A dominant negative granulocyte-macrophage colony-stimulating factor receptor α chain reveals the multimeric structure of the receptor complex

Abstract

The receptor for the hemopoietic growth factor granulocyte-macrophage colony-stimulating factor (GM-CSF) is composed of two chains, both of which belong to the superfamily of cytokine receptors. The α chain confers low affinity binding only, whereas the β chain (β(c)) confers high affinity binding when associated with α. Ectopic expression of both chains of the receptor in murine NIH-3T3 fibroblasts results in signal transduction, mitogenesis, and morphologic transformation. The cytoplasmic domain of the GM-CSF receptor α subunit (GMR-α) comprises 54 amino acids that have been shown to be important for signal transduction through the β chain. The present study was designed to address the possibility of receptor oligomerization and its functional implication. Cross-linking studies with 125I-GM-CSF on NIH-3T3 transfectants is consistent with the presence of α and β(c) dimers and of receptor oligomers. We have, therefore, generated an inert α chain through polymerase chain reaction-mediated truncation of 47 amino acids of the COOH-terminal domain of α (α(t)), and coexpressed α(t), α, and β(c) in NIH-3T3. In cells in which α(t) and a are present in stoichiometric proportion within the GM-CSF-binding complex, we provide evidence that α(t) is dominant negative over wild type α an the basis of two different functional assays: cell proliferation and foci formation. Hence, our results suggest the requirement for at least two functional α chains for signal transduction. Together with the cross-linking studies, our data indicate that the functional GMR is an oligomer that contains at least two α chains.