scholarly journals The Structural and Functional Basis of Cytokine Receptor Activation: Lessons From the Common β Subunit of the Granulocyte-Macrophage Colony-Stimulating Factor, Interleukin-3 (IL-3), and IL-5 Receptors

Blood ◽  
1997 ◽  
Vol 89 (5) ◽  
pp. 1471-1482 ◽  
Author(s):  
Christopher J. Bagley ◽  
Joanna M. Woodcock ◽  
Frank C. Stomski ◽  
Angel F. Lopez
Keyword(s):  
Receptor Activation ◽  
Cytokine Receptor ◽  
Β Subunit ◽  
Colony Stimulating Factor ◽  
Interleukin 3 ◽  
Functional Basis ◽  
Macrophage Colony Stimulating Factor ◽  
The Common ◽  
Macrophage Colony ◽  
Stimulating Factor

scholarly journals The Human Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF ) Receptor Exists as a Preformed Receptor Complex That Can Be Activated by GM-CSF, Interleukin-3, or Interleukin-5

Blood ◽  
1997 ◽  
Vol 90 (8) ◽  
pp. 3005-3017 ◽  
Author(s):  
Joanna M. Woodcock ◽  
Barbara J. McClure ◽  
Frank C. Stomski ◽  
Michael J. Elliott ◽  
Christopher J. Bagley ◽  
...  
Keyword(s):  
Myeloid Cell ◽  
Cytokine Receptor ◽  
Activation Mechanism ◽  
Receptor Complex ◽  
Colony Stimulating Factor ◽  
Interleukin 3 ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Abstract The granulocyte-macrophage colony-stimulating factor (GM-CSF ) receptor is expressed on normal and malignant hematopoietic cells as well as on cells from other organs in which it transduces a variety of functions. Despite the widespread expression and pleiotropic nature of the GM-CSF receptor, little is known about its assembly and activation mechanism. Using a combination of biochemical and functional approaches, we have found that the human GM-CSF receptor exists as an inducible complex, analogous to the interleukin-3 (IL-3) receptor, and also as a preformed complex, unlike the IL-3 receptor or indeed other members of the cytokine receptor superfamily. We found that monoclonal antibodies to the GM-CSF receptor α chain (GMRα) and to the common β chain of the GM-CSF, IL-3, and IL-5 receptors (βc ) immunoprecipitated both GMRα and βc from the surface of primary myeloid cells, myeloid cell lines, and transfected cells in the absence of GM-CSF. Further association of the two chains could be induced by the addition of GM-CSF. The preformed complex required only the extracellular regions of GMRα and βc , as shown by the ability of soluble βc to associate with membrane-anchored GMRα or soluble GMRα. Kinetic experiments on eosinophils and monocytes with radiolabeled GM-CSF, IL-3, and IL-5 showed association characteristics unique to GM-CSF. Significantly, receptor phosphorylation experiments showed that not only GM-CSF but also IL-3 and IL-5 stimulated the phosphorylation of GMRα-associated βc . These results indicate a pattern of assembly of the heterodimeric GM-CSF receptor that is unique among receptors of the cytokine receptor superfamily. These results also suggest that the preformed GM-CSF receptor complex mediates the instantaneous binding of GM-CSF and is a target of phosphorylation by IL-3 and IL-5, raising the possibility that some of the biologic activities of IL-3 and IL-5 are mediated through the GM-CSF receptor complex.

Identification of a 14-3-3 Binding Sequence in the Common β Chain of the Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), Interleukin-3 (IL-3), and IL-5 Receptors That Is Serine-Phosphorylated by GM-CSF

Blood ◽  
1999 ◽  
Vol 94 (6) ◽  
pp. 1933-1942 ◽  
Author(s):  
F.C. Stomski ◽  
M. Dottore ◽  
W. Winnall ◽  
M.A. Guthridge ◽  
J. Woodcock ◽  
...  
Keyword(s):  
Binding Site ◽  
Signaling Molecules ◽  
Colony Stimulating Factor ◽  
Interleukin 3 ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
The Common ◽  
Macrophage Colony ◽  
Β Chain ◽  
Stimulating Factor

Abstract The common β chain (βc) of the granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 receptors is the major signaling subunit of these receptors coupling ligand binding to multiple biological activities. It is thought that these multiple functions arise as a consequence of the recruitment of specific signaling molecules to tyrosine-phosphorylated residues in the cytoplasmic domain of βc. However, the contribution of serine phosphorylation in βc to the recruitment of signaling molecules is not known. We show here the identification of a phosphoserine motif in the cytoplasmic domain of βc that interacts with the adaptor protein 14-3-3ζ. Coimmunoprecipitation and pull-down experiments with a glutathione S-transferase (GST):14-3-3ζ fusion protein showed that 14-3-3 directly associates with βc but not the GM-CSF receptor  chain. C-terminal truncation mutants of βcfurther showed that a region between amino acids 544 and 626 in βc was required for its association with 14-3-3ζ. This region contains the sequence 582HSRSLP587, which closely resembles the RSXSXP (where S is phosphorylated) consensus 14-3-3 binding site identified in a number of signaling molecules, including Raf-1. Significantly, substitution of582HSRSLP587 for EFAAAA completely abolished interaction of βc with GST–14-3-3ζ. Furthermore, the interaction of βc with GST–14-3-3 was greatly reduced in the presence of a peptide containing the 14-3-3 binding site, but only when 585Ser was phosphorylated. Direct binding experiments showed that the peptide containing phosphorylated 585Ser bound 14-3-3ζ with an affinity of 150 nmol/L. To study the regulation of 585S phosphorylation in vivo, we raised antibodies that specifically recognized 585Ser-phosphorylated βc. Using these antibodies, we showed that GM-CSF stimulation strongly upregulated 585Ser phosphorylation in M1 myeloid leukemic cells. The proximity of the SHC-binding site (577Tyr) to the 14-3-3–binding site (582HSRSLP587) and their conservation between mouse, rat, and human βc but not in other cytokine receptors suggest that they form a distinct motif that may subserve specialized functions associated with the GM-CSF, IL-3, and IL-5 receptors.

Identification of a 14-3-3 Binding Sequence in the Common β Chain of the Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), Interleukin-3 (IL-3), and IL-5 Receptors That Is Serine-Phosphorylated by GM-CSF

Blood ◽  
1999 ◽  
Vol 94 (6) ◽  
pp. 1933-1942 ◽  
Author(s):  
F.C. Stomski ◽  
M. Dottore ◽  
W. Winnall ◽  
M.A. Guthridge ◽  
J. Woodcock ◽  
...  
Keyword(s):  
Binding Site ◽  
Signaling Molecules ◽  
Colony Stimulating Factor ◽  
Interleukin 3 ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
The Common ◽  
Macrophage Colony ◽  
Β Chain ◽  
Stimulating Factor

The common β chain (βc) of the granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 receptors is the major signaling subunit of these receptors coupling ligand binding to multiple biological activities. It is thought that these multiple functions arise as a consequence of the recruitment of specific signaling molecules to tyrosine-phosphorylated residues in the cytoplasmic domain of βc. However, the contribution of serine phosphorylation in βc to the recruitment of signaling molecules is not known. We show here the identification of a phosphoserine motif in the cytoplasmic domain of βc that interacts with the adaptor protein 14-3-3ζ. Coimmunoprecipitation and pull-down experiments with a glutathione S-transferase (GST):14-3-3ζ fusion protein showed that 14-3-3 directly associates with βc but not the GM-CSF receptor  chain. C-terminal truncation mutants of βcfurther showed that a region between amino acids 544 and 626 in βc was required for its association with 14-3-3ζ. This region contains the sequence 582HSRSLP587, which closely resembles the RSXSXP (where S is phosphorylated) consensus 14-3-3 binding site identified in a number of signaling molecules, including Raf-1. Significantly, substitution of582HSRSLP587 for EFAAAA completely abolished interaction of βc with GST–14-3-3ζ. Furthermore, the interaction of βc with GST–14-3-3 was greatly reduced in the presence of a peptide containing the 14-3-3 binding site, but only when 585Ser was phosphorylated. Direct binding experiments showed that the peptide containing phosphorylated 585Ser bound 14-3-3ζ with an affinity of 150 nmol/L. To study the regulation of 585S phosphorylation in vivo, we raised antibodies that specifically recognized 585Ser-phosphorylated βc. Using these antibodies, we showed that GM-CSF stimulation strongly upregulated 585Ser phosphorylation in M1 myeloid leukemic cells. The proximity of the SHC-binding site (577Tyr) to the 14-3-3–binding site (582HSRSLP587) and their conservation between mouse, rat, and human βc but not in other cytokine receptors suggest that they form a distinct motif that may subserve specialized functions associated with the GM-CSF, IL-3, and IL-5 receptors.

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