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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.

Granulocyte-colony stimulating factor (G-CSF) binds to a specific cell surface receptor and induces signals for growth and differentiation in cells of granulocyte hematopoietic lineage. In order to understand how G-CSF binding initiates signals into these cells, we have studied its interactions with the entire extracellular domain of the receptor (sG-CSFR). The sG-CSFR was purified from CHO cell conditioned media with a G-CSF affinity column, resting in a preparation fully competent for ligand binding. However, when sG-CSFR was purified by conventional means, i.e., without affinity chromatography, only about half was competent. Therefore, all studies were carried out using affinity-purified material. The sG-CSFR exhibited a weak self-association into a dimer with a dissociation constant of 200microM in the absence of G-CSF. Addition of G-CSF dimerizes the receptor, with a preferred stoichiometry of 2 G-CSF molecules plus 2 receptors. Unexpectedly, receptor-receptor interactions rather than through two receptors binding to the same G-CSF molecule; i.e., G-CSF is a monovalent ligand. G-CSF binding to the receptor monomer occurs with high affinity. The binding of G-CSF also enhances the receptor-receptor dimerization; when G-CSF is bound to both receptors, dimerization is enhanced 2000-fold, while the interaction of a 1:1 receptor-ligand complex with a second ligand-free receptor is enhanced 80-fold. Thus, the mechanism of receptor dimerization is fundamentally different than that of related cytokine receptors such as growth hormone and erythropoietin receptors. Circular dichroic spectra showed a small but significant conformational change of receptor upon binding G-CSF. This is consistent with the idea that G-CSF binding alters the conformation of the receptor, resulting in an increase in receptor-receptor interactions.