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 ␣ chain confers low affinity binding only, whereas the ␤ chain (␤ c ) confers high affinity binding when associated with ␣. 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 ␣ subunit (GMR-␣) comprises 54 amino acids that have been shown to be important for signal transduction through the ␤ chain. The present study was designed to address the possibility of receptor oligomerization and its functional implication. Cross-linking studies with 125 I-GM-CSF on NIH-3T3 transfectants is consistent with the presence of ␣ and ␤ c dimers and of receptor oligomers. We have, therefore, generated an inert ␣ chain through polymerase chain reaction-mediated truncation of 47 amino acids of the COOH-terminal domain of ␣ (␣ t ), and coexpressed ␣ t , ␣, and ␤ c in NIH-3T3. In cells in which ␣ t and ␣ are present in stoichiometric proportion within the GM-CSF-binding complex, we provide evidence that ␣ t is dominant negative over wild type ␣ on the basis of two different functional assays: cell proliferation and foci formation. Hence, our results suggest the requirement for at least two functional ␣ 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 ␣ chains.

Dissection of the cytoplasmic domains of cytokine receptors involved in STAT and Ras dependent proliferation

“…It is well accepted though that the integrity of the a chain is required for optimal function. Deletion studies of the cytoplasmic region of GM-CSF Ra indicate that this domain is absolutely required in GM-CSF functionality (Matsuguchi et al, 1997;Weiss et al, 1993), and thus impairs the proliferative response to GM-CSF (Lia et al, 1996). Also, a mutant of IL-5 Ra lacking its cytoplasmic domain does not respond to IL-5 stimulation (Takaki et al, 1993).…”

Regulation of Proliferation, Differentiation and Survival by the IL-3/IL-5/GM-CSF Receptor Family

“…Recent evidence suggests that a bona-fide receptor is composed of two ␤c molecules and one ␣ chain [19]. However, others have shown that at least two functional ␣ chains are necessary for a signalling-competent receptor [20]. In addition, the GM-CSF receptor (␣ ϩ ␤c) was recently demonstrated to exist in a pre-formed complex in unstimulated cells [21].…”

Identification of primary structural features that define the differential actions of IL-3 and GM-CSF receptors

“…Addition of hGM-CSF will promote formation of the hGM/␤ c complexes that will signal via the cytoplasmic domain of ␤ c because it has been demonstrated that GM-CSF can promote homodimerization of hGM R␣ subunits, without a requirement for the presence of a ␤ c (h␤ c or endogenous murine ␤ c subunits). 40 There has been some controversy over the ability of chimeric hGM/␤ c constructs to signal. The construct of others 17,25 is unable to function unless ␤ c is coexpressed.…”