Human granulocyte-macrophage colonystimulating factor (GM-CSF) is a pleiotropic hemopoietic growth factor and activator of mature myeloid cell function. We have previously shown that residue 21 in the first helix of GM-CSF plays a critical role in both biological activity and high-affinity receptor binding. We have now generated analogues of GM-CSF mutated at residue 21, expressed them in Escherichia coli, and examined them for binding, agonistic, and antagonistic activitles. Binding experiments showed that GM E21A, E21Q, E21F, E21H, E21R, and E21K bound to the GM-CSF receptor a chain with a similar affinity to wild-type GM-CSF and had lost high-affinity binding to the GM-CSF receptor a-chain-common 13-chain complex. From these mutants, only the charge reversal mutants E21R and E21K were completely devoid of agonistic activity. Significantly we found that E21R and E21K antagonized the proliferative effect of GM-CSF on the erythroleukemic cell line TF-1 and primary acute myeloid leukemias, as well as GM-CSF-mediated stimulation of neutrophil superoxide production. This antagonism was specific for GM-CSF in that no antagonism of interleukin 3-mediated TF-1 cell proliferation or tumor necrosis factor a-mediated stimulation of neutrophil superoxide production was observed. E. coli-derived GM E21R and E21K were effective antagonists of both nonglycosylated and glycosylated wild-type GM-CSF. These results show that low-affinity GM-CSF binding can be dissociated from receptor activation and have potential clinical significance for the management of inflammatory diseases and certain leukemias where GM-CSF plays a pathogenic role.

The functional role of the predicted first alpha‐helix of human granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) was analysed by site‐directed mutagenesis and multiple biological and receptor binding assays. Initial deletion mutagenesis pointed to residues 20 and 21 being critical. Substitution mutagenesis showed that by altering Gln20 to Ala full GM‐CSF activity was retained but that by altering Glu21 for Ala GM‐CSF activity and high affinity receptor binding were decreased. Substitution of different amino acids for Glu21 showed that there was a hierarchy in the ability to stimulate the various biological activities of GM‐CSF with the order of potency being Asp21 greater than Ser21 greater than Ala21 greater than Gln21 greater than Lys21 = Arg21. To distinguish whether position 21 was important for GM‐CSF binding to high or low affinity receptors, GM‐CSF (Arg21) was used as a competitor for [125I]GM‐CSF binding to monocytes that express both types of receptor. GM‐CSF (Arg21) exhibited a greatly reduced capacity to compete for binding to high affinity receptors, however, it competed fully for [125I]GM‐CSF binding to low affinity receptors. Furthermore, GM‐CSF (Arg21) was equipotent with wild‐type GM‐CSF in binding to the cloned low affinity alpha‐chain of the GM‐CSF receptor. These results show that (i) this position is critical for high affinity but not for low affinity GM‐CSF receptor binding thus defining two functional parts of the GM‐CSF molecule; (ii) position 21 of GM‐CSF is critical for multiple functions of GM‐CSF; and (iii) stimulation of proliferation and mature cell function by GM‐CSF are mediated through high affinity receptors.

Human interleukin 3 (IL-3) variants generated by site-directed mutgeness were analyzed in multiple biological and binding assays to identify residues critical for IL-3 activity. model where the C terminus of IL-3 interacts with the a chain of the IL-3 receptor, making this region a useful focus for the development of more potent IL-3 agonists or antagonists.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic cytokine that stimulates the production and functional activity of granulocytes and macrophages, properties that have encouraged its clinical use in bone marrow transplantation and in certain infectious diseases. Despite the importance of GM-CSF in regulating myeloid cell numbers and function, little is known about the exact composition and mechanism of assembly of the GM-CSF receptor complex. We have now produced soluble forms of the GM-CSF receptor ␣ chain (sGMR␣) and ␤ chain (s␤c) and utilized GM-CSF, the GM-CSF antagonist E21R (Glu21Arg), and the ␤c-blocking monoclonal antibody BION-1 to define the molecular assembly of the GM-CSF receptor complex. We found that GM-CSF and E21R were able to form low-affinity, binary complexes with sGMR␣, each having a stoichiometry of 1:1. Importantly, GM-CSF but not E21R formed a ternary complex with sGMR␣ and s␤c, and this complex could be disrupted by E21R. Significantly, sizeexclusion chromatography, analytical ultracentrifugation, and radioactive tracer experiments indicated that the ternary complex is composed of one s␤c dimer with a single molecule each of sGMR␣ and of GM-CSF. In addition, a hitherto unrecognized direct interaction between ␤c and GM-CSF was detected that was absent with E21R and was abolished by BION-1. These results demonstrate a novel mechanism of cytokine receptor assembly likely to apply also to interleukin-3 (IL-3) and IL-5 and have implications for our molecular understanding and potential manipulation of GM-CSF activation of its receptor. (Blood. 2003;101: 1308-1315)