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Truncated erythropoietin receptor in a murine erythroleukemia cell line
hr.
J. Biochem.
1357-2725(!B)OO128X
pp. 175-181, 1996 Copyright 0 1996 Elsevicr Science Ltd Printed in Great Britain. All rights reserved Cell Eiol. Vol. 28, No. 2,
1357-2725/96
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Truncated Erythropoietin Receptor in a Murine Erythrdeukemia Cell Line THOMAS BITTORF,” SAMANTHA J. BUSFIELD, S. PETER KLINKEN,? PETA A. TILBROOK Department
of Biochemistry,
University of Western Australia, Nedlands 6009, Australia
The Friend spieen focus forming virus produces a 55 kDa envelope glycoprotein which associates with the erythropoietin receptor. We compared the erythropoiethr receptor in Friend virus transformed mmrineerythroleukemic F4N and 707 cell lines with the J2E erythroid line generated by the 52 retrovims. Reverse transcriptase PCR was used to determine transcript size. Erythropoiethr receptor cDNAs were then sequencedand protein products analysed by Western blotting and hnmunoprecipitation. We show here that the F4N mu&e erythroleukemic cell line had an enlarged erythropoietin receptor mRNA. In contrast, the 707 and J2E cell ihres had normal sizedtranscripts for the receptor. Sequence analysisof the receptor in F4N celis revealed that introus which separate the exons coding for the cytoplasmic domain of the receptor were retained in these transcripts. As a consequence, a premature stop codon had been hrtroduced, leaving only four amino acids in the intraceihdar portion of the receptor molecule. The normal erythropoietiu receptor is approx. 66-7OkDa, but hnmunoprecipitation of [*Slmethionine/cysteine labelled ceii lysates with an antibody to the amino-termiuus of the erythropoietin receptor ident&d a truncated 37 kDa protein in F4N celIs. Despite the severe carboxy-terminal truncation of the erythropoiethr receptor, F4N ceIiscontinued to proliferate like the other murhre erythroleukemia cell lines. This study showsthat faihue to remove h&roes from the erythropoietin receptor mRNA in F4N ceiis has resulted in the production of a smaller protein with vhtuaiiy no cytoplasmic domain. Keywords: Erythropoietin receptor Murine erythroleukemia Int. J. Biochem. Cell Biol. (1996) 28, 175-181
activation of the epo receptor (epo-R) and subsequent induction of intracellular signalling pathways. Apart from stimulation of the epo-R by its natural ligand, the receptor has been shown to be constitutively activated by a point mutation in the extracellular domain (Yoshimura et al., 1990a; Longmore et al., 1994; Longmore and Lodish, 1991). This activating mutation causes homodimerization of the receptor, in the absence of epo, mimicking the proposed ligand-induced dimerization (Watowich et al., 1992, 1994, Miura and Ihle, 1993; Ohashi et al., 1994). The third type of epo-R activation results from interaction with the transmembrane protein product of the spleen focus forming virus (Li et al., 1990). This replication-defective virus and the replication-competent Friend murine leukemia virus together
INTRODUCTION
The glycoprotein erythropoietin (epo) regulates mammalian erythropoiesis by promoting the viability, proliferation and differentiation of erythroid progenitor cells (D’Andrea and Zon, 1990; Youssoutian et al., 1993; Longmore et al., 1993). These functions are realised by hormonal *Present address: Department of Medical Biochemistry, University of Restock, Restock, Germany. tTo whom all correspondence should be addressed at Department of Biochemistry, Royal Perth Hospital, Perth WA 6000, Australia. Received II January 1995; accepted 18 September 1995. Abbreviu~ions: epo, erythropoietin; epo-R, erythropoietin receptor; MEL, murine erythroleukemia; ER, endoplasmic reticulum; PCR, polymerase chain reaction; RT, reverse transcriptase. 175
176
Thomas
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constitute the Friend virus complex, which is known to induce acute erythroleukemia in adult mice (Ben-David and Bernstein, 1991). The initial phase of this disease is caused by the binding of the viral env-gene product, gp55, to the epo-R (Li et al., 1990) producing hormoneindependent proliferation (Li et al., 1990; Ruscetti et al., 1990). The interaction occurs mainly in the endoplasmic reticulum (ER) (Yoshimura et al., 1990b) but only a proportion of the complex is expressed at the cell surface (Casadevall et al., 1991). It appears that the gp55/epo-R complex in the ER is not sufficient to evoke a mitogenic response (Wang et al., 1993), and a strong correlation exists between the ability of the gp55 molecules to leave the ER and elimination of growth factor requirements for the cells (Ferro et al., 1993). Nevertheless, receptor binding of epo is not influenced by the gp55/epo-R interaction as demonstrated by the existence of cross-linked epo/epo-R/gp55 complexes (Casdevall et al., 1991; Ferro et al., 1993). Mutational analysis of chimaeric receptors has shown that the transmembrane regions of the epo-R and gp55 are critical for transformation (Zon et al., 1992; Showers et al., 1993). Additionally, it has been reported that transformation does not occur if the receptor is severely truncated (D’Andrea et al., 1991; Barber et al., 1994). It is not known if the mitogenic signals induced by the gp55-activated receptor and the epo-stimulated receptor are identical. Significantly, the pattern of tyrosine phosphorylation differs between these two modes of receptor stimulation (Showers et al., 1992). Stimulation by epo causes the rapid phosphorylation of intracellular proteins (Miura et al., 1991; Quelle and Wojchowski, 1991a; Kuramochi et al., 1991; Damen et al., 1992) including JAK2 and the receptor itself (Witthuhn et al., 1993; Yoshimura and Lodish, 1992). A decrease in the phosphorylation status of carboxy-terminal truncated receptor mutants correlates with the down modulation of mitogenic response to epo, suggesting a role for this region in negative regulation of the receptor activity (D’Andrea et al., 1991; Yoshimura et al., 199Oa). A region of the cytoplasmic domain, proximal to the transmembrane region is considered to be important for the proliferative and differentiative actions of epo (D’Andrea et al., 1991; Miura et al., 1993; Chiba et al., 1992; Maruyama et al., 1994). Recently a truncated form of the epo-R has been described which is unable to transmit
et al.
a mitogenic signal and has lost the ability to prevent apoptosis (Nakamura et al., 1992; Nakamura and Nakauchi, 1994). However, the extracellular domain may also play a role in determining the specificity of the proliferative signals as chimaeric molecules consisting of an extracellular/transmembrane domain of the epo-R and an intracellular domain of receptors for IL-2 or IL-3, transmitted epo-specific tyrosine phosphorylation of cytosolic proteins (Chiba et al., 1993). In this report we studied the epo-R in a number of erythroid cell lines. The F4N and 707 murine erythroleukemia (MEL) cell lines were transformed by the Friend virus complex (Ostertag et al., 1974; Dube et al., 1975; Friend et al., 1971), whereas the J2E line was generated by infecting erythroid precursors with a raf/myc-containing 52 virus (Klinken et al., 1988). Normal sized receptors were found in 707 and J2E cells. However we describe the discovery of a truncated murine epo-R in the F4N cell line which contained only four amino acids in the intracellular domain, caused by the failure to splice out introns VI and VII. Despite this major truncation, cellular growth in culture was unaffected. MATERIALS
AND
METHODS
Cell lines The F4N (Ostertag et al., 1974; Dube et al., 1975), MEL 707 (Friend et al., 1971) and J2E cell lines (Klinken et al., 1988) were maintained in DMEM + 10% fetal calf serum at 37°C with 5% co*. Southern blots DNA was prepared using the method of Bowtell (1987), as we have used elsewhere (Klinken et al., 1988). The DNA was separated on agarose gels, transferred to nylon membranes (Hybond N, Amersham, Bucks, U.K.) and hybridized to full length 3%abelied epo-R probes (D’Andrea et al., 1989) before autoradiography. Polymerase chain reaction and cDNA sequencing RNA was extracted by the method of Gough (1988) as used previously by us (Busfield and Klinken, 1992), and cDNA was synthesized using random decanucleotides (1: 20; Bresatec, South Australia) and AMV reverse transcripto tase (RT) ( QG WEI the manufacturer’s protocol. EpowR oDNA fragments were ampfiAed by polymerase chain
Erythropoietin
177
receptor truncation in a MEL cell line
reaction (PCR) using 2U Taq polymerase (Promega, Madison, WI) and primers 1 (5’-ACCCTCTCATCTTGACGCT-3’; nt 4526 4544) and 4 (S-TAAGTATGGCTTCACCAA TC-3’; nt 61276108) for the intracellular domain, and primers 5 (5’-TGAGCTTCCTGAAGCTA-GG-3’; nt 1678-1696) and 11 (5’AGCGTCAAGATGAGAGGGT-3’; nt 45444526) for the extracellular domain of the receptor. The epo-R cDNA fragments were sequenced using a deaza-G Taq sequencing kit (Promega, Madison, WI) and primers within the epo-R DNA sequence: primer 1, primer 2 (5’CTGAAGCTTCATCCATAGTC-3’; nt 57575738), primer 4, primer 5, primer 6 (5’-TACAGCTTCTCATACCAGC-3’; nt 19071925), primer 8 (5’-TCCACCACAGACAACCATCA-3’; nt 5505-5486), primer 10 (5’CCAATGTCGTCAACT-TTGTC-3’; nt 48254844), primer 11 and primer 12 (5’-TGTGACTATGGATGAAGCT-3’; nt 5738-5757). The nucleotide numbers of the primers refer to the position of the primer in the genomic sequence (Kuramochi et al., 1990). Western blots Proteins were extracted from cell lines in a buffer of 1% triton X100, 0.5% deoxycholate, 150 mM NaCl, 20 mM Tris pH 7.5, 40 mM Na4P,0,, 0.1 mM Na, V04, 50 mM NaF, 5 mM MgCl,, 1 mM PMSF, 10 pg/ml aprotinin, incubated on ice for 20 min and cleared by centrifugation at lO,OOOg, 10 min, 4°C. Protein (100 pg) was electrophoresed on a 10 or 15% polyacrylamide-SDS gel and transferred to a nitrocellulose membrane (Amersham, Bucks, U.K.). The membrane was blocked in 5% skim milk in TBST (20mM Tris pH 7.5, 150mM NaCl, 0.05% Tween 20) for 1 hr, then incubated with 1: 2000 rabbit anti-carboxyl terminus epo-R antibody # 187 (Watowich et al., 1992) followed by 1: 5000 HRP-anti rabbit (Amersham, Bucks, U.K.) and visualized by ECL (Amersham, Bucks, U.K.). Metabolic labelling Cells were washed and incubated for 1 hr in 1.5 ml methionine free DMEM-2% FCS before addition of 500 pCi/ml [35S]methionine/cysteine (3000 Ci/mmol; NEN DuPont) for 4 hr. Cells were washed in PBS, lysed as described above and immunoprecipitated with anti-amino terminus epo-R # 189 antibodies (Watowich et al., 1992), followed by electrophoresis on a 10% SDS-PAGE gel. The gel was transferred to a
nitrocellulose membrane (Amersham, U.K.) and autoradiographed. RESULTS
AND
Bucks,
DISCUSSION
As different types of epo-R mRNA have been detected within erythroid cells (Nakamura, 1992; D’Andrea et al., 1989; Kuramochi et al., 1990; Ehrenman et al., 1991; Barron et al., 1994), epo-R transcripts were examined in two MEL cell lines (F4N and 707) and the J2E line using RT-PCR. The primers were designed to identify alterations in either the extracellular domain or the intracellular region (Fig. l(A)). After RT-PCR of mRNA from the three lines, fragments were separated on agarose gels. Figure l(B) shows that 707 and J2E cells had extracellular and intracellular fragments of the expected size (D’Andrea et al., 1989; Kuramochi et al., 1990). Although the extracellular domain of F4N cells was normal, as reported previously (Heberlein et al., 1992) the intracellular portion was not of the predicted length. Whereas the normal fragment was
e
PCR 511 k
” f
PCR1-4 fo
-4
7fi 7
Fig. 1. Abnormal epo-R transcript in F4N cells. (A), Intron-sxon structure of the murine epo-R gene. Striped boxes represent the epo-R extracellular domain (exons I-V), the black box the transmembrane domain (exon VI) and the grey boxes the intracellular domain (exons VII-VIII). The location of the PCR and sequencing primers is indicated by the arrows. (B), PCR products of the epo-R cDNAs isolated from murine erythroleukemic cell lines. Epo-R cDNA from J2E (lanes 1 and 5), MEL 707 (lanes 2 and 6) and F4N (lanes 3 and 7) cell lines was synthesized using primers 5 and 11 for the extracellular domain (lanes I-3) and primers 1 and 4 for the intracellular domain (Ianes 5-7) of the receptor. The sizesof the PCR fragments are indicated, and the DNA molecular weight marker (lane 4) is SPPI digested with Eco RI.
Thomas Bittorf et al.
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123456
WIW type q&o-R cDNA
-4.3kb
-T
I ATG
-2.3kb
TAG
-21kb F4N epo-R cDNA
I ATG
5006
5015 1
5418 I
WT
ttgaa-aacac
ccagctaaoac
F4N
ttgaaaaacac
cc-gctg-cat
5mo I
WT F4N
5060 I
Cccatacccth cccat-cccta 5105 I
WT
actccatatgc
F4N
a&cc-tatgc
5443 I
Cgatceagc& tga--gcgcag
5426 I
WT F4N 5453 I
W F4N
5116 I
Fig. 2. Sequence of the mutated epo-R in F4N cells. The striped boxes represent the epo-R extracellular domain (exons I-V), the black box the transmembrane domain (exon VI) and the grey boxes the intracellular domain exons (VII-VIII). The white boxes represent insertions in F4N transcripts labelled c(and /I, which correspond to the introns VI and VII. Alterations in the F4N cDNA sequence from the wild tvue seauence are also shown. with numbers representing the positions of the n~le&Iee in the wild type gene (Kuramochi et al., 1990). .
.
Fig. 3. Genomic structure of epo-R is normal in F4N cells. DNA was prepared from F4N (lanes 2, 4 and 6) and J2E (lanes 1, 3 and 5) cells digested with the enzymes Bum HI (lanes 1 and 2), Bgl II (lanes 3 and 4) and Hind III (lanes 5 and 6). After transfer to nylon membranes, the DNA was hybridized to JZP-labelled epo-R probes, and exposed to X-ray film for autoradiography.
L
829 bp in length, the abnormal transcript in the F4N line was significantly larger and 1597 bp long (see below). To determine the reason for the Wrgement of the F4N transcripts, the PC& products were sequenced using primers shown in Fig. l(A). The nucleotide sequence was then compared with the published sequence of the murine epoR (D’Andrea et al., 1989; Kuramochi et al., 1990). It can be seen in Fig. 2 that numerous alterations to the epo-R transcript had occurred in the F4N line. However, Southern blotting of the epo-R gene using Barn HI, Hind III and BgI ZZ (Kuramochi et al., 1990; Heberiein et al., 1992), failed to identify any gross alterations to the genomic structure (Fig. 3). Others have also not detected alterations in the epo-R gene of F4N cells using EcoRV (Chretien et al., 1994).
Based on the sequence published for the gene it appears that a number of splicing alterations have occurred in this cell line as introns VI and VII had not been removed. Mutations to the splice sites were not the cause of the altered transcripts, as the sequence at these junctions precisely matched those published (D’Andrea et al., 1989; Kuramochi et al., 1990). These changes most probably originated either from a defect in the splicing machinery or represent an alternatively spliced variant. In addition to the insertion of two introns, other minor alterations were detected in these intervening sequencesfive mono- and di-nucleotide insertions and deletions were also found in the F4N transcripts (Fig. 2). In contrast, the sequence of the epo-R in J2E cells was exactly as described by D’Andrea et al. (1989) (Tilbrook et al., submitted). The presence of the introns in F4N epo-R mRNA introduced a putative premature stop codon which would produce a truncated protein (Fig. 2). As a result only 275 of the 507 codons would be translated before termination (Fig. 4).
wild
typa s+Ba.R
.
F4N epo.R
Fig. 4. Intracellular domain of the truncated epo-R in F4N cells. The amino acid sequence of the wild type and F4N epo-R are aligned in the transmembrane (boxed) regions.
Erythropoietin
receptor truncation in a MEL cell line
1234
Fig. 5. Immunoblot of epo-R protein -in murine erythroleukemic cell lines. Protein (100 pg) from NIH 3T3 (lane 1), F4N (lane 2), J2E (lane 3) and MEL 707 (lane 4) cell lines were analysed by Western blotting using an anti-carboxyl terminus epo-R antibody. The lower band indicated is a non-specific band.
179
introns in this case has produced a premature stop codon, resulting in abbreviated protein synthesis. Significantly, a similar transcript retaining intron VI and exposing a cryptic termination codon has been described recently by Barron et al. (1994). These investigators identified this modified form of the epo-R mRNA by PCR analysis of the myeloid 32D cell line, but did not search for an altered protein. We have demonstrated in this study that a smaller epo-R protein is indeed present in F4N cells. It is interesting to speculate on the effect of the epo-R truncation in F4N cells. Only four amino acids of the wild type receptor are left in the cytoplasmic domain, and several studies have demonstrated that critical signalling domains (D’Andrea et al., 1991; Chiba et al., 1992; Marayama et al., 1994; Quelle and Wojchowski, 1991b) are missing from this altered receptor.
Thus a total of four amino acids of the cytoplasmic domain, proximal to the transmembrane region, would be left in this modified receptor. To confirm that a major deletion to the carboxy terminus had occurred in these cells, protein was prepared and immunoblotted with an antibody directed to the C-terminus of the receptor (Watowich et al., 1992). Figure 5 shows that proteins of the appropriate molecu6 A lar weights for wild type epo-R were detected in f%N BE F4N J2E J2E and 707 cells, while no epo-R protein was detected in F4N cells. The inability of carboxyterminal antibodies to detect severely truncated forms of epo-R has been reported previously (Miura and Ihle, 1993). Due to the inadequacy of the amino terminus antibody for immunoblotting, a truncated protein could not be detected in F4N cells using this antibody (data not shown). However, when cells were metabolically labelled and proteins immunoprecipitated with the amino-terminal antibody, a truncated epo-R protein approx. 37 kDa in size was detected in the F4N cells, but not the J2E cells (Fig. 6). We concluded from these data that a premature stop codon had indeed been introduced into the transcript and that the protein was truncated accordingly. From the data presented here it appears that the mutated epo-R has emerged either as a result of a splicing error, or as a differentially spliced variant of the receptor. Several differentially spliced versions of epo-R transcripts have been identified in a variety of erythroid lines (Nakamura et al., 1992; D’Andrea et al., 1989; Kuramochi et al., 1990; Ehrenman et al., 1991; Barron et al., 1994). The splicing machinery CONTROL a 189 must, therefore, be tightly regulated to produce the full-length receptor, soluble epo-R and the Fig. 6. F4N cells contain a truncated epo-R. F4N (lanes 1 and 3) and J2E (lanes 2 and 4) cells were metabolically various truncated forms of the molecule (Nakalabelled with 35S-methionine/cysteine and immunoprecipimura et al., 1992; D’Andrea et al., 1989; tated with either a control rabbit antibody (lanes 1 and 2) Kuramochi et al., 1990; Ehrenman et al., 1991; or an anti-amino terminus epo-R antibody (lanes 3 and 4). Barron et al., 1994). The failure to remove two The epo-R proteins are indicated with small bold rules,
Thomas Bittorf et al.
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However, the transmembrane region of epo-R has been identified as being essential for gp55 activation (Zon et al., 1992) and the transforming activities of gp55 localized to the transmembrane region of this molecule (Showers et al., 1993). It is conceivable, therefore, that signals emanating from the gp55/epo-R interaction in the transmembrane region of F4N cells may be sufficient to maintain cellular proliferation. In contrast with this suggestion, it has been shown that severely truncated epo-Rs will bind gp55, but not transmit mitogenic signals in the Ba/F3 line, rendering these cells resistant to transformation (D’Andrea et al., 1991; Barber et al., 1994). Significantly, truncated epo-Rs have a dominant negative effect over normal receptors with respect to epo, or gp55 signalling (Watowich et al., 1994; Barber et al., 1994; Nakamura et al., 1994). An alternative explanation is that the alteration to the epo-R described here occurred after transformation of these cells. Perhaps sufficient additional genetic errors have accumulated in the F4N line and signalling via the gp55/epo-R complex is no longer essential to maintain a transformed phenotype and continuous proliferation. We have shown that, as expected of MEL cell lines (Ben-David and Bernstein, 1991), ~53 is mutated in F4N cells (unpublished data). To determine which of these possibilities is correct, the truncated epo-R from these cells could be cloned and introduced into cells co-expressing gp55. Acknowledgements-This research was supported by grants from NH&MRC (91-0171 and 93-0098), Raine Medical Research Foundation, Cancer Foundation of WA and Faculty of Medicine (UWA). Thomas Bittorf and Peta A. Tilbrook are the recipients of a German Academic Exchange Service (DAAD) Fellowship and a Richard Walter Gibbon Fellowship, respectively. The anti-epo-R antibody used was a gift from Dr D. Hilton (Walter and Eliza Hall Institute, Melbourne, Australia).
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Wang Y., Kayman S., Li J.-P. and Pinter A. (1993) Erythropoietin receptor (EpoR) dependent mitogenicity of spleen focus forming virus correlates with viral pathogenicity and processing of env protein but not with formation of gp52-EpoR complexes in the endoplasmic reticulum. J. Virol. 67, 1322-1327. Watowich S. S., Yoshimura A., Longmore G. D., Hilton D. J., Yoshimura Y. and Lodish H. F. (1992) Homodimerization and constitutive activation of the erythropoietin receptor. Proc. Nat1 Acad. Sci. V.S.A. 89, 2140-2144. Watowich S. S., Hilton D. J. and Lodish H. F. (1994) Activation and inhibition of erythropoietin receptor function: role of receptor dimerisation. Mol. Cell. Biol. 14, 3535-3549.
Witthuhn B., Quelle F. W., Silvennoinen O., Yi T., Tang B., Miura 0. and Ihle J. N. (1993) JAK2 associates with the erythropoietin receptor and is tyrosine phosphorylated and activated following stimulation with erythropoietin. Cell 74, 227-236.
Yoshimura A., Longmore G. D. and Lodish H. F. (1990a) Point mutation in the exoplasmic domain of the erythropoietin receptor resulting in hormoneindependent activation and tumorigenicity. Nature 348, 647-649.
Yoshimura A., D’Andrea A. D. and Lodish H. F. (1990b) Friend spleen focus-forming virus glycoprotein gp55 interacts with the erythropoietin receptor in the endoplasmic reticulum and affects receptor metabolism. Proc. Nat1 Acad. Sci. V.S.A. 87, 41394143. Yoshimura A. and Lodish H. F. (1992) In vitro phosphorylation of the erythropoietin receptor and an associated protein ~~130. Mol. Cell. Biol. 12, 706-715. Youssoufian H., Longmore G. D., Neumann D., Yoshimura A. and Lodish H. F. (1993) Structure, function and activation of the erythropoietin receptor. Blood 81, 2223-2236.
Zon L. I., Moreau J.-F., Koo J.-W., Mathey-Prevot B. and D’Andrea A. D. (1992) The erythropoietin receptor transmembrane region is necessary for activation by the Friend spleen focus forming virus gp55 glycoprotein. Mol. Cell.
Biol.
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J. Biochem.
1357-2725(!B)OO128X
pp. 175-181, 1996 Copyright 0 1996 Elsevicr Science Ltd Printed in Great Britain. All rights reserved Cell Eiol. Vol. 28, No. 2,
1357-2725/96
$15.00 + 0.00
Truncated Erythropoietin Receptor in a Murine Erythrdeukemia Cell Line THOMAS BITTORF,” SAMANTHA J. BUSFIELD, S. PETER KLINKEN,? PETA A. TILBROOK Department
of Biochemistry,
University of Western Australia, Nedlands 6009, Australia
The Friend spieen focus forming virus produces a 55 kDa envelope glycoprotein which associates with the erythropoietin receptor. We compared the erythropoiethr receptor in Friend virus transformed mmrineerythroleukemic F4N and 707 cell lines with the J2E erythroid line generated by the 52 retrovims. Reverse transcriptase PCR was used to determine transcript size. Erythropoiethr receptor cDNAs were then sequencedand protein products analysed by Western blotting and hnmunoprecipitation. We show here that the F4N mu&e erythroleukemic cell line had an enlarged erythropoietin receptor mRNA. In contrast, the 707 and J2E cell ihres had normal sizedtranscripts for the receptor. Sequence analysisof the receptor in F4N celis revealed that introus which separate the exons coding for the cytoplasmic domain of the receptor were retained in these transcripts. As a consequence, a premature stop codon had been hrtroduced, leaving only four amino acids in the intraceihdar portion of the receptor molecule. The normal erythropoietiu receptor is approx. 66-7OkDa, but hnmunoprecipitation of [*Slmethionine/cysteine labelled ceii lysates with an antibody to the amino-termiuus of the erythropoietin receptor ident&d a truncated 37 kDa protein in F4N celIs. Despite the severe carboxy-terminal truncation of the erythropoiethr receptor, F4N ceIiscontinued to proliferate like the other murhre erythroleukemia cell lines. This study showsthat faihue to remove h&roes from the erythropoietin receptor mRNA in F4N ceiis has resulted in the production of a smaller protein with vhtuaiiy no cytoplasmic domain. Keywords: Erythropoietin receptor Murine erythroleukemia Int. J. Biochem. Cell Biol. (1996) 28, 175-181
activation of the epo receptor (epo-R) and subsequent induction of intracellular signalling pathways. Apart from stimulation of the epo-R by its natural ligand, the receptor has been shown to be constitutively activated by a point mutation in the extracellular domain (Yoshimura et al., 1990a; Longmore et al., 1994; Longmore and Lodish, 1991). This activating mutation causes homodimerization of the receptor, in the absence of epo, mimicking the proposed ligand-induced dimerization (Watowich et al., 1992, 1994, Miura and Ihle, 1993; Ohashi et al., 1994). The third type of epo-R activation results from interaction with the transmembrane protein product of the spleen focus forming virus (Li et al., 1990). This replication-defective virus and the replication-competent Friend murine leukemia virus together
INTRODUCTION
The glycoprotein erythropoietin (epo) regulates mammalian erythropoiesis by promoting the viability, proliferation and differentiation of erythroid progenitor cells (D’Andrea and Zon, 1990; Youssoutian et al., 1993; Longmore et al., 1993). These functions are realised by hormonal *Present address: Department of Medical Biochemistry, University of Restock, Restock, Germany. tTo whom all correspondence should be addressed at Department of Biochemistry, Royal Perth Hospital, Perth WA 6000, Australia. Received II January 1995; accepted 18 September 1995. Abbreviu~ions: epo, erythropoietin; epo-R, erythropoietin receptor; MEL, murine erythroleukemia; ER, endoplasmic reticulum; PCR, polymerase chain reaction; RT, reverse transcriptase. 175
176
Thomas
Bittorf
constitute the Friend virus complex, which is known to induce acute erythroleukemia in adult mice (Ben-David and Bernstein, 1991). The initial phase of this disease is caused by the binding of the viral env-gene product, gp55, to the epo-R (Li et al., 1990) producing hormoneindependent proliferation (Li et al., 1990; Ruscetti et al., 1990). The interaction occurs mainly in the endoplasmic reticulum (ER) (Yoshimura et al., 1990b) but only a proportion of the complex is expressed at the cell surface (Casadevall et al., 1991). It appears that the gp55/epo-R complex in the ER is not sufficient to evoke a mitogenic response (Wang et al., 1993), and a strong correlation exists between the ability of the gp55 molecules to leave the ER and elimination of growth factor requirements for the cells (Ferro et al., 1993). Nevertheless, receptor binding of epo is not influenced by the gp55/epo-R interaction as demonstrated by the existence of cross-linked epo/epo-R/gp55 complexes (Casdevall et al., 1991; Ferro et al., 1993). Mutational analysis of chimaeric receptors has shown that the transmembrane regions of the epo-R and gp55 are critical for transformation (Zon et al., 1992; Showers et al., 1993). Additionally, it has been reported that transformation does not occur if the receptor is severely truncated (D’Andrea et al., 1991; Barber et al., 1994). It is not known if the mitogenic signals induced by the gp55-activated receptor and the epo-stimulated receptor are identical. Significantly, the pattern of tyrosine phosphorylation differs between these two modes of receptor stimulation (Showers et al., 1992). Stimulation by epo causes the rapid phosphorylation of intracellular proteins (Miura et al., 1991; Quelle and Wojchowski, 1991a; Kuramochi et al., 1991; Damen et al., 1992) including JAK2 and the receptor itself (Witthuhn et al., 1993; Yoshimura and Lodish, 1992). A decrease in the phosphorylation status of carboxy-terminal truncated receptor mutants correlates with the down modulation of mitogenic response to epo, suggesting a role for this region in negative regulation of the receptor activity (D’Andrea et al., 1991; Yoshimura et al., 199Oa). A region of the cytoplasmic domain, proximal to the transmembrane region is considered to be important for the proliferative and differentiative actions of epo (D’Andrea et al., 1991; Miura et al., 1993; Chiba et al., 1992; Maruyama et al., 1994). Recently a truncated form of the epo-R has been described which is unable to transmit
et al.
a mitogenic signal and has lost the ability to prevent apoptosis (Nakamura et al., 1992; Nakamura and Nakauchi, 1994). However, the extracellular domain may also play a role in determining the specificity of the proliferative signals as chimaeric molecules consisting of an extracellular/transmembrane domain of the epo-R and an intracellular domain of receptors for IL-2 or IL-3, transmitted epo-specific tyrosine phosphorylation of cytosolic proteins (Chiba et al., 1993). In this report we studied the epo-R in a number of erythroid cell lines. The F4N and 707 murine erythroleukemia (MEL) cell lines were transformed by the Friend virus complex (Ostertag et al., 1974; Dube et al., 1975; Friend et al., 1971), whereas the J2E line was generated by infecting erythroid precursors with a raf/myc-containing 52 virus (Klinken et al., 1988). Normal sized receptors were found in 707 and J2E cells. However we describe the discovery of a truncated murine epo-R in the F4N cell line which contained only four amino acids in the intracellular domain, caused by the failure to splice out introns VI and VII. Despite this major truncation, cellular growth in culture was unaffected. MATERIALS
AND
METHODS
Cell lines The F4N (Ostertag et al., 1974; Dube et al., 1975), MEL 707 (Friend et al., 1971) and J2E cell lines (Klinken et al., 1988) were maintained in DMEM + 10% fetal calf serum at 37°C with 5% co*. Southern blots DNA was prepared using the method of Bowtell (1987), as we have used elsewhere (Klinken et al., 1988). The DNA was separated on agarose gels, transferred to nylon membranes (Hybond N, Amersham, Bucks, U.K.) and hybridized to full length 3%abelied epo-R probes (D’Andrea et al., 1989) before autoradiography. Polymerase chain reaction and cDNA sequencing RNA was extracted by the method of Gough (1988) as used previously by us (Busfield and Klinken, 1992), and cDNA was synthesized using random decanucleotides (1: 20; Bresatec, South Australia) and AMV reverse transcripto tase (RT) ( QG WEI the manufacturer’s protocol. EpowR oDNA fragments were ampfiAed by polymerase chain
Erythropoietin
177
receptor truncation in a MEL cell line
reaction (PCR) using 2U Taq polymerase (Promega, Madison, WI) and primers 1 (5’-ACCCTCTCATCTTGACGCT-3’; nt 4526 4544) and 4 (S-TAAGTATGGCTTCACCAA TC-3’; nt 61276108) for the intracellular domain, and primers 5 (5’-TGAGCTTCCTGAAGCTA-GG-3’; nt 1678-1696) and 11 (5’AGCGTCAAGATGAGAGGGT-3’; nt 45444526) for the extracellular domain of the receptor. The epo-R cDNA fragments were sequenced using a deaza-G Taq sequencing kit (Promega, Madison, WI) and primers within the epo-R DNA sequence: primer 1, primer 2 (5’CTGAAGCTTCATCCATAGTC-3’; nt 57575738), primer 4, primer 5, primer 6 (5’-TACAGCTTCTCATACCAGC-3’; nt 19071925), primer 8 (5’-TCCACCACAGACAACCATCA-3’; nt 5505-5486), primer 10 (5’CCAATGTCGTCAACT-TTGTC-3’; nt 48254844), primer 11 and primer 12 (5’-TGTGACTATGGATGAAGCT-3’; nt 5738-5757). The nucleotide numbers of the primers refer to the position of the primer in the genomic sequence (Kuramochi et al., 1990). Western blots Proteins were extracted from cell lines in a buffer of 1% triton X100, 0.5% deoxycholate, 150 mM NaCl, 20 mM Tris pH 7.5, 40 mM Na4P,0,, 0.1 mM Na, V04, 50 mM NaF, 5 mM MgCl,, 1 mM PMSF, 10 pg/ml aprotinin, incubated on ice for 20 min and cleared by centrifugation at lO,OOOg, 10 min, 4°C. Protein (100 pg) was electrophoresed on a 10 or 15% polyacrylamide-SDS gel and transferred to a nitrocellulose membrane (Amersham, Bucks, U.K.). The membrane was blocked in 5% skim milk in TBST (20mM Tris pH 7.5, 150mM NaCl, 0.05% Tween 20) for 1 hr, then incubated with 1: 2000 rabbit anti-carboxyl terminus epo-R antibody # 187 (Watowich et al., 1992) followed by 1: 5000 HRP-anti rabbit (Amersham, Bucks, U.K.) and visualized by ECL (Amersham, Bucks, U.K.). Metabolic labelling Cells were washed and incubated for 1 hr in 1.5 ml methionine free DMEM-2% FCS before addition of 500 pCi/ml [35S]methionine/cysteine (3000 Ci/mmol; NEN DuPont) for 4 hr. Cells were washed in PBS, lysed as described above and immunoprecipitated with anti-amino terminus epo-R # 189 antibodies (Watowich et al., 1992), followed by electrophoresis on a 10% SDS-PAGE gel. The gel was transferred to a
nitrocellulose membrane (Amersham, U.K.) and autoradiographed. RESULTS
AND
Bucks,
DISCUSSION
As different types of epo-R mRNA have been detected within erythroid cells (Nakamura, 1992; D’Andrea et al., 1989; Kuramochi et al., 1990; Ehrenman et al., 1991; Barron et al., 1994), epo-R transcripts were examined in two MEL cell lines (F4N and 707) and the J2E line using RT-PCR. The primers were designed to identify alterations in either the extracellular domain or the intracellular region (Fig. l(A)). After RT-PCR of mRNA from the three lines, fragments were separated on agarose gels. Figure l(B) shows that 707 and J2E cells had extracellular and intracellular fragments of the expected size (D’Andrea et al., 1989; Kuramochi et al., 1990). Although the extracellular domain of F4N cells was normal, as reported previously (Heberlein et al., 1992) the intracellular portion was not of the predicted length. Whereas the normal fragment was
e
PCR 511 k
” f
PCR1-4 fo
-4
7fi 7
Fig. 1. Abnormal epo-R transcript in F4N cells. (A), Intron-sxon structure of the murine epo-R gene. Striped boxes represent the epo-R extracellular domain (exons I-V), the black box the transmembrane domain (exon VI) and the grey boxes the intracellular domain (exons VII-VIII). The location of the PCR and sequencing primers is indicated by the arrows. (B), PCR products of the epo-R cDNAs isolated from murine erythroleukemic cell lines. Epo-R cDNA from J2E (lanes 1 and 5), MEL 707 (lanes 2 and 6) and F4N (lanes 3 and 7) cell lines was synthesized using primers 5 and 11 for the extracellular domain (lanes I-3) and primers 1 and 4 for the intracellular domain (Ianes 5-7) of the receptor. The sizesof the PCR fragments are indicated, and the DNA molecular weight marker (lane 4) is SPPI digested with Eco RI.
Thomas Bittorf et al.
178
123456
WIW type q&o-R cDNA
-4.3kb
-T
I ATG
-2.3kb
TAG
-21kb F4N epo-R cDNA
I ATG
5006
5015 1
5418 I
WT
ttgaa-aacac
ccagctaaoac
F4N
ttgaaaaacac
cc-gctg-cat
5mo I
WT F4N
5060 I
Cccatacccth cccat-cccta 5105 I
WT
actccatatgc
F4N
a&cc-tatgc
5443 I
Cgatceagc& tga--gcgcag
5426 I
WT F4N 5453 I
W F4N
5116 I
Fig. 2. Sequence of the mutated epo-R in F4N cells. The striped boxes represent the epo-R extracellular domain (exons I-V), the black box the transmembrane domain (exon VI) and the grey boxes the intracellular domain exons (VII-VIII). The white boxes represent insertions in F4N transcripts labelled c(and /I, which correspond to the introns VI and VII. Alterations in the F4N cDNA sequence from the wild tvue seauence are also shown. with numbers representing the positions of the n~le&Iee in the wild type gene (Kuramochi et al., 1990). .
.
Fig. 3. Genomic structure of epo-R is normal in F4N cells. DNA was prepared from F4N (lanes 2, 4 and 6) and J2E (lanes 1, 3 and 5) cells digested with the enzymes Bum HI (lanes 1 and 2), Bgl II (lanes 3 and 4) and Hind III (lanes 5 and 6). After transfer to nylon membranes, the DNA was hybridized to JZP-labelled epo-R probes, and exposed to X-ray film for autoradiography.
L
829 bp in length, the abnormal transcript in the F4N line was significantly larger and 1597 bp long (see below). To determine the reason for the Wrgement of the F4N transcripts, the PC& products were sequenced using primers shown in Fig. l(A). The nucleotide sequence was then compared with the published sequence of the murine epoR (D’Andrea et al., 1989; Kuramochi et al., 1990). It can be seen in Fig. 2 that numerous alterations to the epo-R transcript had occurred in the F4N line. However, Southern blotting of the epo-R gene using Barn HI, Hind III and BgI ZZ (Kuramochi et al., 1990; Heberiein et al., 1992), failed to identify any gross alterations to the genomic structure (Fig. 3). Others have also not detected alterations in the epo-R gene of F4N cells using EcoRV (Chretien et al., 1994).
Based on the sequence published for the gene it appears that a number of splicing alterations have occurred in this cell line as introns VI and VII had not been removed. Mutations to the splice sites were not the cause of the altered transcripts, as the sequence at these junctions precisely matched those published (D’Andrea et al., 1989; Kuramochi et al., 1990). These changes most probably originated either from a defect in the splicing machinery or represent an alternatively spliced variant. In addition to the insertion of two introns, other minor alterations were detected in these intervening sequencesfive mono- and di-nucleotide insertions and deletions were also found in the F4N transcripts (Fig. 2). In contrast, the sequence of the epo-R in J2E cells was exactly as described by D’Andrea et al. (1989) (Tilbrook et al., submitted). The presence of the introns in F4N epo-R mRNA introduced a putative premature stop codon which would produce a truncated protein (Fig. 2). As a result only 275 of the 507 codons would be translated before termination (Fig. 4).
wild
typa s+Ba.R
.
F4N epo.R
Fig. 4. Intracellular domain of the truncated epo-R in F4N cells. The amino acid sequence of the wild type and F4N epo-R are aligned in the transmembrane (boxed) regions.
Erythropoietin
receptor truncation in a MEL cell line
1234
Fig. 5. Immunoblot of epo-R protein -in murine erythroleukemic cell lines. Protein (100 pg) from NIH 3T3 (lane 1), F4N (lane 2), J2E (lane 3) and MEL 707 (lane 4) cell lines were analysed by Western blotting using an anti-carboxyl terminus epo-R antibody. The lower band indicated is a non-specific band.
179
introns in this case has produced a premature stop codon, resulting in abbreviated protein synthesis. Significantly, a similar transcript retaining intron VI and exposing a cryptic termination codon has been described recently by Barron et al. (1994). These investigators identified this modified form of the epo-R mRNA by PCR analysis of the myeloid 32D cell line, but did not search for an altered protein. We have demonstrated in this study that a smaller epo-R protein is indeed present in F4N cells. It is interesting to speculate on the effect of the epo-R truncation in F4N cells. Only four amino acids of the wild type receptor are left in the cytoplasmic domain, and several studies have demonstrated that critical signalling domains (D’Andrea et al., 1991; Chiba et al., 1992; Marayama et al., 1994; Quelle and Wojchowski, 1991b) are missing from this altered receptor.
Thus a total of four amino acids of the cytoplasmic domain, proximal to the transmembrane region, would be left in this modified receptor. To confirm that a major deletion to the carboxy terminus had occurred in these cells, protein was prepared and immunoblotted with an antibody directed to the C-terminus of the receptor (Watowich et al., 1992). Figure 5 shows that proteins of the appropriate molecu6 A lar weights for wild type epo-R were detected in f%N BE F4N J2E J2E and 707 cells, while no epo-R protein was detected in F4N cells. The inability of carboxyterminal antibodies to detect severely truncated forms of epo-R has been reported previously (Miura and Ihle, 1993). Due to the inadequacy of the amino terminus antibody for immunoblotting, a truncated protein could not be detected in F4N cells using this antibody (data not shown). However, when cells were metabolically labelled and proteins immunoprecipitated with the amino-terminal antibody, a truncated epo-R protein approx. 37 kDa in size was detected in the F4N cells, but not the J2E cells (Fig. 6). We concluded from these data that a premature stop codon had indeed been introduced into the transcript and that the protein was truncated accordingly. From the data presented here it appears that the mutated epo-R has emerged either as a result of a splicing error, or as a differentially spliced variant of the receptor. Several differentially spliced versions of epo-R transcripts have been identified in a variety of erythroid lines (Nakamura et al., 1992; D’Andrea et al., 1989; Kuramochi et al., 1990; Ehrenman et al., 1991; Barron et al., 1994). The splicing machinery CONTROL a 189 must, therefore, be tightly regulated to produce the full-length receptor, soluble epo-R and the Fig. 6. F4N cells contain a truncated epo-R. F4N (lanes 1 and 3) and J2E (lanes 2 and 4) cells were metabolically various truncated forms of the molecule (Nakalabelled with 35S-methionine/cysteine and immunoprecipimura et al., 1992; D’Andrea et al., 1989; tated with either a control rabbit antibody (lanes 1 and 2) Kuramochi et al., 1990; Ehrenman et al., 1991; or an anti-amino terminus epo-R antibody (lanes 3 and 4). Barron et al., 1994). The failure to remove two The epo-R proteins are indicated with small bold rules,
Thomas Bittorf et al.
180
However, the transmembrane region of epo-R has been identified as being essential for gp55 activation (Zon et al., 1992) and the transforming activities of gp55 localized to the transmembrane region of this molecule (Showers et al., 1993). It is conceivable, therefore, that signals emanating from the gp55/epo-R interaction in the transmembrane region of F4N cells may be sufficient to maintain cellular proliferation. In contrast with this suggestion, it has been shown that severely truncated epo-Rs will bind gp55, but not transmit mitogenic signals in the Ba/F3 line, rendering these cells resistant to transformation (D’Andrea et al., 1991; Barber et al., 1994). Significantly, truncated epo-Rs have a dominant negative effect over normal receptors with respect to epo, or gp55 signalling (Watowich et al., 1994; Barber et al., 1994; Nakamura et al., 1994). An alternative explanation is that the alteration to the epo-R described here occurred after transformation of these cells. Perhaps sufficient additional genetic errors have accumulated in the F4N line and signalling via the gp55/epo-R complex is no longer essential to maintain a transformed phenotype and continuous proliferation. We have shown that, as expected of MEL cell lines (Ben-David and Bernstein, 1991), ~53 is mutated in F4N cells (unpublished data). To determine which of these possibilities is correct, the truncated epo-R from these cells could be cloned and introduced into cells co-expressing gp55. Acknowledgements-This research was supported by grants from NH&MRC (91-0171 and 93-0098), Raine Medical Research Foundation, Cancer Foundation of WA and Faculty of Medicine (UWA). Thomas Bittorf and Peta A. Tilbrook are the recipients of a German Academic Exchange Service (DAAD) Fellowship and a Richard Walter Gibbon Fellowship, respectively. The anti-epo-R antibody used was a gift from Dr D. Hilton (Walter and Eliza Hall Institute, Melbourne, Australia).
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