Characterization of the receptor binding determinants of granulocyte colony stimulating factor.
We performed a series of experiments using alanine-scanning mutagenesis to locate side chains within human granulocyte colony-stimulating factor (G-CSF) that are involved in human G-CSF receptor binding. We constructed a panel of 28 alanine mutants that examined all surface exposed residues on helices A and D, as well as all charged residues on the surface of G-CSF. The G-CSF mutants were expressed in a transiently transfected mammalian cell line and quantitated by a sensitive biosensor method. We measured the activity of mutant proteins using an in vitro proliferation assay and an ELISA binding competition assay. These studies show that there is a region of five charged residues on helices A and C employed by G-CSF in binding its receptor, with the most important residue in this binding patch being Glu 19. Both wild-type G-CSF and the E19A mutant were expressed in E. coli. The re-folded proteins were found to have proliferative activities similar to the analogous proteins from mammalian cells: furthermore, biophysical analysis indicated that the E19A mutation does not cause gross structural perturbations in G-CSF. Although G-CSF is likely to signal through receptor homo-dimerization, we found no compelling evidence for a second receptor binding region. We also found no evidence of self-antagonism at high G-CSF concentrations, suggesting that, in contrast to human growth hormone (hGH) and erythropoietin (EPO), G-CSF probably does not signal via a pure 2:1 receptor ligand complex. Thus, G-CSF, while having a similar tertiary structure to hGH and EPO, uses different areas of the four helix bundle for high-affinity interaction with its receptor.
Young
D C
DC
Department of Molecular Biology, Arris Pharmaceutical Corporation, South San Francisco, California 94080, USA.
Zhan
H
H
Cheng
Q L
QL
Hou
J
J
Matthews
D J
DJ
D000409:Alanine / Q000235:genetics; D001665:Binding Sites; D002455:Cell Division / Q000187:drug effects; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D016179:Granulocyte Colony-Stimulating Factor / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D016188:Receptors, Granulocyte Colony-Stimulating Factor / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology
D016188:Receptors, Granulocyte Colony-Stimulating Factor; D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor; D000409:Alanine