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A cytokine-cytokine interaction in the assembly of higher-order structure and activation of the interleukine-3:receptor complex.

Dey R, Ji K, Liu Z, Chen L - PLoS ONE (2009)

Bottom Line: These observations are consistent with structure-function studies of the GM-CSF:receptor complex showing that formation of the higher-order cytokine:receptor complex is required for signaling.However, a key question not answered from previous studies is how cytokine binding facilitates the assembly of the higher-order complex.Our studies here reveal a potential cytokine-cytokine interaction that participates in the assembly of the dodecamer complex, thus linking cytokine binding to receptor activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Molecular and Computational Biology, University of Southern California, Los Angeles, California, United States of America.

ABSTRACT
Interleukine-3 (IL-3) binds its receptor and initiates a cascade of signaling processes that regulate the proliferation and differentiation of hematopoietic cells. To understand the detailed mechanisms of IL-3 induced receptor activation, we generated a homology model of the IL-3:receptor complex based on the closely related crystal structure of the GM-CSF:receptor complex. Model-predicted interactions between IL-3 and its receptor are in excellent agreement with mutagenesis data, which validate the model and establish a detailed view of IL-3:receptor interaction. The homology structure reveals an IL-3:IL-3 interaction interface in a higher-order complex modeled after the dodecamer of the GM-CSF:receptor complex wherein an analogous GM-CSF:GM-CSF interface is also identified. This interface is mediated by a proline-rich hydrophobic motif (PPLPLL) of the AA' loop that is highly exposed in the structure of isolated IL-3. Various experimental data suggest that this motif is required for IL-3 function through receptor-binding independent mechanisms. These observations are consistent with structure-function studies of the GM-CSF:receptor complex showing that formation of the higher-order cytokine:receptor complex is required for signaling. However, a key question not answered from previous studies is how cytokine binding facilitates the assembly of the higher-order complex. Our studies here reveal a potential cytokine-cytokine interaction that participates in the assembly of the dodecamer complex, thus linking cytokine binding to receptor activation.

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Related in: MedlinePlus

Activation mechanism of the IL-3:receptor complex.(A) In the absence of cytokine, the receptor subunits are scattered on the cell surface, where the beta dimer, which binds JAK2 through its cytoplasmic region, form transient tetramer through interface IV that may contribute to residual phosphoryaltion of JAK2. (B) In the presence of IL-3, the cytokine and receptor subunits assembly into a dodecamer that is stabilized by IL-3:IL-3 interaction, leading to robust signal activation through JAK2 trans-phosphorylation. (C) A variety of experimental data showed that modifications of the AA′ loop of IL-3, including deletion, loss-of-function mutations, and antibody binding, diminished the activity IL-3, whereas gain-of-function mutations are also frequently observed in this region that enhanced the IL-3 without affecting the receptor binding affinity.
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pone-0005188-g007: Activation mechanism of the IL-3:receptor complex.(A) In the absence of cytokine, the receptor subunits are scattered on the cell surface, where the beta dimer, which binds JAK2 through its cytoplasmic region, form transient tetramer through interface IV that may contribute to residual phosphoryaltion of JAK2. (B) In the presence of IL-3, the cytokine and receptor subunits assembly into a dodecamer that is stabilized by IL-3:IL-3 interaction, leading to robust signal activation through JAK2 trans-phosphorylation. (C) A variety of experimental data showed that modifications of the AA′ loop of IL-3, including deletion, loss-of-function mutations, and antibody binding, diminished the activity IL-3, whereas gain-of-function mutations are also frequently observed in this region that enhanced the IL-3 without affecting the receptor binding affinity.

Mentions: Though far away from the receptor binding sites, the PPLPLL motif (Pro30-Leu35) has been shown to be a functionally important site on IL-3 by a number of experiments. In mapping functional sites of IL-3 using monoclonal antibodies that bind IL-3 and inhibit its activity, Lokker et al. showed that the epitopes of two neutralizing antibodies were located between Leu32 and Asp36 [26], [35]. Deletion of a significant portion of this region, such as Pro30-Leu33 (del-Pro30-Leu33) and Pro30-Leu34 (del-Pro30-Leu34), diminished the activity of IL-3. Site-specific mutations of residues in this region affected the activity of IL-3 both positively and negatively. While the Leu34Glu mutation showed little effect on the function of IL-3, the Leu34Gly mutant showed much reduced activity than the wild type IL-3. On the other hand, substitution of Pro33 with Asn (Pro33Asn) and Gly (Pro33Gly) enhanced the specific activity of IL-3. Gain-of-function mutations of IL-3 have also been identified in the PPLPLL motif in a saturation mutagenesis study [31], where Leu32Arg, Leu34Ser and Leu34Met showed increased activity. Many of the loss-of-function mutations such as del-Pro30-Leu33, del-Pro30-Leu34, and Leu34Gly also showed decreased affinity for the receptor [35]. How these modifications affect the binding of IL-3 to its receptor is not clear since the AA′ loop does not make direct contact to either subunits of the receptor in the modeled structure (Figure 3A and 3C). While these negative results seem to contradict the model of interface V mediated signal activation through dodecamer assembly, it must be kept in mind that loss-of-function of mutations often involve complex but trivial mechanisms, such as protein mis-folding or aggregation, especially considering the hydrophobic nature of the AA′ loop (see discussion below). By contrast, the gain-of-function mutations can often be much more mechanistically revealing. Here these mutations enhanced the specific activity of IL-3 substantially with little to modest effect on the binding affinity for the receptor. Most notably, the Pro33Gly mutant showed 14-fold increase in activity but virtually the same receptor affinity as compared with the wild type protein in cell-based assays, indicating that modifications in the PPLPLL motif of the AA′ loop can affect the function of IL-3 through mechanisms independent of receptor binding. Animal studies of an IL-3 triple mutant (Pro33Gly/Trp104Glu/Asn105Asp) also showed that modifications of the PPLPLL motif and its nearby regions could enhance the specific activity of IL-3 significantly under physiological conditions (Kunmei Ji and Zhigang Liu, data not shown). Mutations on interface IV have also been shown to disrupt the function of the receptor without affecting the binding of the cytokine [34]. These observations suggest that interface V identified here contribute to the activation of the receptor through a similar mechanism to interface IV, namely the assembly of the dodecameric cytokine:receptor complex (Figure 7).


A cytokine-cytokine interaction in the assembly of higher-order structure and activation of the interleukine-3:receptor complex.

Dey R, Ji K, Liu Z, Chen L - PLoS ONE (2009)

Activation mechanism of the IL-3:receptor complex.(A) In the absence of cytokine, the receptor subunits are scattered on the cell surface, where the beta dimer, which binds JAK2 through its cytoplasmic region, form transient tetramer through interface IV that may contribute to residual phosphoryaltion of JAK2. (B) In the presence of IL-3, the cytokine and receptor subunits assembly into a dodecamer that is stabilized by IL-3:IL-3 interaction, leading to robust signal activation through JAK2 trans-phosphorylation. (C) A variety of experimental data showed that modifications of the AA′ loop of IL-3, including deletion, loss-of-function mutations, and antibody binding, diminished the activity IL-3, whereas gain-of-function mutations are also frequently observed in this region that enhanced the IL-3 without affecting the receptor binding affinity.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2662821&req=5

pone-0005188-g007: Activation mechanism of the IL-3:receptor complex.(A) In the absence of cytokine, the receptor subunits are scattered on the cell surface, where the beta dimer, which binds JAK2 through its cytoplasmic region, form transient tetramer through interface IV that may contribute to residual phosphoryaltion of JAK2. (B) In the presence of IL-3, the cytokine and receptor subunits assembly into a dodecamer that is stabilized by IL-3:IL-3 interaction, leading to robust signal activation through JAK2 trans-phosphorylation. (C) A variety of experimental data showed that modifications of the AA′ loop of IL-3, including deletion, loss-of-function mutations, and antibody binding, diminished the activity IL-3, whereas gain-of-function mutations are also frequently observed in this region that enhanced the IL-3 without affecting the receptor binding affinity.
Mentions: Though far away from the receptor binding sites, the PPLPLL motif (Pro30-Leu35) has been shown to be a functionally important site on IL-3 by a number of experiments. In mapping functional sites of IL-3 using monoclonal antibodies that bind IL-3 and inhibit its activity, Lokker et al. showed that the epitopes of two neutralizing antibodies were located between Leu32 and Asp36 [26], [35]. Deletion of a significant portion of this region, such as Pro30-Leu33 (del-Pro30-Leu33) and Pro30-Leu34 (del-Pro30-Leu34), diminished the activity of IL-3. Site-specific mutations of residues in this region affected the activity of IL-3 both positively and negatively. While the Leu34Glu mutation showed little effect on the function of IL-3, the Leu34Gly mutant showed much reduced activity than the wild type IL-3. On the other hand, substitution of Pro33 with Asn (Pro33Asn) and Gly (Pro33Gly) enhanced the specific activity of IL-3. Gain-of-function mutations of IL-3 have also been identified in the PPLPLL motif in a saturation mutagenesis study [31], where Leu32Arg, Leu34Ser and Leu34Met showed increased activity. Many of the loss-of-function mutations such as del-Pro30-Leu33, del-Pro30-Leu34, and Leu34Gly also showed decreased affinity for the receptor [35]. How these modifications affect the binding of IL-3 to its receptor is not clear since the AA′ loop does not make direct contact to either subunits of the receptor in the modeled structure (Figure 3A and 3C). While these negative results seem to contradict the model of interface V mediated signal activation through dodecamer assembly, it must be kept in mind that loss-of-function of mutations often involve complex but trivial mechanisms, such as protein mis-folding or aggregation, especially considering the hydrophobic nature of the AA′ loop (see discussion below). By contrast, the gain-of-function mutations can often be much more mechanistically revealing. Here these mutations enhanced the specific activity of IL-3 substantially with little to modest effect on the binding affinity for the receptor. Most notably, the Pro33Gly mutant showed 14-fold increase in activity but virtually the same receptor affinity as compared with the wild type protein in cell-based assays, indicating that modifications in the PPLPLL motif of the AA′ loop can affect the function of IL-3 through mechanisms independent of receptor binding. Animal studies of an IL-3 triple mutant (Pro33Gly/Trp104Glu/Asn105Asp) also showed that modifications of the PPLPLL motif and its nearby regions could enhance the specific activity of IL-3 significantly under physiological conditions (Kunmei Ji and Zhigang Liu, data not shown). Mutations on interface IV have also been shown to disrupt the function of the receptor without affecting the binding of the cytokine [34]. These observations suggest that interface V identified here contribute to the activation of the receptor through a similar mechanism to interface IV, namely the assembly of the dodecameric cytokine:receptor complex (Figure 7).

Bottom Line: These observations are consistent with structure-function studies of the GM-CSF:receptor complex showing that formation of the higher-order cytokine:receptor complex is required for signaling.However, a key question not answered from previous studies is how cytokine binding facilitates the assembly of the higher-order complex.Our studies here reveal a potential cytokine-cytokine interaction that participates in the assembly of the dodecamer complex, thus linking cytokine binding to receptor activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Molecular and Computational Biology, University of Southern California, Los Angeles, California, United States of America.

ABSTRACT
Interleukine-3 (IL-3) binds its receptor and initiates a cascade of signaling processes that regulate the proliferation and differentiation of hematopoietic cells. To understand the detailed mechanisms of IL-3 induced receptor activation, we generated a homology model of the IL-3:receptor complex based on the closely related crystal structure of the GM-CSF:receptor complex. Model-predicted interactions between IL-3 and its receptor are in excellent agreement with mutagenesis data, which validate the model and establish a detailed view of IL-3:receptor interaction. The homology structure reveals an IL-3:IL-3 interaction interface in a higher-order complex modeled after the dodecamer of the GM-CSF:receptor complex wherein an analogous GM-CSF:GM-CSF interface is also identified. This interface is mediated by a proline-rich hydrophobic motif (PPLPLL) of the AA' loop that is highly exposed in the structure of isolated IL-3. Various experimental data suggest that this motif is required for IL-3 function through receptor-binding independent mechanisms. These observations are consistent with structure-function studies of the GM-CSF:receptor complex showing that formation of the higher-order cytokine:receptor complex is required for signaling. However, a key question not answered from previous studies is how cytokine binding facilitates the assembly of the higher-order complex. Our studies here reveal a potential cytokine-cytokine interaction that participates in the assembly of the dodecamer complex, thus linking cytokine binding to receptor activation.

Show MeSH
Related in: MedlinePlus