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The Axl receptor tyrosine kinase was identified as a protein encoded by a transforming gene from primary human myeloid leukaemia cells by DNA-mediated transformation of NIH 3T3 cells. Axl is the founding member of a family of related receptors that includes Eyk, encoded by a chicken proto-oncogene originally described as a retroviral transforming gene, and c-Mer, encoded by a human proto-oncogene expressed in neoplastic B- and T-cell lines. The transforming activity of Axl demonstrates that the receptor can drive cellular proliferation. The function of Axl in non-transformed cells and tissues is unknown, but may involve the stimulation of cell proliferation in response to an appropriate signal, namely a ligand that activates the receptor. We report here the purification of an Axl stimulatory factor, and its identification as the product of growth-arrest-specific gene 6 (ref. 6). This is, to our knowledge, the first description of a ligand for the Axl family of receptors.

A protein ligand for the ECK receptor protein-tyrosine kinase has been isolated by using the extracellular domain (ECK-X) of the receptor as an affinity reagent. Initially, concentrated cell culture supernatants were screened for receptor binding activity using immobilized ECK-X in a surface plasmon resonance detection system. Subsequently, supernatants from selected cell lines were fractionated directly by receptor affinity chromatography, resulting in the single-step purification of B61, a protein previously identified as the product of an early response gene induced by tumour necrosis factor-alpha. We report here that recombinant B61 induces autophosphorylation of ECK in intact cells, consistent with B61 being an authentic ligand for ECK. ECK is a member of a large orphan receptor protein-tyrosine kinase family headed by EPH, and we suggest that ligands for other members of this family will be related to B61, and can be isolated in the same way.

The present study shows that recombinant human megakaryocyte growth and development factor (r-HuMGDF) behaves both as a megakaryocyte colony stimulating factor and as a differentiation factor in human progenitor cell cultures. Megakaryocyte colony formation induced with rHuMGDF is synergistically affected by stem cell factor but not by interleukin 3. Megakaryocytes stimulated with rHuMGDF demonstrate progressive cytoplasmic and nuclear maturation. Measurable levels of megakaryocyte growth and development factor in serum from patients undergoing myeloablative therapy and transplantation are shown to be elaborated in response to thrombocytopenic stress. These data support the concept that megakaryocyte growth and development factor is a physiologically regulated cytokine that is capable of supporting several aspects of megakaryopoiesis. (J. Clin. Invest. 1995Invest. . 95:2973Invest. -2978

No abstract

Platelet formation, occurring from bone marrow or lung megakaryocytes, has been difficult to study mechanistically. Recombinant human megakaryocyte growth and development factor (rHuMGDF), a recently described cytokine, has now been used to establish an in vitro system in which this important and little understood process occurs. CD34+ cells cultured with rHuMGDF develop into megakaryocytes which form long cytoplasmic extensions (proplatelets) that fragment into platelet-sized particles (in vitro platelets). Morphologically, in vitro and human plasma-derived platelets (control platelets) are virtually identical with respect to size, dense granule distribution and ultrastructural features. Functionally, in vitro and control platelets have similar aggregation and activation responses, and similarly incorporate mepacrine into dense granules. These findings suggest that rHuMGDF is sufficient to generate platelet-synthesizing megakaryocytes from CD34+ cells and provide an experimental setting in which the study of human platelet formation can be adequately performed.

c-kit encodes the transmembrane receptor tyrosine kinase (Kit) for the recently described ligand stem cell factor (SCF). We have developed an enzyme-linked immunosorbent assay for measuring soluble human Kit and we have used the assay to show high levels of soluble Kit in human serum. The distribution of soluble Kit levels was investigated among 112 normal human serum donors. The mean serum level (+/- SD) was found to be 324 +/- 105 ng/mL with the values falling between 163 ng/mL and 788 ng/mL. No correlation between soluble Kit levels and the sexes or ages of the donors was found. Partial purification using immunoaffinity chromatography allowed us to characterize the soluble Kit from pooled human serum. Antibodies generated to a 497-amino acid recombinant human soluble Kit corresponding to the N-terminal extracellular domain of the receptor recognized the serum-derived soluble Kit by immunoblotting. We found that the serum-derived soluble Kit is glycosylated, with mostly N- linked but also O-linked carbohydrate, and with terminal sialic acid residues. When compared with the recombinant human soluble Kit, the serum-derived material was similar both in size and glycosylation pattern. CNBr cleavage of the isolated serum-derived material followed by amino terminal sequencing confirmed the presence of five peptides expected for the extracellular portion of the Kit molecule. The immunoaffinity purified serum-derived soluble Kit inhibited binding of [125I]SCF to membrane-bound receptor in an in vitro assay. These results indicate that soluble Kit could modulate the activity and functions of SCF in vivo.

The cytotoxicity of pertussis toxin, a multisubunit exotoxin produced byBordeteUapertussis, is believed to be due to the ADP-ribosyltransferase activity of the Si subunit. We have previously described the recombinant expression of each of the five individual pertussis toxin subunits in Escherichia coli and the production of an enzymaticaily deficient form of the Si subunit by site-directed mutagenesis. We now report the in vitro assembly of holotoxin from native pertussis toxin B oligomer and recombinant Si subunits, the latter purified and refolded from insoluble inclusion bodies. Holotoxin assembled with recombinant S1 of authentic amino acid sequence was indistinguishable from native pertussis toxin in its electrophoretic migration and ability to elicit a cytopathic response in cultured Chinese hamster ovary cells; in contrast, holotoxin assembled with the genetically deactivated analog of recombinant Si displayed greatly diminished cytopathicity.These results verify that the in vitro cytopathic effects of pertussis toxin are the result of the enzymatic activity of the S1 subunit and illustrate the potential for constructing complex quaternary protein structures in vitro from insoluble, unfolded polypeptides derived from expression in recombinant systems.In an effort to create a fully efficacious yet less reactogenic pertussis vaccine, we have focused on the recombinant production of PTX subunit proteins, detoxification of the Si moiety by site-specific mutagenic inactivation of enzyme activity, and the in vitro assembly of a genetic "holotoxoid." For these purposes, the individual subunit polypeptides were expressed at high levels in Escherichia coli (36), and a region of the recombinant Si subunit (Val8-Pro'5) was identified as both necessary for enzyme activity and critical for the formation of its neutralizing, protective epitope (37). Selective site-directed mutagenesis of the subcloned S1 cistronic element permitted us to derive and analog polypeptide (Arg9 -k Lys) retaining the protective epitope, but with strikingly reduced ADP-ribosyltransferase activity (38). We wished to assess the biological activity of this genetically deactivated S1 mutant subunit and evaluate its potential to participate in the production of a recombinant holotoxoid. To this end, we have assembled holotoxin molecules in vitro that are composed of native PTX B oligomer and recombinant SI subunits of either native or mutant sequence and evaluated their relative abilities to elicit cytotoxic responses in cultured cells.Pertussis toxin (PTX) is both a major virulence factor of Bordetella pertussis (1), the etiologic agent of whooping cough (2), and an important antigenic element in vaccines for immunoprotection against disease (3-7). Certain clinical manifestations of infection (1,8,9) and minor reactions to vaccination (10,11) are attributable to the effects of this complex exotoxin. The origin of the extremely rare adverse neurologic events temporally related to pertussis immunization remains controversial (11-15). Chemic...