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Inactivation of Erythropoietin Receptor Function by Point Mutations in a Region Having Homology with Other Cytokine Receptors OSAMU MIURA,'T JOHN L

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Inactivation of Erythropoietin Receptor Function by Point Mutations in a Region Having Homology with Other Cytokine Receptors OSAMU MIURA,'T JOHN L MOLECULAR AND CELLULAR BIOLOGY, Mar. 1993, p. 1788-1795 Vol. 13, No. 3 0270-7306/93/031788-08$02.00/0 Copyright © 1993, American Society for Microbiology Inactivation of Erythropoietin Receptor Function by Point Mutations in a Region Having Homology with Other Cytokine Receptors OSAMU MIURA,'t JOHN L. CLEVELAND,' AND JAMES N. IHLEl,2* Department ofBiochemistry, St. Jude Children's Research Hospital, Memphis, Tennessee 31051,1 and Department ofBiochemistry, University of Tennessee, Memphis, Tennessee 3816332 Received 13 July 1992/Returned for modification 12 August 1992/Accepted 21 December 1992 The cytoplasmic domain of the erythropoietin receptor (EpoR) contains a region, proximal to the transmembrane domain, that is essential for function and has homology with other members of the cytokine receptor family. To explore the functional significance of this region and to identify critical residues, we introduced several amino acid substitutions and examined their effects on erythropoietin-induced mitogenesis, tyrosine phosphorylation, and expression of immediate-early (c-fos, c-myc, and egr-1) and early (ornithine decarboxylase and T-cell receptor 'y) genes in interleukin-3-dependent cell lines. Amino acid substitution of W-282, which is strictly conserved at the middle portion of the homology region, completely abolished all the functions of the EpoR. Point mutation at L-306 or E-307, both of which are in a conserved LEVL motif, drastically impaired the function of the receptor in all assays. Other point mutations, introduced into less conserved amino acid residues, did not significantly impair the function of the receptor. These results demonstrate that conserved amino acid residues in this domain of the EpoR are required for mitogenesis, stimulation of tyrosine phosphorylation, and induction of immediate-early and early genes. The growth and differentiation of hematopoietic cells are Although hematopoietic growth factor receptors induce regulated by various cytokines which act through specific tyrosine phosphorylation, little is known about the tyrosine receptors (3, 22). Most of the hematopoietic growth factor kinases that may associate with the receptors and be in- receptors, including the receptor for erythropoietin (EpoR), volved in mitogenesis. The simplest hypothesis is that a belong to a cytokine receptor family (2, 6). Members of this tyrosine kinase physically associates with members of the family characteristically have four conserved cysteine resi- cytokine receptor family. The similar response that is in- dues and the WSXWS motif in the extracellular domain. The duced by various growth factors has been interpreted to WSXWS motif has been suggested to be critical for the indicate that common, or highly related, tyrosine kinases folding of the extracellular domain of the interleukin-2 re- associate with different receptors. Recently several studies ceptor Pi subunit (IL-2,BR) (26). In contrast, the intracellular have shown an association of Ick with IL-213R (13) or domains of these receptors are quite different and do not activation of Ick kinase activity following IL-2 binding (16). contain motifs that would indicate potential functions in However, Ick associates with a region of the receptor that is limited signal transduction. Recently, however, sequence not required for a mitogenic response (13), and IL-2PR similarity has been noted within the cytoplasmic domains of induces mitogenesis in cells that do not express Ick. There- several of the hematopoietic growth factor receptors (11, 26, fore, it is likely that other domains within the receptor may 27). The functional significance of these regions has not been associate with different tyrosine kinases to initiate a mito- established. genic response. The signal-transducing events that are activated by stim- Several studies have begun to define the domains of ulation of factor have been hematopoietic growth receptors hematopoietic growth factors that are required for mitogen- extensively studied and have provided evidence for a role for esis. These studies have shown that the carboxyl half of the tyrosine phosphorylation in growth regulation. In particular, cytoplasmic domain is not required for a mitogenic response constitutively activated tyrosine kinases abrogate the re- interleukin-6 of cells for factors for the receptor (IL-6R)-associated gp130 (27), quirement hematopoietic growth (21, 28), granulocyte colony-stimulating factor (G-CSF) receptor (11), and temperature-sensitive mutants confer a conditional re- quirement for growth factors (4, 18). A number of hemato- IL-213R (14), or EpoR (8, 24, 29). However, in Ba/F3 cells, poietic growth factors, including Epo, have been shown to the cytoplasmic tail of the EpoR can inhibit the mitogenic rapidly induce tyrosine phosphorylation (24, 30). Strikingly, response (8), and in FDC-P1 cells, it down-modulates the different growth factors, such as Epo and interleukin-3 action of the granulocyte-macrophage colony-stimulating (IL-3), induce the phosphorylation of a comparable set of factor receptor (29). Internal deletions and point mutations substrates (24). A correlation has been shown between have begun to focus attention on a region of the cytoplasmic mitogenesis and the ability to induce tyrosine phosphoryla- domain that shows limited sequence identity among a num- ber of tion among a series of carboxyl and internal deletions of the hematopoietic growth factor receptors (11, 26, 27). EpoR (24). This region is of particular interest since many of the receptors mediate comparable events in cells and are thus speculated to associate with common signal-transducing * Corresponding author. proteins. Consistent with this view, some chimeric receptors t Present address: Department of First Internal Medicine, Tokyo have been shown to retain the ability to induce mitogenesis Medical and Dental University, Tokyo, Japan. (37). Deletion of a 20-amino-acid or a 46-amino-acid segment 1788 VOL. 13, 1993 INACTIVATION OF THE EpoR BY POINT MUTATIONS 1789 within the conserved region in the EpoR (24) or IL-2PR (14), chemical), cells were washed free of IL-3, cultured over- respectively, inactivates these receptors. Moreover muta- night, and left unstimulated as a control or stimulated with a tions in conserved sequences in this region of the IL-2PR saturating concentration of Epo or IL-3. Cells were then (26) or the IL-6R-associated gp130 (27) can inactivate recep- lysed in the lysis buffer previously described for analysis of tor function. To extend these studies, we have constructed a phosphotyrosyl proteins by immunoprecipitation (24). After series of mutants of the EpoR containing point mutations in clarification by spinning at 15,000 rpm for 20 min in a conserved amino acids and demonstrate that these changes refrigerated microcentrifuge, lysates were mixed with equal can inactivate receptor function, as assessed by the ability to volumes of 2x Laemmli's sodium dodecyl sulfate (SDS) induce mitogenesis, to stimulate tyrosine phosphorylation, sample buffer and heated at 100°C for 5 min. A sample from and to induce immediate-early and early response genes. 4 x 105 cells was applied to each well of SDS-8.5% polyac- rylamide gels, electrophoresed, and blotted onto nitrocellu- MATERIALS AND METHODS lose membranes. Membranes were probed with the 4G10 antibody followed by detection with an ECL Western immu- Cells and reagents. DA-3 cells, an IL-3-dependent-cell line noblotting detection system (Amersham). derived from a primary Moloney murine leukemia virus- To examine the tyrosine phosphorylation of the EpoR, the induced leukemia (17), were maintained as described previ- receptors were first purified by immunoprecipitation with a ously (24). DA3/EpoR-Wt, a clone of DA-3 cells expressing rabbit antiserum raised against a bacterial recombinant pro- the EpoR obtained by transfecting the wild-type (wt) EpoR tein containing the cytoplasmic region of the EpoR. The cDNA, has been previously described (24). A clone of 32Dcl receptors were then electrophoresed on SDS-polyacrylam- cells that has been previously described (23) was used in ide gels and blotted onto nitrocellulose membranes. Mem- these studies. An expression plasmid for the murine EpoR, branes were first probed with the 4G10 antibody as described pXM-EpoR, and a rabbit polyclonal antipeptide antiserum above. The filters were then treated with stripping buffer directed against the amino terminus of the murine EpoR (100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCI have also been previously described (7, 35). Recombinant [pH 6.7]) at 50°C for 30 min and were subsequently probed human Epo was kindly provided by Amgen Biological with the antiserum against the recombinant EpoR. (Thousand Oaks, Calif.). All other reagents were purchased Isolation and analysis of RNA. Cells were washed free of from commercial sources unless otherwise noted. IL-3, cultured for 24 h, and then stimulated with IL-3 or Epo Construction of expression plasmids for EpoR mutants and for various periods. Total RNA was extracted from cells and transfection into DA-3 cells. Site-directed mutagenesis of analyzed by Northern (RNA) blotting as described previ- amino acids within the region of the EpoR showing homol- ously (4). The DNA probes used for analyses were a 950-bp ogy with the IL-23 receptor chain was carried out by XbaI-SstI mouse c-myc exon 2 fragment, a 1.6-kb BamHI- primer-mediated mutagenesis using the polymerase chain EcoRI mouse ornithine decarboxylase (ODC) cDNA frag- reaction (PCR) method as described by Higuchi (15). In ment, and a 1.8-kb PstI fragment of the chicken ,-actin brief, two complementary primers having a mutation to be cDNA. The probe for egr-1 was a 1.4-kb EcoRI fragment of introduced were synthesized and used in combination with the murine cDNA. The probe for the nonrearranged y T-cell one of two primers on either side of the cloning site of the receptor locus (TCR-y) was a PstI 600-bp murine genomic pXM vector for the first-step PCR. After purification from fragment that detects the constant region of TCRy1-3 (34).
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