A dominant negative granulocyte-macrophage colony-stimulating factor receptor alpha chain reveals the multimeric structure of the receptor complex.
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 alpha chain confers low affinity binding only, whereas the beta chain (betac) confers high affinity binding when associated with alpha. 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 alpha subunit (GMR-alpha) comprises 54 amino acids that have been shown to be important for signal transduction through the beta 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 alpha and betac dimers and of receptor oligomers. We have, therefore, generated an inert alpha chain through polymerase chain reaction-mediated truncation of 47 amino acids of the COOH-terminal domain of alpha (alphat), and coexpressed alphat, alpha, and betac in NIH-3T3. In cells in which alphat and alpha are present in stoichiometric proportion within the GM-CSF-binding complex, we provide evidence that alphat is dominant negative over wild type alpha on the basis of two different functional assays: cell proliferation and foci formation. Hence, our results suggest the requirement for at least two functional alpha 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 alpha chains.
Clinical Research Institute of Montreal, Montreal, Québec, Canada H2W 1R7. hoangt@ircm.umontreal.ca
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D016475:3T3 Cells; D000818:Animals; D002455:Cell Division; D003593:Cytoplasm / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor / Q000737:chemistry*; D014162:Transfection
D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor