Characterization of the receptor binding determinants of granulocyte colony stimulating factor

Protein Science : a Publication of the Protein Society
D C YoungD J Matthews

Abstract

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 secon...Continue Reading

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Citations

May 22, 2002·Protein Science : a Publication of the Protein Society·Anton V FilikovBassil I Dahiyat
Apr 23, 2002·Protein Science : a Publication of the Protein Society·Peizhi LuoBassil I Dahiyat
Sep 24, 1999·Biological Chemistry·J GrötzingerS Rose-John
Feb 24, 2006·Proceedings of the National Academy of Sciences of the United States of America·Taro TamadaRyota Kuroki
Jul 30, 2014·Journal of the American Society for Mass Spectrometry·Daisuke TsuchidaSatoko Akashi
Nov 28, 2013·Langmuir : the ACS Journal of Surfaces and Colloids·Ivan M KempsonClive A Prestidge
Apr 30, 2003·Protein Science : a Publication of the Protein Society·Margaret Speed RicciDavid N Brems
Jun 3, 2004·Chembiochem : a European Journal of Chemical Biology·Sebastian KrauseKarlheinz Friedrich
Jun 15, 2001·The Journal of Biological Chemistry·B BishopL Regan
Oct 16, 2018·Current Computer-aided Drug Design·Maryam G SiahmazgiMehdi Zeinoddini
Dec 23, 2020·PLoS Biology·Birte Hernandez AlvarezMohammad ElGamacy
Jul 7, 1998·Journal of Molecular Biology·A A Bogan, K S Thorn

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