Characterization of specific functional receptors for HuIFN‐α on a human megakaryocytic cell line (Dami): expression related to differentiation

Summary Interferon‐alpha (IFN‐α) treatment has been shown to be highly effective in inhibiting human megakaryocytopoiesis and controlling thrombocytosis in patients with myeloproliferative disorders. These observations suggest that IFN‐α might play some role in the biological feature of the megakaryocytic lineage and led us to investigate the presence of specific receptors for IFN‐α on human megakaryocytic cells, i.e. the Dami cell line, and to study the regulation of their expression. Our study demonstrates that [125I]‐recombinant human IFN‐α ([125I]rHu‐IFN‐α) binds to high‐affinity specific receptor on these cells. Scatchard analysis of binding data indicates the presence of homogeneous binding sites estimated in the range of 3000–5000. with an apparent equilibrium dissociation constant, Kd, of 1–2 × 109m. Also, [125I]rHuIFN‐α binding capacity decreased in Dami cells incubated with unlabelled rHuIFN‐α. This down‐regulation which was dose‐dependent appeared to result from a reduction of IFN‐α cell surface receptors and was observed at doses that elicited antiproliferative effects in Dami cells. Crosslinking of [125I]rHuIFN‐α to Dami membrane proteins using a bifunctional reagent yielded to a radioactive complex of 150,000 kD on SDS‐PAGE. Furthermore, in response to PMA, which induces the differentiation/maturation of the Dami cells as evaluated by surface marker and ploidy analysis, a 3‐fold increase of the number of specific membrane receptors for IFN‐α was observed, without any modification of either the affinity or the Mr value of the cross‐linked complex. Such an increase appeared to be restricted to IFN'‐a receptors; actually it was not observed in [125I]IFN‐y binding experiments. Transcript analysis indicated that down‐regulation and increased expression of the IFN‐α receptor after PMA treatment are post‐transcriptional events.