CHARACTERIZATION OF SPECIFIC FUNCTIONAL RECEPTORS FOR HUIFN-ALPHA ON A HUMAN MEGAKARYOCYTIC CELL-LINE (DAMI) - EXPRESSION RELATED TO DIFFERENTIATION

Interferon-alpha (IFN-alpha) treatment has been shown to be highly eff ective in inhibiting human megakaryocytopoiesis and controlling thromb ocytosis in patients with myeloproliferative disorders. These observat ions suggest that IFN-cr might play some role in the biological featur e of the megakaryocytic lineage and led us to investigate the presence of specific receptors for IFN-alpha on human megakaryocytic cells, i. e. the Dami cell line, and to study the regulation of their expression . Our study demonstrates that [I-125]-recombinant human IFN-alpha (I-1 25]rHu-IFN-alpha binds to high-affinity specific receptor on these cel ls. Scatchard analysis of binding data indicates the presence of homog eneous binding sites estimated in the range of 3000-5000, with an appa rent equilibrium dissociation constant, K-d of 1-2 x 10(-9) M, Also, [ I-125]rHuIFN-alpha binding capacity decreased in Dami cells incubated with unlabelled rHulFN-alpha. This down-regulation which was dose-depe ndent appeared to result from a reduction of IFN-alpha( cell surface r eceptors and was observed at doses that elicited antiproliferative eff ects in Dami cells. Crosslinking of [I-125]rHuIFN-alpha to Dami membra ne proteins using a bifunctional reagent yielded to a radioactive comp lex of approximate to 150,000 kD on SDS-PAGE. Furthermore, in response to PMA, which induces the differentiation/maturation of the Dami cell s as evaluated by surface marker and ploidy analysis, a 3-fold increas e of the number of specific membrane receptors for IFN-alpha was obser ved, without any modification of either the affinity or the M(r) value of the cross-linked complex. Such an increase appeared to be restrict ed to IFN-a receptors; actually it was not observed in [I-125]IFN-gamm a binding experiments. Transcript analysis indicated that down-regulat ion and increased expression of the IFN-cr receptor after PMA treatmen t are post-transcriptional events.