Requirement for the immunoglobulin-like domain of granulocyte colony-stimulating factor receptor in formation of a 2:1 receptor-ligand complex.
The extracellular portion of the granulocyte colony-stimulating factor (G-CSF) receptor has a mosaic structure of six domains (each approximately 100 amino acid residues) consisting of an immunoglobulin-like (Ig) domain, a cytokine receptor homologous region subdivided into amino-terminal (BN) and carboxyl-terminal (BC) domains, and three fibronectin type III repeats. In the present study, we expressed the Ig-BN and the BN-BC regions and purified them to homogeneity as monomers using G-CSF affinity column chromatography. Using gel filtration high performance liquid chromatography, we investigated the molecular composition of receptor-ligand complexes formed between G-CSF and purified BN-BC or Ig-BN domains. In contrast to the well characterized example of the human growth hormone (GH) receptor, in which the BN-BC.GH complex shows a 2:1 receptor-ligand complex stoichiometry, the BN-BC domain of the G-CSF receptor formed a 1:1 complex. The isolated Ig-BN domain also formed a 1:1 complex with G-CSF. However, in the presence of both Ig-BN and BN-BC domains, we detected a 1:1:1 Ig-BN.G-CSF.BN-BC complex corresponding to the 2:1 receptor: ligand stoichiometry. These results suggest that 1) the Ig domain and both the BN and the BC domains are required for oligomerization of the G-CSF receptor, 2) G-CSF contains two binding sites for its receptor, and 3) there are two ligand binding sites on the G-CSF receptor, one site on the BN-BC domain and one on the Ig-BN domain.
Hiraoka
O
O
Protein Engineering Research Institute, Osaka, Japan.
Anaguchi
H
H
Asakura
A
A
Ota
Y
Y
D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D016179:Granulocyte Colony-Stimulating Factor / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013006:Growth Hormone / Q000378:metabolism; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics; D008024:Ligands; D008956:Models, Chemical; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017924:Nucleopolyhedroviruses / Q000235:genetics; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016188:Receptors, Granulocyte Colony-Stimulating Factor / Q000235:genetics / Q000378:metabolism*; D011986:Receptors, Somatotropin / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D007074:Immunoglobulin G; D008024:Ligands; D010446:Peptide Fragments; D016188:Receptors, Granulocyte Colony-Stimulating Factor; D011986:Receptors, Somatotropin; D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor; D013006:Growth Hormone