Hematopoietic activity of granulocyte/macrophage colony-stimulating factor is dependent upon two distinct regions of the molecule: functional analysis based upon the activities of interspecies hybrid growth factors.

Abstract

Granulocyte/macrophage colony-stimulating factor (GM-CSF) is an acidic glycoprotein that stimulates hematopoiesis in vitro and in vivo. Despite a high degree of sequence homology, the GM-CSFs from human and murine sources fail to crossreact in their respective colony-forming assays. On the basis of this rinding, a series of hybrid molecules containing various proportions of human-and murine-specific amino acid sequences were generated by recombinant DNA techniques and assayed for species-specific activity against human and murine marrow target cells. Two regions of GM-CSF, residues 38-48 and residues 95-111, were found to be critical for hematopoietic function. These regions are structurally characterized by an amphiphilic helix and by a disulfide-bonded loop, respectively, and are homologous in position in the human and murine growth factors. In addition, competition assays suggested that, together, these regions bind to the GM-CSF receptor.

High-affinity autoantibodies specifically eliminate granulocyte-macrophage colony-stimulating factor activity in the lungs of patients with idiopathic pulmonary alveolar proteinosis

“…This epitope has been reported to be an important functional domain of GM-CSF. 27,28,36,37 Thus, though autoantibodies can recognize various regions of the GM-CSF molecule, the consistent, strong targeting of an epitope known to be important for GM-CSF function strongly supports the notion that autoantibodies specifically neutralize GM-CSF in patients with iPAP.…”

“…First, this region of GM-CSF is known to be functionally important. 27,28,36,37 Second, identifying an epitope that is consistently targeted by autoantibodies in iPAP patients suggests that disease severity may relate to epitope targeting by autoantibodies in a given iPAP patient rather than simply autoantibody levels. Alternatively, residues 78 to 94 may simply represent a hot spot, the targeting of which is necessary for neutralizing GM-CSF and thus for physiological function of the autoantibodies.…”

Aspects of Receptor Binding and Signalling of Interleukin-4 Investigated by Site-directed Mutagenesis and NMR Spectroscopy

“…An example is the comparison of mutations on helix C, where the IL-4 mutants R88Q/D show very different binding properties, and the substitutions R85Q and W91R are rather neutral. The experiments with the chimeric proteins on GM-CSF show that residues in the region 78 to 94 (GM-CSF numbering) (Kaushansky et al, 1989) are responsible for the activity, but the active site cannot be localized further.…”

“…For other mutagenesis experiments on GM-CSF the effects are more difficult to explain in terms of receptor-hormone interactions, because they involve either tripeptide deletions (Shanafelt & Kastelein, 1989), the synthesis of chimeric mouse/human proteins (Kaushansky et al, 1989) or proline scanning (Altmann et al, 1991). In all cases, larger structural rearrangements are expected.…”

Receptor binding properties of four‐helix‐bundle growth factors deduced from electrostatic analysis

“…The spatial conservation of both the cytokines and their receptors suggests that the 3D structure of their complexes may also be conserved. Immunochemical experiments and experiments on recombinant analogs of murine (Zurawski & Zurawski, 1989, 1992Zurawski et al, 1990) and human IL-2 (Liang et al, 1986;Weigel et al, 1989;Landgraf et al, 1992), IL-4 (Kruse et al, 1991(Kruse et al, , 1992(Kruse et al, , 1993Ramanathan et al, 1993), and CM-CSF (Kaushansky et al, 1989;Lopez et al, 1992;Meropol et al, 1992) suggest that all of them are likely to interact with their receptors in an orientation similar to hGH (de Vos et al, 1992), i.e., with the surface involving the A, C, and D helices. Mapping of functionally important residues onto the crystal structures of IL-2 and IL-4 indicates that a region near the C-terminus is one of the potential receptor binding sites.…”