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Introduction of erythropoietin receptors (EpoRs) into the interleukin-3 (IL-3)-dependent murine hemopoietic cell line, Ba/F3, enables these cells to not only proliferate, after an initial lag in G1, but also to increase beta-globin mRNA levels in response to erythropoietin (Epo). With IL-3 and Epo costimulation, IL-3-induced signaling appears to be dominant since no increase in beta-globin mRNA occurs. Differentiation and proliferation signals may be uncoupled since EpoRs lacking all eight intracellular tyrosines were compromised in proliferative signaling but retained erythroid differentiation ability. Intriguingly, a chimeric receptor of the extracellular domain of the EpoR and the transmembrane and intracellular domains of IL-3RbetaIL-3 chain (EpoR/IL-3RbetaIL-3) was capable of Epo-induced proliferative and differentiating signaling, suggesting either the existence of a second EpoR subunit responsible for differentiation or that the alpha subunit of the IL-3 receptor (IL-3R) prevents it. Arguing against the former, a truncated EpoR lacking an intracellular domain was incapable of promoting proliferation or differentiation. An EpoR/IL-3Ralpha chimera, in contrast, was capable of transmitting a weak Epo-induced proliferative signal but failed to stimulate accumulation of beta-globin mRNA. Most significantly, coexpression of the EpoR/IL-3Ralpha chimera with either EpoR/IL-3Rbeta or wild-type EpoRs suppressed Epo-induced beta-globin mRNA accumulation. Taken together, these results suggest an active role for the IL-3Ralpha subunit in inhibiting EpoR-specific differentiating signals.

In this report we describe the purification of the murine interleukin 3 receptor (mIL-3R) to apparent homogeneity using a two-step procedure involving biotinylated mIL-3 (B-mIL-3) and affinity binding to immobilized antiphosphotyrosine and streptavidin agarose (SA). Purification was monitored using an assay for detergent solubilized-mIL-3Rs that utilized unglycosylated 125I-mIL-3 and concanavalin A (ConA)-Sepharose beads. The final material consisted of a 140-kDa tyrosine and serine phosphorylated protein that was greater than 98% pure as assessed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of either [35S]methionine-labeled, silver-stained, or radioiodinated preparations. Characterization of the purified receptor revealed that it migrated identically under reducing and nonreducing conditions in SDS gels, possessed 10 kDa of N-linked carbohydrate, and was cleaved upon storage at 4 degrees C to a 70-kDa form. These properties suggested that the purified mIL-3R was identical to that identified by cross-linking studies. The KD of the purified receptor was 1-5 nM, similar to estimates obtained using intact normal mouse bone marrow cells and mIL-3-dependent cell lines. The two-step purification procedure also isolated a 120-kDa serine phosphorylated but nontyrosine phosphorylated mIL-3R species. Apart from phosphorylation differences, the 140- and 120-kDa species were apparently identical, yielding, after alkaline phosphatase treatment, the same molecular mass on SDS gels and similar chymotryptic peptide maps. Amino acid sequences and composition data obtained from the more abundant and more stable serine phosphorylated 120-kDa mIL-3R, further purified by SDS-polyacrylamide gel electrophoresis, suggested that the purified mIL-3R may be identical to the predicted sequence of the recently isolated cDNA clone AIC2A. This was further suggested by comparing chymotryptic maps of the 120-kDa mIL-3R with the Aic2A protein and using antibodies corresponding to the amino and carboxyl termini of the AIC2A cDNA product. However, the Aic2A protein, when expressed on the surface of COS or 3T3 cells or following detergent solubilization and partial purification with biotinylated mIL-3 and SA, displayed a substantially lower affinity for mIL-3.

Molecular cloning, mapping, and DNA sequencing techniques have been used to characterize the functional human dihydrofolate reductase (DHFR) gene. The gene is about 30 kilobases in length. Its coding portions are separated into 6 exons, the intron-exon boundaries of which are identical to those of the previously characterized mouse DHFR gene. The 5 introns vary in length from 362 to 12,000 base pairs. The position of the DHFR gene promoter was identified as being shortly upstream from the initiation codon in an in vitro transcription reaction by polymerase II. A DHFR minigene was constructed in a plasmid expression vector by combining a DNA fragment containing exon 1, intron I, and a small part of exon 2 from the functional gene, with a second DNA fragment containing exons 2-6 from a processed intronless gene, the coding sequences of which are identical to those of the normal locus. Transcription initiation from the DHFR promoter was localized to a position 71 +/- 2 base pairs upstream from the initiation codon, both in monkey kidney cells transfected with vectors containing the DHFR minigene, and in human HeLa cells. This single transcription start and the three previously identified polyadenylation sites account for the 800-, 1,000-, and 3,800-nucleotide DHFR mRNA species found in human cells. On comparison of the mouse and human DHFR genes, sequence homology was shown to be limited to the coding regions and 100 base pairs of the 3' untranslated region up to the first polyadenylation site of both genes. In addition, there is fairly extensive homology in the 5' flanking region, although the quadruply repeated 48-base pair sequence found in the mouse genome is represented only once in human DNA.

Monoclonal antibody 114/A10, raised against the murine multipotential hemopoietic progenitor cell line B6SUtA, identifies an antigen highly expressed by primary myeloid progenitor cells, the myelomonocytic leukemia cell line WEHI-3, and various interleukin-3 (IL-3)-dependent cell lines. Western blotting studies indicate that the 114/A10 antigen has a mean relative molecular mass of 150,000 but varies greatly in size range between different cell types. cDNA clones encoding this protein were isolated from a plasmid-based expression vector library prepared from B6SUtA RNA. Three clones corresponding in size to the two major mRNA species detected in IL-3-dependent cell lines (3.0 and 2.2 kilobase pairs) and differing in their utilization of alternative polyadenylation signals were obtained. These clones contain a single long open reading frame of 573 amino acids possessing the typical characteristics of an integral membrane protein. A particularly striking feature of this sequence is the presence at the extracellular amino terminus of a series of eight highly conserved 27-amino acid, serine/threonine-rich (55%) tandem repeats that may serve as sites of extensive glycosylation. The extracellular domain also contains three epidermal growth factor-like cysteine-rich repeats. The distribution and structural characteristics of the 114/A10 antigen suggest a possible regulatory role in the cellular response to IL-3.