DIMERIZATION OF THE EXTRACELLULAR DOMAIN OF GRANULOCYTE-COLONY-STIMULATING FACTOR-RECEPTOR BY LIGAND-BINDING - A MONOVALENT LIGAND INDUCES 2 2 COMPLEXES/

Granulocyte-colony stimulating factor (G-CSF) binds to a specific cell surface receptor and induces signals for growth and differentiation i n cells of granulocyte hematopoietic lineage. In order to understand h ow G-CSF binding initiates signals into these cells, we have studied i ts interaction with the entire extracellular domain of the receptor (s G-CSFR). The sG-CSFR was purified from CHO cell. conditioned media wit h a G-CSF affinity column, resulting 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 com petent. Therefore, all studies were carried out using affinity-purifie d material. The sG-CSFR exhibited a weak self-association into a dimer with a dissociation constant of 200 mu M in the absence of G-CSF. Add ition of G-CSF dimerizes the receptor, with a preferred stoichiometry of 2 G-CSF molecules plus 2 receptors, Unexpectedly, receptor dimeriza tion appears to occur through receptor-receptor interactions rather th an through two receptors binding to the same G-CSF molecule; i.e., G-C SF is a monovalent ligand. G-CSF binding to the receptor monomer occur s with high affinity. The binding of G-CSF also enhances the receptor- receptor dimerization; when G-CSF is bound to both receptors, dimeriza tion is enhanced 2000-fold, while the interaction of a 1:1 receptor-li gand complex with a second ligand-free receptor is enhanced 80-fold. T hus, the mechanism of receptor dimerization is fundamentally different than that of related cytokine receptors such as growth hormone and er ythropoietin receptors. Circular dichroic spectra showed a small but s ignificant 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 intera ctions.