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Crucial role of the residue R280 at the F'-G' loop of the human granulocyte/macrophage colony-stimulating factor receptor alpha chain for ligand recognition.

Rajotte D, Cadieux C, Haman A, Wilkes BC, Clark SC, Hercus T, Woodcock JA, Lopez A, Hoang T - J. Exp. Med. (1997)

Bottom Line: The GM-CSF receptor (GMR)-alpha chain confers low affinity binding only (5-10 nM), whereas the other chain, betac, does not bind GM-CSF by itself but confers high affinity binding when associated with GMR-alpha (25-100 pM).Second, a mutant GM-CSF with charge reversal mutation at position Asp112 exhibited a 1,000-fold decrease in affinity in receptor binding, whereas charge ablation or conservative mutations were the least affected (10-20-fold).Sequence alignment and modeling of interleukin (IL)-3R and IL-5R identified an arginine residue at the tip of a beta turn in a highly divergent context at the F'-G' loop, close to a conserved structural element, the WSXWS motif, suggesting the possibility of a ligand association mechanism similar to the one described herein for GMR.

View Article: PubMed Central - PubMed

Affiliation: Clinical Research Institute of Montréal, Laboratory of Hemopoiesis and Leukemia, Montréal, Quebec, Canada.

ABSTRACT
The receptor for granulocyte/macrophage colony-stimulating factor (GM-CSF) is composed of two chains, alpha and betac. Both chains belong to the superfamily of cytokine receptors characterized by a common structural feature, i.e., the presence of at least two fibronectin-like folds in the extracellular domain, which was first identified in the growth hormone receptor. The GM-CSF receptor (GMR)-alpha chain confers low affinity binding only (5-10 nM), whereas the other chain, betac, does not bind GM-CSF by itself but confers high affinity binding when associated with GMR-alpha (25-100 pM). The present study was designed to define the assembly of the GMR complex at the molecular level through site-directed mutagenesis guided by homology modeling with the growth hormone receptor complex. In our three-dimensional model, R280 of GMR-alpha, located in the F'-G' loop and close to the WSSWS motif, is in the vicinity of the ligand Asp112, suggesting the possibility of electrostatic interaction between these two residues. Through site directed mutagenesis, we provide several lines of evidence indicating the importance of electrostatic interaction in ligand-receptor recognition. First, mutagenesis of GMR-alphaR280 strikingly ablated ligand binding in the absence of beta common (betac); ligand binding was restored in the presence of betac with, nonetheless, a significant shift from high (26 pM) toward low affinity (from 2 to 13 nM). The rank order of the dissociation constant for the different GMR-alphaR280 mutations where Lys > Gln > Met > Asp, suggesting the importance of the charge at this position. Second, a mutant GM-CSF with charge reversal mutation at position Asp112 exhibited a 1,000-fold decrease in affinity in receptor binding, whereas charge ablation or conservative mutations were the least affected (10-20-fold). Third, removal of the charge at position R280 of GMR-alpha introduced a 10-fold decrease in the association rate constant and only a 2-fold change in the dissociation rate constant, suggesting that R280 is implicated in ligand recognition, possibly through interaction with Asp112 of GM-CSF. For all R280 mutants, the half-efficient concentrations of GM-CSF required for membrane (receptor binding) to nuclear events (c-fos promoter activation) and cell proliferation (thymidine incorporation) were in the same range, indicating that the threshold for biologic activity is governed mainly by the affinity of ligand-receptor interaction. Furthermore, mutation of other residues in the immediate vicinity of R280 was less drastic. Sequence alignment and modeling of interleukin (IL)-3R and IL-5R identified an arginine residue at the tip of a beta turn in a highly divergent context at the F'-G' loop, close to a conserved structural element, the WSXWS motif, suggesting the possibility of a ligand association mechanism similar to the one described herein for GMR.

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The F′–G′ loop of the second fibronectin domain of the GM-CSF, IL-3, and -5 α chain receptors: sequence alignment and three-dimensional models and homology modeling of the erythropoietin receptor complex with its ligand. The alignment is adapted from Bazan (14) and Goodall  et al. (19). Residues in bold are shown through site-directed mutagenesis to be important for proper ligand binding (humgmcsfr, GMR-α, shown herein;  humil6r, IL-6R-α (17); fn10, fibronectin type III tenth repeat (16); humepor, EpoR (41, 42). Conserved residues within the F′ and G′ strands are boxed,  as well as the conserved WSXWS sequence. Comparison of the F′–G′ loop (yellow) facing helix D (red) of the ligand in the GMR-α, IL-3R-α, and IL-5R-α  models, highlights a possible electrostatic interaction between a positively charged residue, Arg (green), and a negatively charged residue, Asp or Glu (blue).  In GMR-α, R280 is at the tip of the β turn pointing towards the ligand, whereas the aspartic acid (D278, green) is in the vicinity of a lysine (K191, magenta) located in the linker region between the two fibronectin domains. Note that in the IL-3 model, an aromatic residue on the F′–G′ loop, Tyr261, is  close to another aromatic residue, Phe113 from helix D. In the lower right panel, the extracellular domains of the two Epo receptor chains are shown  complexed with the modelized Epo hormone. The Epo–EpoR2 complex is based on the structure of the GH–GHR2 complex, and was generated as described in the Materials and Methods. The π-charge interaction in EpoR consisting of one arginine (white) and two tryptophan (green) from the WSXWS  motif is illustrated. The two disulfide bridges formed between the four conserved cysteine are shown in blue.
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Figure 6: The F′–G′ loop of the second fibronectin domain of the GM-CSF, IL-3, and -5 α chain receptors: sequence alignment and three-dimensional models and homology modeling of the erythropoietin receptor complex with its ligand. The alignment is adapted from Bazan (14) and Goodall et al. (19). Residues in bold are shown through site-directed mutagenesis to be important for proper ligand binding (humgmcsfr, GMR-α, shown herein; humil6r, IL-6R-α (17); fn10, fibronectin type III tenth repeat (16); humepor, EpoR (41, 42). Conserved residues within the F′ and G′ strands are boxed, as well as the conserved WSXWS sequence. Comparison of the F′–G′ loop (yellow) facing helix D (red) of the ligand in the GMR-α, IL-3R-α, and IL-5R-α models, highlights a possible electrostatic interaction between a positively charged residue, Arg (green), and a negatively charged residue, Asp or Glu (blue). In GMR-α, R280 is at the tip of the β turn pointing towards the ligand, whereas the aspartic acid (D278, green) is in the vicinity of a lysine (K191, magenta) located in the linker region between the two fibronectin domains. Note that in the IL-3 model, an aromatic residue on the F′–G′ loop, Tyr261, is close to another aromatic residue, Phe113 from helix D. In the lower right panel, the extracellular domains of the two Epo receptor chains are shown complexed with the modelized Epo hormone. The Epo–EpoR2 complex is based on the structure of the GH–GHR2 complex, and was generated as described in the Materials and Methods. The π-charge interaction in EpoR consisting of one arginine (white) and two tryptophan (green) from the WSXWS motif is illustrated. The two disulfide bridges formed between the four conserved cysteine are shown in blue.

Mentions: Primary sequence alignment of the three closest members of the cytokine receptors superfamily, IL-3R-α, -5R-α, and GMR-α highlights the presence of one Arg at the F′–G′ loop in an otherwise diverging context (Fig. 6). Interestingly, the IL-3 and -5 receptor complex models reveal that a negatively charged residue (Glu or Asp) on the ligand is in close contact with the Arg of the F′–G′ loop, indicating a potential electrostatic interaction. Thus, the contribution of the Arg residue to ligand recognition may also be extended to these receptors. Sequence divergence in this loop among the GM-CSF, IL-3, and -5 α chain receptors suggest that this region is likely to be important for the observed specificity of the corresponding receptor subunit.


Crucial role of the residue R280 at the F'-G' loop of the human granulocyte/macrophage colony-stimulating factor receptor alpha chain for ligand recognition.

Rajotte D, Cadieux C, Haman A, Wilkes BC, Clark SC, Hercus T, Woodcock JA, Lopez A, Hoang T - J. Exp. Med. (1997)

The F′–G′ loop of the second fibronectin domain of the GM-CSF, IL-3, and -5 α chain receptors: sequence alignment and three-dimensional models and homology modeling of the erythropoietin receptor complex with its ligand. The alignment is adapted from Bazan (14) and Goodall  et al. (19). Residues in bold are shown through site-directed mutagenesis to be important for proper ligand binding (humgmcsfr, GMR-α, shown herein;  humil6r, IL-6R-α (17); fn10, fibronectin type III tenth repeat (16); humepor, EpoR (41, 42). Conserved residues within the F′ and G′ strands are boxed,  as well as the conserved WSXWS sequence. Comparison of the F′–G′ loop (yellow) facing helix D (red) of the ligand in the GMR-α, IL-3R-α, and IL-5R-α  models, highlights a possible electrostatic interaction between a positively charged residue, Arg (green), and a negatively charged residue, Asp or Glu (blue).  In GMR-α, R280 is at the tip of the β turn pointing towards the ligand, whereas the aspartic acid (D278, green) is in the vicinity of a lysine (K191, magenta) located in the linker region between the two fibronectin domains. Note that in the IL-3 model, an aromatic residue on the F′–G′ loop, Tyr261, is  close to another aromatic residue, Phe113 from helix D. In the lower right panel, the extracellular domains of the two Epo receptor chains are shown  complexed with the modelized Epo hormone. The Epo–EpoR2 complex is based on the structure of the GH–GHR2 complex, and was generated as described in the Materials and Methods. The π-charge interaction in EpoR consisting of one arginine (white) and two tryptophan (green) from the WSXWS  motif is illustrated. The two disulfide bridges formed between the four conserved cysteine are shown in blue.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196330&req=5

Figure 6: The F′–G′ loop of the second fibronectin domain of the GM-CSF, IL-3, and -5 α chain receptors: sequence alignment and three-dimensional models and homology modeling of the erythropoietin receptor complex with its ligand. The alignment is adapted from Bazan (14) and Goodall et al. (19). Residues in bold are shown through site-directed mutagenesis to be important for proper ligand binding (humgmcsfr, GMR-α, shown herein; humil6r, IL-6R-α (17); fn10, fibronectin type III tenth repeat (16); humepor, EpoR (41, 42). Conserved residues within the F′ and G′ strands are boxed, as well as the conserved WSXWS sequence. Comparison of the F′–G′ loop (yellow) facing helix D (red) of the ligand in the GMR-α, IL-3R-α, and IL-5R-α models, highlights a possible electrostatic interaction between a positively charged residue, Arg (green), and a negatively charged residue, Asp or Glu (blue). In GMR-α, R280 is at the tip of the β turn pointing towards the ligand, whereas the aspartic acid (D278, green) is in the vicinity of a lysine (K191, magenta) located in the linker region between the two fibronectin domains. Note that in the IL-3 model, an aromatic residue on the F′–G′ loop, Tyr261, is close to another aromatic residue, Phe113 from helix D. In the lower right panel, the extracellular domains of the two Epo receptor chains are shown complexed with the modelized Epo hormone. The Epo–EpoR2 complex is based on the structure of the GH–GHR2 complex, and was generated as described in the Materials and Methods. The π-charge interaction in EpoR consisting of one arginine (white) and two tryptophan (green) from the WSXWS motif is illustrated. The two disulfide bridges formed between the four conserved cysteine are shown in blue.
Mentions: Primary sequence alignment of the three closest members of the cytokine receptors superfamily, IL-3R-α, -5R-α, and GMR-α highlights the presence of one Arg at the F′–G′ loop in an otherwise diverging context (Fig. 6). Interestingly, the IL-3 and -5 receptor complex models reveal that a negatively charged residue (Glu or Asp) on the ligand is in close contact with the Arg of the F′–G′ loop, indicating a potential electrostatic interaction. Thus, the contribution of the Arg residue to ligand recognition may also be extended to these receptors. Sequence divergence in this loop among the GM-CSF, IL-3, and -5 α chain receptors suggest that this region is likely to be important for the observed specificity of the corresponding receptor subunit.

Bottom Line: The GM-CSF receptor (GMR)-alpha chain confers low affinity binding only (5-10 nM), whereas the other chain, betac, does not bind GM-CSF by itself but confers high affinity binding when associated with GMR-alpha (25-100 pM).Second, a mutant GM-CSF with charge reversal mutation at position Asp112 exhibited a 1,000-fold decrease in affinity in receptor binding, whereas charge ablation or conservative mutations were the least affected (10-20-fold).Sequence alignment and modeling of interleukin (IL)-3R and IL-5R identified an arginine residue at the tip of a beta turn in a highly divergent context at the F'-G' loop, close to a conserved structural element, the WSXWS motif, suggesting the possibility of a ligand association mechanism similar to the one described herein for GMR.

View Article: PubMed Central - PubMed

Affiliation: Clinical Research Institute of Montréal, Laboratory of Hemopoiesis and Leukemia, Montréal, Quebec, Canada.

ABSTRACT
The receptor for granulocyte/macrophage colony-stimulating factor (GM-CSF) is composed of two chains, alpha and betac. Both chains belong to the superfamily of cytokine receptors characterized by a common structural feature, i.e., the presence of at least two fibronectin-like folds in the extracellular domain, which was first identified in the growth hormone receptor. The GM-CSF receptor (GMR)-alpha chain confers low affinity binding only (5-10 nM), whereas the other chain, betac, does not bind GM-CSF by itself but confers high affinity binding when associated with GMR-alpha (25-100 pM). The present study was designed to define the assembly of the GMR complex at the molecular level through site-directed mutagenesis guided by homology modeling with the growth hormone receptor complex. In our three-dimensional model, R280 of GMR-alpha, located in the F'-G' loop and close to the WSSWS motif, is in the vicinity of the ligand Asp112, suggesting the possibility of electrostatic interaction between these two residues. Through site directed mutagenesis, we provide several lines of evidence indicating the importance of electrostatic interaction in ligand-receptor recognition. First, mutagenesis of GMR-alphaR280 strikingly ablated ligand binding in the absence of beta common (betac); ligand binding was restored in the presence of betac with, nonetheless, a significant shift from high (26 pM) toward low affinity (from 2 to 13 nM). The rank order of the dissociation constant for the different GMR-alphaR280 mutations where Lys > Gln > Met > Asp, suggesting the importance of the charge at this position. Second, a mutant GM-CSF with charge reversal mutation at position Asp112 exhibited a 1,000-fold decrease in affinity in receptor binding, whereas charge ablation or conservative mutations were the least affected (10-20-fold). Third, removal of the charge at position R280 of GMR-alpha introduced a 10-fold decrease in the association rate constant and only a 2-fold change in the dissociation rate constant, suggesting that R280 is implicated in ligand recognition, possibly through interaction with Asp112 of GM-CSF. For all R280 mutants, the half-efficient concentrations of GM-CSF required for membrane (receptor binding) to nuclear events (c-fos promoter activation) and cell proliferation (thymidine incorporation) were in the same range, indicating that the threshold for biologic activity is governed mainly by the affinity of ligand-receptor interaction. Furthermore, mutation of other residues in the immediate vicinity of R280 was less drastic. Sequence alignment and modeling of interleukin (IL)-3R and IL-5R identified an arginine residue at the tip of a beta turn in a highly divergent context at the F'-G' loop, close to a conserved structural element, the WSXWS motif, suggesting the possibility of a ligand association mechanism similar to the one described herein for GMR.

Show MeSH
Related in: MedlinePlus