRP-2 form is that it is expressed only in the pituitary gland and that its

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expression is down regulated by T, (Hodin et al., 1989, 1990). In the chicken, temporal and tissue-specific expression has been reported, suggesting specific developmental functions for TRP (Forrest et al., 1990a). In human, the syndrome of generalized thyroid hormone resistance has been tightly linked to the TRP gene and multiple mutations in T R P have been reported in affected kindreds (Sakurai et al., 1989; Usala et al., 1990, 1991a,b). V. Structural Differences between v-erbA and c-erbA

Examination of the v-ErbA amino acid sequence reveals that the v-ErbA protein represents a heavily mutated version of the chicken c-ErbA/TRa (Fig. 4; Sap et al., 1986). In the AEV viral genome the erbA-

A 1

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55 GDKATGYHYR CITCEGCKGF FRRTIQKNLH PTYSCKYDGC CVIDKITRNQ CQLCRFKKCI 295 GDKATGYHYR CITCEGCKSF FRRTIQKNLH PTYSCTYDGC CVIDKITRNQ CQLCRFKKCI

c-erbA v-erbA

115 SVGMAMDLVL DDSKRVAKRK LIEENRERRR KEEMJKSLQH RPSPSAEEWE LIHVVTEAHR 355 SVGMAMDLVL DDSKRVAKRK LIEENRERRR KEEMIKSLQH RPSPSAEEWE LIHVVTEAHR

c-erbA v-erbA

1 7 5 STNAQGSHWK QKRKFLPEDI GQSPMASMPD GDKVDLFAFS EFTKIITPAI TRVVDFAKKL 415 STNAQGSHWK QRRKFLLEDI GQSPMASMLD GDKVDLEAFS EFTKIITPAI TRVVDFAKNL

c-erbA v-erbA

235 PMFSELPCED QIILLKGCCM EIMSLRAAVR YDPESETLTL SGEMAVKREQ LKNGGLGWS

c-erbA v-erbA

295 DAIFDLGKSL SAFNLDDTEV ALLQAVLLMS SDRTGLICVD KIEKCQETYL LAF'EHYINYR 535 DAIFDLGKSL SAFNLDDTEV ALLQAVLLMS SDRTGLICVD KIEKCQESYL LAFEHYINYR

c-erbA v-erbA

355 KHNIPHFWPK LLMKVTDLRM IGACHASRFL HMKVECPTEL FPPLFLEVFE DQEV 595 KHNIPHFWSK LLMKVADLRM IGAYHASRFL HMKVECPTEL Sip-------- -QEV

MEQK PSTLDPLSEP EDTRWLDGKR KRKSSQCLVK SSMSGYIPSY LDKDEQCWC EDTRWLDGKE KRXSSQCLVK SSMSGYIPSC LDKDEQCWC

y v m w r m EGPAWTPLEP

475 PMFSELPCED QIILLKGCCM EIMSLRAAVR YDPESETLTL SGEMAVKREQ LKNGGLGWS

FIG. 4. (A) Schematic organization of chicken c-ErhA and v-ErhA protein sequences. DNA and T3/T4 refer to the DNA- and hormone-binding domains, respectively. (B) Amino acid differences between c-ErhA and v-ErbA. Amino acid sequences of chicken c-ErbA and v-ErbA are compared in the one-letter code. Differences are indicated by hold letters and shading;-, deletions.

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specific sequences form one long open reading frame together with 5' gag sequences to code for a protein of 75 kDa (p75gUg-"lbA). The chicken c-erhA/TRa gene product is a protein of 46 kDa. In p75gag-erbAthe 253 N-terminal amino acids derived from the retroviral gag gene are fused in frame to chicken T R a sequences starting at amino acid 13. A comparison between the c-erbA/TRa coding sequence (Debuire et al., 1984; Damm et al., 1987; Sap et al., 1986) and the gag sequences of Rous sarcoma virus (Schwartz et al., 1983) demonstrated a region with 19 of22 nucleotide identities that bridges the junction between the gag and erbA domains in p75ga:afi-erhA. This suggests that c-erbA/TRa was fused to gag either by homologous recombination at the DNA level or during retrotranscription of c-erhA/TRa mRNA packaged into virions. The coding sequence of the erbA-specific part of the molecule shows 17 point mutations in v-erbA as compared to the c-rrbA/TRa sequence (Damm et al., 1987; Sap et al., 1986). These mutations lead to 12 amino acid substitutions, 2 of which are located between the gag and the DNA-binding domains, two within the DNA-binding domain, and the remaining eight in the region corresponding to the ligand-binding domain. Finally, ~75g"g-~*b* exhibits a nine-amino acid deletion three amino acids from the carboxy terminus. As a functional consequences of the mutations in the ligand-binding domain, the v-ErbA product is defective in binding thyroid hormones (Sap et al., 1986).

VI. Functional Properties of the ErbA Proteins Steroid/thyroid hormone receptors act as transcriptional regulatory proteins whose ability to control gene expression is dependent on the binding of their specific. ligand (for review, see Evans, 1988; Beato, 1989). Regulation of transcription results from the specific interaction of the hormone-receptor complex with "hormone response elements" in the promoter region of target genes. T h e cloning of the c-erhA gene provided the first opportunity to dissect the structure and functional properties of both the oncogene (v-erbA) and protooncogene (c-erbA /TR). Using cotransfection assays, the thyroid hormone receptors encoded by the c-erbA genes have been shown to act as hormone-inducible trans-acting factors similar to other hormone receptors of this class (Umesono et al., 1988; Koenig et al., 1988; Damm et al., 1989; Thompson and Evans, 1989). T h e most exciting development in our understanding of v-ErbA and c-ErbA/TR activity, however, has been the demonstration that v-ErbA and unliganded thyroid hormone receptors can bind thyroid hormone response elements (TREs) and may act as constitutive negative regulators of genes containing these elements (Damm et d.,

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1989; Sap et al., 1989). Mutational analysis and structural comparisons of the different members of the steroid hormone receptor family identified specific domains responsible for hormone binding, DNA binding, and transcriptional activation (for review, see Evans, 1988; Green and Chambon, 1988; Beato, 1989). Similar domains and properties can be found in the v-erbAlc-erbAITR subfamily. A. HORMONE BINDING The initial characterization of in vitro-translated c-ErbA protein demonstrated that it can bind thyroid hormones with affinities (K = 0.2 nM) similar to the values obtained with thyroid hormone receptors in whole cells and in nuclear extracts (Sap et al., 1986; Weinberger et al., 1986). Sap et al. also tested chicken embryo fibroblasts containing either c-ErbA or v-ErbA for hormone binding. The nuclei from cultures of c-ErbA-expressing cells bound T, at levels similar to that found in GH1 cells, a rat pituitary cell line expressing endogenous thyroid hormone receptors. In contrast, only a low amount of specific T, binding was found in v-ErbA-expressing cells, demonstrating the defectiveness of the oncogene product for binding the ligand (Sap et al., 1986). To identify which of the mutations in v-ErbA conferred the inability to bind hormone, Mufioz et al. (1988) characterized a series of recombinants between the viral and cellular genes. Recombinant proteins synthesized in reticular lysates revealed that as expected the c-ErbA product binds T, 12 times better than v-ErbA. A progressive reduction in hormonebinding ability results from introducing into c-ErbA the mutations present in the ligand-binding domain of v-ErbA. These results reveal that the point mutations and the nine-amino acid deletion close to the C terminus act together in abolishing hormone binding and suggest that hormone-independent action by v-ErbA provided a selective advantage to AEV during its selection as a highly and acutely oncogenic virus strain (Mufioz et al., 1988). However, the cellular environment or external factors may also influence binding activity since it was recently shown that in the yeast Saccharomyces cerevisiae the v-ErbA protein responds to high concentrations of the thyroid hormone derivative TRIAC (Privalsky et al., 1990). Thus, cellular components influencing the ability of the v-ErbA product to respond to hormone may be important in the oncogenicity of v-ErbA.

-

B. DNA BINDING The identification of thyroid hormone response elements (TREs) in the promoter regions of regulated genes made it possible to demonstrate

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sequence-specific DNA binding by both c-ErbA and v-ErbA proteins. Putative TREs have been identified in the rat growth hormone (Glass et al., 1987; Koenig et al., 1987; Bonde and Privalsky, 1990; Sap et al., 1990), a-myosin heavy chain (Izumo and Mahdavi, 1988), Moloney leukemia virus long terminal repeat (LTR) (Sap et al., 1989), the malic enzyme (Petty et al., 1990), and in the erythrocyte specific carbonic anhydrase I1 (Disela et al., 1991) genes. A variation of the rat growth hormone TRE that is characterized by the palindromic motif 5’-TCAGGTCATGACCTGA-3’ represents a very effective TRE (Glass el al., 1988; Umesono et al., 1988) and was used in the experiments by Damm et al. (1989) described below. When whole-cell extracts of COS cells expressing c-ErbA were incubated with a 32P-labeled TRE and separated in a nondenaturing acrylamide gel, a protein-DNA complex migrating more slowly than the free DNA was observed, regardless of whether the cells were cultured in the absence or presence of T,. Similarly, T, had no effect on the appearance of the retarded complex when it was added to extracts of transfected COS cells that were not exposed to the hormone in uivo. Hormone-independent formation of retarded complexes was also observed with extracts from cells expressing v-ErbA (Damm et al., 1989; Sap et al., 1989). Thus, the v-ErbA protein is able to selectively bind to specific DNA sequences in uitro, demonstrating that the amino acid changes in the DNA-binding domain of v-ErbA do not grossly interfere with DNA binding activity. However, these amino acid differences might nevertheless influence binding affinity and target gene specificity of v-ErbA (Damm et al., 1989; Sap et al., 1989; Bonde and Privalsky, 1990) because the first of the amino acid substitutions in the DNAbinding domain is located just after the first zinc finger and represents one of the three amino acids that are important in discriminating the sequence of the hormone response element (Umesono and Evans, 1989; Mader et al., 1989; Danielsen et al., 1989). The second amino acid substitution is in between the two cysteine residues at the base of the second zinc finger, a region that stands as a potential interface of receptor dimerization (Umesono and Evans, 1989). C. ONCOGENE (v-erbA) AND PROTOONCOGENE (c-erbA) ACT AS TRANSCRIPTION FACTORS The transcriptional activity of both c-ErbA and v-ErbA products was assessed by their ability to regulate expression of thyroid hormoneresponsive reporter genes (Damm et al., 1989). These constructs contain oligonucleotides corresponding to previously identified thyroid hormone response elements linked to a heterologous promoter and the

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chloramphenicol acetyltransferase (CAT) gene (Umesono et al., 1988). In a cotransfection assay, expression plasmids containing c-erbA (rTRa cDNA) or the v-erbA oncogene under the transcriptional control of the Rous sarcoma virus (RSV) long terminal repeat were cotransfected with one of the reporter plasmids into CV1 cells, which lack significant levels of endogenous TR. Transfection of RSV-rTRa together with AMTVTREp-CAT, containing a palindromic response element, resulted in a hormone-dependent 20-fold stimulation of reporter gene expression, demonstrating that the c-ErbA/TR product is, as expected, a potent, hormone-dependent transcriptional activator (Fig. 5A).This functional assay enabled also a direct determination of the putative transcriptional activity of the v-erbA oncogene product. Since v-ErbA has lost its ability to bind thyroid hormone but retains an intact DNA-binding domain, it seems logical that it would function as a constitutively active TR. Unexpectedly, no constitutive, hormone-independent stimulation of transcription could be observed when AMTV-TREp-CAT was cotransfected with a v-erbA expression plasmid. However, when a different reporter construct, containing the same response element (TREp) but a different promoter (tk), was used in this cotransfection paradigm, novel regula-

A

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FIG. 5. (A) Trans-activation assay for ErbA proteins. CVl cells were transfected with the reporter construct AM-TREp-CAT and the respective expression plasmid. Thyroid hormone was added as indicated. Induction values are typically 20- to 30-fold using either c-ErbA/TRa or while no activity was observed with v-ErbA. (B) Repression of basal promoter level. CV 1 cells were transfected with the reporter construct tk-TREpP-CAT and the respective expression plasmid. All experiments were performed in the absence of thyroid hormone. c-ErbA and v-ErbA caused an -80% reduction of basal promoter activity. (C) Competition of T3 induction. The reporter gene AM-TREp-CAT was cotransfected into CVl cells with 1 pg c-ErbA expression vector and increasing quantities of the non-hormone-binding competitor v-ErbA. An increase in v-ErbA expression leads to a decrease in the hormone-dependent trans-activation by c-ErbA.

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tory properties of both v-ErbA and c-ErbA were revealed (Fig. 5B). Transfection of tk-TREp-CAT alone resulted in a relative high basal level of CAT activity whereas cotransfection with the v-erbA expression vector resulted in an 80% decrease in basal CAT activity that could not be relieved by the addition of T,. This marked effect on tk-TREp-CAT expression demonstrated that v-ErbA provokes a ligand-independent inhibitory effect on transcription and can act as a constitutive repressor of TRE-containing genes. Surprisingly, the c-ErbA protein, in the absence of ligand, induced a similar repression of the reporter construct. In the case of c-ErbA, however, addition of thyroid hormone resulted in a 20-fold induction of transcription, suggesting that the primary effect of the hormone is to trigger a conformational change in the DNA-bound receptor that relieves the repression activity and reveals or induces the activation function. In this context, v-ErbA acts as a constitutive repressor of gene transcription because the point mutations in the ligand binding domain as well as the C-terminal deletion impaired hormone binding and transcriptional activation functions. D. COTRANSFECTION A N D COMPETITION Chicken erythroid progenitor cells were shown to contain c-ErbA products, suggesting that these proteins are involved in erythroid differentiation (Hentzen et al., 1987; Bigler and Eisenman, 1988). Therefore, in AEV-infected erythroid cells, v-erbA and c-erbA might be coexpressed. It seems likely that v-ErbA occupies c-ErbA binding sites on the DNA, thereby interfering with the function of the endogenous c-ErbA and acting as a constitutive repressor of gene transcription because it has lost the ability to bind and thus to be regulated by T,. This type of dominant negative oncogene function has been described on theoretical grounds by Herskowitz (1987) and was proposed by Bishop (1986) as a possible mechanism of v-ErbA function. To evaluate this property of v-ErbA, CV 1 cells were cotransfected with the AMTV-TREp-CAT reporter plash i d and a c-erbAlrTRol-expression construct (Damm et al., 1989). The levels of expression of v-ErbA"' competitor proteins were varied by titrating the amounts of the respective expression plasmids transfected into the recipient cells. These experiments revealed that v-ErbA serves as a negative regulator of thyroid hormone action since the transcriptional response to thyroid hormone is drastically reduced as the proportion of v-ErbA protein is increased (Fig. 5C). A 1: 1 ratio of c-ErbA and v-ErbAwtresulted in a 70% inhibition and a 3: 1 ratio completely blunted the hormonal response (Damm et al., 1989). Using a TRE identified in the Moloney leukemia virus LTR, Sap et al. (1989) also found a v-ErbA-

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induced inhibition of the T, response. Because an intact DNA-binding domain is essential for v-ErbA function and biological activity (Damm et al., 1989; Privalsky et al., 1988), the competition or antagonism apparently occurs at the level of the response element by blocking the binding of c-ErbA to its template. T h e effectiveness of v-ErbA in blocking c-ErbA function may be a consequence of altered DNA-binding properties, e.g., a higher affinity for or a lower off rate from the DNA. On the other hand, c-ErbA is thought to bind DNA as a dimer and thus might form heterodimers with the v-ErbA product. If these c-Erb/v-ErbA heterodimers are inactive then, at a 1:l ratio, 75% of the homo- and heterodimers in a cell would be of the inhibitory type. Since the DNA-binding domain has been implicated in dimer formation of the receptor molecules it might well be a combination of the two mechanistic explanations that make v-ErbA function so effectively. These results place v-er6A as a dominant negative oncogene, dominant because its phenotype is manifested in the presence of the wild-type gene, and as it inactivates at least one wild-type gene function, it acts negatively. VII. Mutations Affecting the Biological Activity of v-erbA The functional properties of a biologically inactive v-erbA gene derived from the mutant AEVtd359virus (Damm et al., 1987) directly link the transforming potential of v-er6A to its ability to act as a negative regulator of transcription (Damm et al., 1992). First, when v-ErbAfdwas tested for its ability to act as a repressor of the basal promoter level, the mutant protein, in contrast to v-ErbAwt,failed to reduce promoter activity significantly. Second, v-ErbAtd also showed a reduced ability to act as a dominant negative inhibitor of the hormone-activated c-erbA. A 1: 1 plasmid ratio of c-erbA and the mutant v-erbAtd introduced into CV1 cells resulted in no significant reduction of the transcriptional response, whereas cotransfection of v-er6AWtunder identical conditions resulted in a 70% inhibition. Furthermore, v-ErbAr12, a natural revertant of v-ErbAtd which regained its biological activity (Damm et al., 1987), antagonizes the c-er6A activation as well as v-ErbAwfdoes. T h e defectiveness of the v-ErbAtd protein was not due to an impairment of its ability to interact with a thyroid hormone response element (TRE), since in an electrophoretic mobility shift assay retarded protein-DNA complexes were observed with v-ErbAfd-, v-ErbAwf-,and c-ErbA/TRa-containing extracts but not with untransfected control extract. Thus, the Pro+ Arg change that is responsible for the biological defectiveness of v-ErbAtd, and which is reverted in v-ErbAr12 (Damm et al., 1987), severely and

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specifically affects the negative regulatory functions and the ability of v-ErbA to act as a dominant negative inhibitor of the thyroid hormone receptor. To identify the contribution of the different mutations in v-ErbAwtto the altered properties of this protein, to confirm the deleterious effect of the arginine mutation in v-ErbAtd, and to further dissect the processes of activation and repression, chimeric receptors of the v-erbA oncogene and the rat c-erbA/TRa were constructed and analyzed (Fig. 6; Damm et al., 1989). From amino acids 154 to 410, v-ErbAwfdiffers from rat c-ErbA/TRa in 26 amino acids and a deletion of 9 amino acids close to the carboxy terminus. Substitution of this region between rTRa and v-ErbA"' gives rise to the hybrid TR( 1 54)Awt,whose properties are virtually identical to the v-ErbAWthomolog. Thus, the mutations of the DNA-binding domain and flanking amino acids are not crucial to the v-ErbA phenotype. The mutations responsible for the functional conversion of c-ErbA into v-ErbA are localized to the ultimate C terminus, since replacement of the carboxy-terminal93 amino acids of rat c-ErbA/TRa with the corresponding sequence of v-ErbAwtyielded the hybrid protein TR(31 7)Awt,with suppressor properties identical to the viral oncogene product. These observations were supported in several subsequent transformation studies that demonstrated the importance of the v-ErbAwtC terminus for biological function and transcriptional repression in erythroid cells (Privalsky et al., 1988; Boucher and Privalsky,

T

R

C

c-erbA/TR

+

+

+

v-erbA

-

+

+

v-erbA-td

-

( 154/3 16)A

+

+

+

(1 54/31 6)A-td

+

-

-

-

FIG.6. Structure and activity of c/v-ErbA chimeric proteins. The origin of the different domains in the chimeras is indicated by different shading; R depicts the P-to-R mutation in v-ErbALd.Hormone-dependent trans-activation (T), repression of basal promoter activity (R), and competition of hormone induction (C) were performed as described in Fig. 5. Ts-binding derivatives were tested in the C-assay for competition against the retinoic acid receptor.

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1990; Forrest et al., 1990b; Zenke et al., 1990). However, other regions in the ligand-binding domain have also been implicated in v-ErbA transcriptional and biological control functions. The hybrid TR( 154)Atd, containing the Pro + Arg mutation found in v-ErbAfd but otherwise structurally similar to TR( 154)Awt,exhibited properties that are in striking contrast to TR(154)Awt. Similar to v-ErbAtd, TR(154)Atd did not lower the basal transcription level of tk-TREp2-CAT and did not inhibit the hormone-dependent trans-activation by c-ErbA, demonstrating that the structural integrity of the region harboring the mutation, the socalled hinge region linking the DNA to the ligand-binding domain, seems to be critical for the correct function of the ErbA proteins. This is further supported by additional chimeric constructs in which a substitution of an internal region of the ligand-binding domain, amino acids 154-3 16, creates T,-responsive hybrids [TR(154/316)Awt and TR( 154/316)Atd]that exhibit activation properties similar to c-ErbA. However, in the absence of ligand TR( 154/316)Atd does not lower the basal promoter level. This is again in contrast to the equivalent TR( 154/316)Awtproduct and demonstrates clearly that the Pro + Arg change selectively abolishes the negative regulatory properties of the ErbA proteins (Damm et al., 1992). VIII. c-ErbA Regulation of Erythroid Differentiation and Gene Expression

T h e question of how v-ErbA, c-ErbA, and chimeras of both exert specific effects in erythroblasts was approached by using recombinant retroviruses to introduce the various ErbA derivatives into target cells (Zenke et al., 1988, 1990; Boucher and Privalsky, 1990; Privalsky et al., 1988; Forest et al., 1990a; Schroeder et al., 1990; Gandrillon et al., 1989; Pain et al., 1990). These studies revealed that v-ErbA by itself is sufficient to arrest differentiation (Gandrillon et al., 1989; Schroeder et al., 1990) and to selectively suppress the activity of three erythrocyte genes: the erythrocyte-specific carbonic anhydrase I1 (CAII) gene, the anion transporter gene (Band 3), and the erythrocyte version of the &aminolevulinic acid synthase (ALA-S) gene (Zenke et al., 1988; Schroeder et al., 1990). When the c-erbA gene was expressed as a Cag-c-ErbA fusion protein in erythroblasts, two effects were observed. In the absence of T,, Gag-c-ErbA arrested differentiation and reduced the levels of CAII, Band 3, and ALA-S mRNA. In the presence of T,, however, the erythroblasts expressed elevated levels of the three mRNAs and underwent abnormal differentiation (Zenke et al., 1990). Combination of the DNA-binding domain of v-ErbA with the hormone-binding domain of

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105

c-ErbA resulted in a protein that bound T, with affinity similar to that of the Gag-c-ErbA protein and led to a similar T,-dependent regulation of the three erythrocyte-specific genes (Zenke et al., 1990). However, the transformation phenotypes induced were distinct since this chimeric protein caused only a partial inhibition of differentiation in the absence of hormone, while permitting normal or even accelerated differentiation into red cells in the presence of T,. This suggests that the amino acid substitutions in the v-ErbA DNA-binding region affect its activity in erythroid cells. A chimeric protein carrying the C-terminal deletion of v-ErbA in the context of the c-ErbA hormone-binding domain was unresponsive to T, but retained the ability to suppress differentiation and erythrocyte gene expression in a constitutive fashion, thus confirming the results of the hybrid chimeras studied in the transient transfection assays (Damn1 et al., 1989; Zenke et al., 1990). That the C-terminal domain is critical for v-ErbA biological and biochemical properties has also been demonstrated in erythroblasts and functional assays (Privalsky et al., 1988; Boucher and Privalsky, 1990; Forrest et al., 1990a). A specific role for the N terminus is suggested by the fact that c-ErbA and v-ErbA proteins might also be susceptible to posttranslational modifications such as phosphorylation by CAMP-dependent kinases or protein kinases C that modulate their function as transcriptional regulators (Goldberg et al., 1988; Glineur el al., 1989, 1990).

IX. c-erbA: Protooncogene or Growth Suppressor Gene? T h e results of the transient transfection experiments described above generated a new model of how the v-~rbAoncoprotein acts to block differentiation: it may actively repress the transcription of differentiation-specific genes as well as inhibit the function of its normal endogenous counterpart, resulting in a loss of hormone responsiveness and probably hormone-induced differentiation. Here we have a parallel to the recessive oncogene (or growth suppressor gene), where it is also a loss of function that induces the transformation process (for review, see Klein, 1987). Among the natural targets of c-erbA appear to be genes that play important parts during erythroid differentiation (Zenke et al., 1988, 1990). Thyroid hormones reveal or induce the activating function of c-erbA, causing the respective genes to be transcribed at maximum efficiency and allowing the differentiation to proceed. In this way, c-erbA acts as a growth suppressor since the resulting differentiated cells irreversibly lose proliferative potential. c-erbA might also fulfill another criterion usually implicated with growth suppressor genes: if both alleles of

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the c-erbA gene are inactivated, the result would be a loss of hormone responsiveness and most likely a failure of the cells to differentiate. On the other hand, if one allele were converted by somatic mutation into a dominant negative version, differentiation would be blocked also and the cells would be free to proliferate. Thus, gain of function, such as the activation of c-erbA as a dominant negative similar to v-erbA, and the loss of function by inactivation of both alleles, might both be involved in neoplastic transformation. If that model is true, are there somatic mutations that inactivate the c-erbA gene, or convert one allele into a dominant negative derivative? A series of experiments, described in the next section, were performed to substantiate this idea (Damm et al., 1991).

X. Mutations Affecting c-erbA Function The constitutive negative phenotype of v-ErbA is due to mutations found in the ultimate C terminus, a region where v-ErbA and the thyroid hormone receptors exhibit a high degree of homology. The removal of as few as 14 amino acids from the 3’ end of the full-length c-ErbA/TRa and c-ErbA/TRP created proteins that exhibit the “v-ErbA phenotype” in cotransfection assays: they act as potent repressors of the basal transcription level, lose all hormone-dependent trans-activation properties, and act as highly effective dominant negative inhibitors of the c-ErbA-mediated hormone response (Fig. 7). C-terminal deletions extending up to about 30 amino acids into the ligand-binding domain do not change this phenotype whereas derivatives with more severe truncations are inactive. Thus, despite an intact DNA-binding domain and the ability to bind to TREs in vitro, these mutants are unable to repress basal promoter level or inhibit the trans-activation through c-ErbA. Interestingly, and in contrast to the glucocorticoid and other steroid receptors (Evans, 1988; Beato, 1989), none of these receptor derivatives showed hormone-independent, constitutive trans-activation functions. Characterization of the mutant derivatives revealed that amino acid sequences implicated in receptor dimerization (Glass et al., 1989, 1990)are absolutely required for the negative regulatory properties of c-ErbA. Therefore, the dominant negative phenotype may, in part, be mediated through the formation of inactive heterodimers between wild-type and mutant receptors. The C-terminal amino acids, which are deleted in v-ErbA and the dominant negative mutants, seem to be involved in hormone binding and in the hormone-dependent relief of the negative regulatory properties (Damm et al., 1989; MuAoz et al., 1988; Forrest et al., 1990b; Zenke et al., 1990). More importantly, this domain might be involved in hormone-dependent transcriptional regulatory functions

c-erbA:

PROTOONCOGENE OR GROWTH SUPPRESSOR GENE?

Z

R

C

TR-A1

-

+

+

TRA2

-

+

+

TR-A3

-

_

_

TR-mi

+

+

+

TR-rn2

-

+

+

TR-rn3

-

+

+

TR-rn4

+

+

*

TR-m5

107

- - *

FIG.7. Schematic structure and activity of c-ErbA/TR mutant proteins. In-frame linker insertions (arrows) in different parts of the ligand-binding domain were analyzed for functional properties as described in Fig. 5. Deletion of up to 30 amino acids (TR-Al) or more than 35 amino acids (TR-A2 and -A3) from the C terminus of c-ErbA results in mutant products with the indicated functional properties.

and thus in the interaction with other factors of the transcription machinery. In the natural splicing variant TRol2, which is identical to TRa in the first 370 amino acids, this domain is not present. Using the TREp as response element, TRa2 does not exhibit any repression ability and does not inhibit the hormone response (Damm et al., 1991), results that are not consistent with those reported by Koenig et al. (1989) and Lazar et al. (1989a). It seems likely that the variant C-terminal region may alter DNA binding by allosteric or other conformational effects and thus change DNA-binding specificity and affinity. In summary, these data demonstrate that most of the ligand-binding domain of c-ErbA is needed for the negative regulatory properties and that the ultimate C terminus is involved in hormone-mediated transcriptional activation processes. However, a single mutational event, e.g., the conversion of a coding nucleotide triplet near the C terminus into a termination codon, might lead to the creation of a mutant c-ErbA product with a dominant negative phenotype and potential oncogenic properties. What effects can we expect from possible point mutations in other regions of the c-erbA gene? The characterization of the biologically inactive revealed that a single amino acid change can have profound effects on transcriptional regulatory functions (Damm et d.,1987; Damm et al., 1992). By using the technique of oligonucleotide-linker mutagenesis a series of mutant c-ErbA proteins with small in-frame

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insertions at different positions in the ligand-binding domain were created (Damm et al., 1992). In the cotransfection assays these mutant proteins exhibited a wide range of biological activity, from mutants indistinguishable from c-ErbA/TR to those completely defective in the functional assays (Fig. 7). An insertion in the N-terminal part of the ligand-binding domain, close to where the arginine mutation in v-ErbAtd is located, had no measurable effect on receptor function whereas a linker insertion closer to the C terminus completely inactivated the resulting protein. Insertions at two different positions within the ligand-binding domain induced the dominant negative phenotype exemplified by v-ErbA, that is, a loss of hormone-dependent transcriptional activation but the retention of the inhibitory potential. Another mutant showed the v-ErbAtd phenotype in that the protein retained hormone-binding and trans-activation capability but lost its negative regulatory properties. This mutational analysis supports the essential role of the C terminus in c-ErbA/TR activity and provides further evidence suggesting that this “ligand-binding domain” is a complex structure exhibiting hormone binding, dimerization, as well as positive and negative transcriptional regulatory functions. XI. Current Concepts and Open Questions In the last years a number of receptors for retinoid acid (RA, vitamin A) and 1,25-dihydroxycholecalciferol(vitamin D,) have been characterized (Giguere et al., 1987; Petkovich et al., 1987; Baker et al., 1988; Brand et al., 1988; Benbrook et al., 1988; Krust et al., 1989; Ishikawa et al., 1990; Mangelsdorf et al., 1990) and together with v-ErbA and c-ErbA/TR classified as a subgroup of the steroid hormone receptor superfamily. This classification is based on structural similarities between the receptor proteins and the finding that they display cross-recognition of their response elements. For example, the palindromic thyroid hormone response element (Glass et al., 1988)described above also serves as an efficient RA response element for the three isoforms of the retinoic acid receptors (RARs) and the retinoid X receptors (Umesono et al., 1988; Ishikawa et al., 1990; Mangelsdorf et al., 1990). The ability of v-ErbA to form nonproductive protein-DNA complexes with this response element suggests that v-ErbA might also interfere with RAR activity. Indeed, in transient transfection experiments v-ErbA acts as a potent inhibitor of the RAR-mediated retinoic acid response (Damm et al., 1992). Retinoic acid is known to be involved in hematopoiesis and thus it is tempting to speculate that v-ErbA might possibly interfere with RA-induced differentiation in vivo. Final proof, however, awaits the

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109

identification of RA-induced hematopoietic genes and the demonstration of their regulation by v-ErbA. Alternatively, the RAR genes might turn out to be protooncogenes themselves. Rearrangements and altered expression patterns of RARa have been reported in several cases of acute promyelocytic leukemia (APL) (de The et al., 1990; Borrow et al., 1990). How these mutated receptors act in APL is unclear at present; however, in embryonal carcinoma cells and in vitro cotransfection assays it was recently demonstrated that mutant RAR proteins might act in a fashion similar to v-ErbA as dominant negative inhibitors of their normal counterparts (Pratt et al., 1990; Damm et al., 1992). v-ErbA and mutated RARs are not the only examples of dominant negative inhibition. A growing number of oncogenes turn out to encode mutated transcription factors that act through a dominant negative mechanism (Ballard et al., 1990; Inoue et d., 1991; Nakabeppu and Nathans, 1991); however, whether their normal counterparts may also act as growth suppressor genes remains to be demonstrated. ACKNOWLEDGMENTS I would like to thank the many colleagues who have provided stimulating discussions and comments while these data were generated, with special thanks to Drs. Hartmut Beug, Thomas Graf, and Bjorn Vennstrom, in whose laboratories at the European Molecular Biology Laboratory the AEV work originated, and Dr. Ron Evans at the Salk Institute for his generous support and many insightful discussions in the course of deciphering the molecular basis of v-erbA oncogenicity.

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c-erbA: protooncogene or growth suppressor gene?

c-erbA: PROTOONCOGENE OR GROWTH SUPPRESSOR GENE? Klaus Damm Gene Expression Laboratory, The Salk Institute for Biological Studies, La Jolla, Californi...
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