Evidence for the ligand-induced conversion from a dimer to a tetramer of the granulocyte colony-stimulating factor receptor

• Published in: FEBS letters 1994-12, Vol.356 (2), p.255-260
• Main Authors: Hiraoka, Osamu, Anaguchi, Hiroyuki, Ota, Yoshimi
• Format: Article
• Language: English
• Subjects: Animals; Autographa californica; Base Sequence; Blotting, Western; Cell Line; CRH, cytokine receptor homologous region; DNA Primers; Extracellular portion; G-CSF, granulocyte colony-stimulating factor; Genetic Vectors; GH, growth hormone; Granulocyte colony-stimulating factor receptor; HPLC, high performance liquid chromatography; Ig, immunoglobulin-like; Insect cell; Insecta; kDa, kilodalton; Ligands; Macromolecular Substances; Mice; Molecular Sequence Data; nuclear polyhedrosis virus; Nucleopolyhedrovirus; PAGE, polyacrylamide gel electrophoresis; Polymerase Chain Reaction; Receptors, Cytokine - chemistry; Receptors, Granulocyte Colony-Stimulating Factor - chemistry; Receptors, Granulocyte Colony-Stimulating Factor - isolation & purification; Receptors, Granulocyte Colony-Stimulating Factor - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; SDS, sodium dodecyl sulfate; Sequence Homology, Amino Acid; Tetramer; Transfection; Trichoplusia ni
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/0014-5793(94)01278-4

An extracellular portion of granulocyte colony-stimulating factor (G-CSF) receptor, which contains an immunoglobulin-like (Ig) domain and cytokine receptor homologous (CRH) region, was secreted into the medium using Trichoplusia ni-Autographa californica nuclear polyhedrosis virus system. The gene product was purified to homogeneity mainly as a dimer (85 kDa) using G-CSF affinity column chromatography and gel filtration HPLC, although the product existed as a monomer (45 kDa) in the medium. Scatchard analyses suggested that only the dimer had high affinity ligand binding ( K d = about 100 pM), which is comparable with the K d value of the cell surface receptor. The binding of G-CSF to Ig-CRH induced its tetramerization (200–250 kDa). The molecular composition of the tetrameric complex showed a stoichiometry of four ligands bound to four Ig-CRH. These results suggested that the oligomeric mechanism of the G-CSF receptor differs from that reported for growth hormone (GH) receptor, although CD spectrum spectroscopy suggested that the Ig-CRH has a GH receptor-like structure.