Structure and transforming properties ofH4/PDGFβR and related mutants.

(A) Schematic diagram of H4/PDGFβR fusion and related mutants. H4/PDGFβR (wt) is the wild-type full-length cDNA. H4/PDGFβR-ΔEX lacks amino acids 101 to 368 of H4; H4/PDGFβR-ΔLZ lacks the leucine zipper domain from amino acids 55 to 93 of H4 moiety;H4/PDGFβR-WW contains 2 W → A substitutions at positions 566/593 of the PDGFβR gene;H4/PDGFβR-634R is a kinase-inactive mutant; andH4/PDGFβR-F2 contains 2 Y → F mutations of residues 579/581 of the PDGFβR portion of the fusion. (B) H4/PDGFβR transformation of Ba/F3 cells. H4/PDGFβR was retrovirally transduced by retrovirus into Ba/F3 cells, followed by selection with G418 (1 mg/mL) in the presence of IL-3. After 7 days, the cells were washed and IL-3 was withdrawn. Cells were counted every 24 hours with the use of trypan blue solution (0.4%) to distinguish between viable and nonviable cells. None of the H4/PDGFβR mutants tested were able to efficiently induce IL-3–independent growth, although longer term culture allowed for eventual outgrowth of IL-3–independent clones (data not shown). Control experiments demonstrated that TEL/JAK2, but not a PGK-neo empty vector, conferred factor independence to Ba/F3 cells. (C) Expression of H4/PDGFβR and related mutants. Expression of H4/PDGFβR and related mutants was confirmed by Western blot analysis of Ba/F3 whole cell lysates by means of anti-PDGFβR antibody as described in “Materials and methods.” Expected band sizes are H4/PDGFβR, 110 kd; H4/PDGFβR ΔEX, 85 kd; and H4/PDGFβR ΔLZ, 105 kd.