Extracellular domain of granulocyte-colony stimulating factor receptor. Interaction with its ligand and identification of a domain in close proximity of ligand-binding region.
An extracellular domain of human granulocyte-colony stimulating factor (G-CSF) receptor was expressed in and purified from Chinese hamster ovary cells. Complex formation between G-CSF and the receptor was studied by size exclusion chromatography, followed by chemical cross-linking. The receptor-ligand complex contained an equimolar ratio of each protein. Crosslinking experiments using disucciniimide suberate revealed that the native complex contained at least two types of cross-linked complexes; one form contained one or two G-CSF molecules per receptor molecule, whereas another form contained one or two G-CSF per two receptor molecules. The tryptic peptide map of the cross-linked complex provided a unique peptide peak which was not found in a peptide map of the original protein. Sequence analysis and mass spectrometry of the peptide indicated that two peptides were covalently linked by cross-linker, one peptide from G-CSF and the other from the receptor. In the cross-linked peptide, Lys-242 of the receptor cross-linked the amino terminal Met of G-CSF through the cross-linker. It was also shown that the N-terminal Met of G-CSF was readily acetylated in the receptor-ligand complex, indicating that it was not directly involved in receptor binding. The results show that the N-terminal Met of G-CSF is located at a distance of approximately 11 A from a reactive Lys-242 of the receptor in the ligand-receptor complex.
Haniu
M
M
Department of Protein Structure, Amgen, Inc., Thousand Oaks, California 91320, USA.
Horan
T
T
Arakawa
T
T
Le
J
J
Katta
V
V
Rohde
M F
MF
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D016179:Granulocyte Colony-Stimulating Factor / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D016188:Receptors, Granulocyte Colony-Stimulating Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017421:Sequence Analysis; D013820:Thermolysin
D003432:Cross-Linking Reagents; D010446:Peptide Fragments; D016188:Receptors, Granulocyte Colony-Stimulating Factor; D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor; D013820:Thermolysin