scholarly journals Three conserved motifs in the extracellular domain of the human granulocyte-macrophage colony-stimulating factor receptor subunit are essential for ligand binding and surface expression

Blood ◽  
1994 ◽  
Vol 84 (8) ◽  
pp. 2539-2553 ◽  
Author(s):  
PD Doshi ◽  
JF DiPersio
Keyword(s):  
Ligand Binding ◽  
Extracellular Domain ◽  
Surface Expression ◽  
Colony Stimulating Factor ◽  
Cos Cells ◽  
Gm Csf ◽  
Alpha Subunits ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

The receptor for the human granulocyte-macrophage colony-stimulating factor (GM-CSF) (GM-R) is a heterodimeric complex consisting of two subunits, GM-R alpha and GM-R beta. Structural analyses have shown a number of highly conserved amino acid motifs present in both GM-R alpha and GM-R beta. These motifs include QYFLY, CXW, XW, and WSXWS motifs in the extracellular domain; a conserved cysteine in the transmembrane domain; and the entire cytoplasmic domain, including the LXVLX box in the carboxy terminal region of the cytoplasmic domain. We have investigated the role of these motifs in GM-R alpha by examining the effects of specific motif mutations on ligand binding and surface expression. Transient expression of these mutant GM-R alpha subunits in COS cells shows that these extracellular motis are essential for ligand binding. Alterations of the cytoplasmic region of GM-R alpha do not alter GM-CSF binding or the reconstitution of high-affinity receptors when coexpressed with GM-R beta. Permeabilization and immunostaining of cells transfected with mutant GM-R alpha subunits yields data suggesting that each of the mutant subunits is present in the cytoplasm. Immunostaining of both intact and permeabilized COS cells transiently transfected with wild-type or mutant GM-R alpha s showed that extracellular domain mutants accumulated in the cytoplasm and were not efficiently transported to the cell surface.

Three conserved motifs in the extracellular domain of the human granulocyte-macrophage colony-stimulating factor receptor subunit are essential for ligand binding and surface expression

Blood ◽  
1994 ◽  
Vol 84 (8) ◽  
pp. 2539-2553 ◽  
Author(s):  
PD Doshi ◽  
JF DiPersio
Keyword(s):  
Ligand Binding ◽  
Extracellular Domain ◽  
Surface Expression ◽  
Colony Stimulating Factor ◽  
Cos Cells ◽  
Gm Csf ◽  
Alpha Subunits ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Abstract The receptor for the human granulocyte-macrophage colony-stimulating factor (GM-CSF) (GM-R) is a heterodimeric complex consisting of two subunits, GM-R alpha and GM-R beta. Structural analyses have shown a number of highly conserved amino acid motifs present in both GM-R alpha and GM-R beta. These motifs include QYFLY, CXW, XW, and WSXWS motifs in the extracellular domain; a conserved cysteine in the transmembrane domain; and the entire cytoplasmic domain, including the LXVLX box in the carboxy terminal region of the cytoplasmic domain. We have investigated the role of these motifs in GM-R alpha by examining the effects of specific motif mutations on ligand binding and surface expression. Transient expression of these mutant GM-R alpha subunits in COS cells shows that these extracellular motis are essential for ligand binding. Alterations of the cytoplasmic region of GM-R alpha do not alter GM-CSF binding or the reconstitution of high-affinity receptors when coexpressed with GM-R beta. Permeabilization and immunostaining of cells transfected with mutant GM-R alpha subunits yields data suggesting that each of the mutant subunits is present in the cytoplasm. Immunostaining of both intact and permeabilized COS cells transiently transfected with wild-type or mutant GM-R alpha s showed that extracellular domain mutants accumulated in the cytoplasm and were not efficiently transported to the cell surface.

scholarly journals Molecular cloning and in vivo evaluation of canine granulocyte- macrophage colony-stimulating factor

Blood ◽  
1991 ◽  
Vol 78 (4) ◽  
pp. 930-937 ◽  
Author(s):  
RA Nash ◽  
F Schuening ◽  
F Appelbaum ◽  
WP Hammond ◽  
T Boone ◽  
...  
Keyword(s):  
Large Animal Model ◽  
Large Animal ◽  
Colony Stimulating Factor ◽  
In Vivo Evaluation ◽  
Cos Cells ◽  
Gm Csf ◽  
Mammalian Expression ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Abstract Canine granulocyte-macrophage colony-stimulating factor (caGM-CSF) was cloned and expressed to allow further investigation of GM-CSF in a large animal model. The cDNA is 850 base pairs (bp) long and encodes a peptide of 144 amino acids. The nucleotide and amino acid sequence homology between caGM-CSF and human GM-CSF (hGM-CSF) is 80% and 70%, respectively. A mammalian expression vector pCMV/CAGM was constructed and used to transfect COS cells for expression of caGM-CSF. Supernatant from transfected COS cells enriched with caGM-CSF was shown to have significant stimulating activity in granulocyte-macrophage colony forming unit (CFU-GM) assays of canine marrow. caGM-CSF, expressed from bacteria, was used to treat seven dogs at varying doses twice daily subcutaneously (sc) for 14 to 16 days. Circulating blood neutrophils and monocytes increased significantly. The increase in circulating eosinophils was variable. Thrombocytopenia developed during administration of caGM-CSF but corrected rapidly after cessation of treatment. Evaluation of survival times of 51Cr-labeled autologous platelets suggested increased consumption as the primary reason for thrombocytopenia. A species-specific GM-CSF will be a useful tool for hematologic or immunologic studies in dogs.

scholarly journals Human granulocyte-macrophage colony-stimulating factor receptor signal transduction requires the proximal cytoplasmic domains of the alpha and beta subunits

Blood ◽  
1993 ◽  
Vol 82 (11) ◽  
pp. 3298-3306 ◽  
Author(s):  
M Weiss ◽  
C Yokoyama ◽  
Y Shikama ◽  
C Naugle ◽  
B Druker ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Beta Subunits ◽  
Colony Stimulating Factor ◽  
Wild Type ◽  
Gm Csf ◽  
High Affinity ◽  
Alpha Subunits ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Abstract Human granulocyte-macrophage colony-stimulating factor (GM-CSF) controls the production, maturation, and function of cells in multiple hematopoietic lineages. These effects are mediated by a cell-surface receptor (GM-R) composed of alpha and beta subunits, each containing 378 and 881 amino acids, respectively. Whereas the alpha subunit exists as several isoforms that bind GM-CSF with low affinity, the beta common subunit (beta c) does not bind GM-CSF itself, but acts as a high- affinity converter for GM-CSF, interleukin-3 (IL-3), and IL-5 receptor alpha subunits. The cytoplasmic region of GM-R alpha consists of a membrane-proximal conserved region shared by the alpha 1 and alpha 2 isoforms and a C-terminal variable region that is divergent between alpha 1 and alpha 2. The cytoplasmic region of beta c contains membrane proximal serine and acidic domains. To investigate the amino acid sequences that influence signal transduction by this receptor complex, we constructed a series of cytoplasmic truncation mutants of the alpha 2 and beta subunits. To study these truncations, we stably transfected the IL-3-dependent murine cell line Ba/F3 with wild-type or mutant cDNAs. We found that the wild-type and mutant alpha subunits conferred similar low-affinity binding sites for human GM-CSF to Ba/F3, and the wild-type or mutant beta subunit converted some of these sites to high- affinity; the cytoplasmic domain of beta was unnecessary for this high- affinity conversion. Proliferation assays showed that the membrane- proximal conserved region of GM-R alpha and the serine-acidic domain of beta c are required for both cell proliferation and ligand-dependent phosphorylation of a 93-kD cytoplasmic protein. We suggest that these regions may represent an important signal transduction motif present in several cytokine receptors.

Kinetic Resolution of Two Mechanisms for High-Affinity Granulocyte-Macrophage Colony-Stimulating Factor Binding to Its Receptor

Blood ◽  
1999 ◽  
Vol 94 (11) ◽  
pp. 3748-3753 ◽  
Author(s):  
Linghao Niu ◽  
David W. Golde ◽  
Juan Carlos Vera ◽  
Mark L. Heaney
Keyword(s):  
Ligand Binding ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Receptor Complexes ◽  
High Affinity ◽  
High Affinity Receptor ◽  
Macrophage Colony Stimulating Factor ◽  
Affinity Receptor ◽  
Macrophage Colony ◽  
Stimulating Factor

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is an important hematopoietic cytokine that exerts its effects by interaction with the GM-CSF receptor (GMR) on the surface of responsive cells. The GM-CSF receptor consists of two subunits: GMR, which binds GM-CSF with low affinity, and GMRβ, which lacks intrinsic ligand-binding capability but complexes with GMR to form a high-affinity receptor (GMR/β). We conducted dynamic kinetic analyses of GM-CSF receptors to define the role of GMRβ in the interaction of ligand and receptor. Our data show that GMR/β exhibits a higher kon than GMR, indicating that GMRβ facilitates ligand acquisition to the binding pocket. Heterogeneity with regard to GM-CSF dissociation from GMR/β points to the presence of loose and tight ligand-receptor complexes in high-affinity binding. Although the loose complex has a koff similar to GMR, the lower koffindicates that GMRβ inhibits GM-CSF release from the tight receptor complex. The two rates of ligand dissociation may provide for discrete mechanisms of interaction between GM-CSF and its high-affinity receptor. These results show that the β subunit functions to stabilize ligand binding as well as to facilitate ligand acquisition.

Identification of the ligand-binding regions in the macrophage colony-stimulating factor receptor extracellular domain

1993 ◽  
Vol 13 (9) ◽  
pp. 5348-5359
Author(s):  
Z E Wang ◽  
G M Myles ◽  
C S Brandt ◽  
M N Lioubin ◽  
L Rohrschneider
Keyword(s):  
Ligand Binding ◽  
Extracellular Domain ◽  
Colony Stimulating Factor ◽  
High Affinity ◽  
Baculovirus Expression ◽  
Baculovirus Expression Vector ◽  
Macrophage Colony Stimulating Factor ◽  
Polymerase Chain ◽  
Macrophage Colony ◽  
Stimulating Factor

The c-fms gene encodes the receptor for the macrophage colony-stimulating factor (M-CSF), and its extracellular domain consists of five immunoglobulin-like subdomains. To identify which of the five immunoglobulin-like regions are involved in ligand binding, we polymerase chain reaction-cloned five segments of the extracellular domain of the murine c-fms gene, each starting with the normal initiation codon and containing successive additions of the immunoglobulin-like subdomains. These protein segments are designated A, B, C, D, and E and contain, from the N-terminal end, either one, two, three, four, or all five immunoglobulin-like subdomains, respectively. Each segment was expressed as a secreted soluble protein from a baculovirus expression vector in Sf9 insect cells. In addition, segments A, B, C, and E were produced as soluble alkaline phosphatase fusion proteins, as was a segment containing only the fourth and fifth immunoglobulin domains. These segments of the Fms extracellular domain were used to assess M-CSF binding by competition radioimmunoassays, plate binding immunoassays, and immunoprecipitation analyses. The results indicated that the first two N-terminal immunoglobulin-like domains did not interact with M-CSF but, in combination with the third immunoglobulin-like domain, provided high-affinity M-CSF binding. The fourth and fifth immunoglobulin-like domains near the cell membrane did not exhibit M-CSF binding and may inhibit interaction of M-CSF with the first three immunoglobulin domains. These results suggest that the three N-terminal immunoglobulin-like domains constitute the high-affinity M-CSF binding region and that the fourth and fifth immunoglobulin-like domains may perform functions other than ligand binding.

scholarly journals Granulocyte-macrophage colony-stimulating factor induces sequential activation and deactivation of binding via a low-affinity IgG Fc receptor, hFc gamma RII, on human eosinophils

Blood ◽  
1993 ◽  
Vol 81 (9) ◽  
pp. 2413-2419
Author(s):  
L Koenderman ◽  
SW Hermans ◽  
PJ Capel ◽  
JG van de Winkel
Keyword(s):  
Ligand Binding ◽  
Binding Activity ◽  
Immune Defense ◽  
Common Mechanism ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Kinetics Of ◽  
Stimulating Factor

Eosinophils are important in antibody-mediated immune defense against parasites based on interaction with Ig receptors (FcR). Of the three classes of IgG FcR in humans, hFc gamma RI, II, and III, solely hFc gamma RII (CD32) is expressed on freshly isolated eosinophils. Despite an expression level similar to that found on monocytes and polymorphonuclear granulocytes, binding activity of hFc gamma RII on eosinophils is constitutively low. Freshly isolated eosinophils had a negligible ability to form rosettes with IgG-sensitized erythrocytes (EA-IgG). Addition of granulocyte-macrophage colony-stimulating factor (GM-CSF) caused an approximately threefold increase in EA-IgG rosettes. This increase was maximal after 35 minutes, and declined upon further incubation at 37 degrees C. Analysis of hFc gamma RII expression levels showed no significant changes and neither was the expression of other hFc gamma R classes induced. Blocking studies with anti-Fc gamma receptor monoclonal antibody (MoAb) proved hFc gamma RII specificity of enhanced IgG complex binding. These phenomena were not restricted to GM- CSF action, because the addition of interleukin-3 or interleukin-5 similarly enhanced EA-IgG binding. The kinetics of activation of hFc gamma RII binding activity were paralleled by the binding of EA-C3bi to CR3 on eosinophils. In contrast to the stable expression of hFc gamma RII during activation with GM-CSF, CR3 expression increased slowly. Ligand binding via both types of opsonin receptors proved receptor specific. However, the kinetics of enhanced binding via hFc gamma RII and CR3 suggested the possibility of a common mechanism underlying the enhancement of ligand binding via hFc gamma RII and CR3. This hypothesis was supported by the fact that binding via hFc gamma RII proved sensitive to both high concentrations of F(ab')2 fragments of anti-CD11b MoAb MO1 and chelation of bivalent cations with EDTA. In conclusion, our studies indicate that cytokines can induce a transient enhancement of hFc gamma RII binding activity. Qualitative, and not quantitative, changes in this receptor appear to underly the modulation of binding activity, which may be linked to changes in CR3 activity.

Kinetic Resolution of Two Mechanisms for High-Affinity Granulocyte-Macrophage Colony-Stimulating Factor Binding to Its Receptor

Blood ◽  
1999 ◽  
Vol 94 (11) ◽  
pp. 3748-3753 ◽  
Author(s):  
Linghao Niu ◽  
David W. Golde ◽  
Juan Carlos Vera ◽  
Mark L. Heaney
Keyword(s):  
Ligand Binding ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Receptor Complexes ◽  
High Affinity ◽  
High Affinity Receptor ◽  
Macrophage Colony Stimulating Factor ◽  
Affinity Receptor ◽  
Macrophage Colony ◽  
Stimulating Factor

Abstract Granulocyte-macrophage colony-stimulating factor (GM-CSF) is an important hematopoietic cytokine that exerts its effects by interaction with the GM-CSF receptor (GMR) on the surface of responsive cells. The GM-CSF receptor consists of two subunits: GMR, which binds GM-CSF with low affinity, and GMRβ, which lacks intrinsic ligand-binding capability but complexes with GMR to form a high-affinity receptor (GMR/β). We conducted dynamic kinetic analyses of GM-CSF receptors to define the role of GMRβ in the interaction of ligand and receptor. Our data show that GMR/β exhibits a higher kon than GMR, indicating that GMRβ facilitates ligand acquisition to the binding pocket. Heterogeneity with regard to GM-CSF dissociation from GMR/β points to the presence of loose and tight ligand-receptor complexes in high-affinity binding. Although the loose complex has a koff similar to GMR, the lower koffindicates that GMRβ inhibits GM-CSF release from the tight receptor complex. The two rates of ligand dissociation may provide for discrete mechanisms of interaction between GM-CSF and its high-affinity receptor. These results show that the β subunit functions to stabilize ligand binding as well as to facilitate ligand acquisition.

scholarly journals Granulocyte-macrophage colony-stimulating factor induces sequential activation and deactivation of binding via a low-affinity IgG Fc receptor, hFc gamma RII, on human eosinophils

Blood ◽  
1993 ◽  
Vol 81 (9) ◽  
pp. 2413-2419 ◽  
Author(s):  
L Koenderman ◽  
SW Hermans ◽  
PJ Capel ◽  
JG van de Winkel
Keyword(s):  
Ligand Binding ◽  
Binding Activity ◽  
Immune Defense ◽  
Common Mechanism ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Kinetics Of ◽  
Stimulating Factor

Abstract Eosinophils are important in antibody-mediated immune defense against parasites based on interaction with Ig receptors (FcR). Of the three classes of IgG FcR in humans, hFc gamma RI, II, and III, solely hFc gamma RII (CD32) is expressed on freshly isolated eosinophils. Despite an expression level similar to that found on monocytes and polymorphonuclear granulocytes, binding activity of hFc gamma RII on eosinophils is constitutively low. Freshly isolated eosinophils had a negligible ability to form rosettes with IgG-sensitized erythrocytes (EA-IgG). Addition of granulocyte-macrophage colony-stimulating factor (GM-CSF) caused an approximately threefold increase in EA-IgG rosettes. This increase was maximal after 35 minutes, and declined upon further incubation at 37 degrees C. Analysis of hFc gamma RII expression levels showed no significant changes and neither was the expression of other hFc gamma R classes induced. Blocking studies with anti-Fc gamma receptor monoclonal antibody (MoAb) proved hFc gamma RII specificity of enhanced IgG complex binding. These phenomena were not restricted to GM- CSF action, because the addition of interleukin-3 or interleukin-5 similarly enhanced EA-IgG binding. The kinetics of activation of hFc gamma RII binding activity were paralleled by the binding of EA-C3bi to CR3 on eosinophils. In contrast to the stable expression of hFc gamma RII during activation with GM-CSF, CR3 expression increased slowly. Ligand binding via both types of opsonin receptors proved receptor specific. However, the kinetics of enhanced binding via hFc gamma RII and CR3 suggested the possibility of a common mechanism underlying the enhancement of ligand binding via hFc gamma RII and CR3. This hypothesis was supported by the fact that binding via hFc gamma RII proved sensitive to both high concentrations of F(ab')2 fragments of anti-CD11b MoAb MO1 and chelation of bivalent cations with EDTA. In conclusion, our studies indicate that cytokines can induce a transient enhancement of hFc gamma RII binding activity. Qualitative, and not quantitative, changes in this receptor appear to underly the modulation of binding activity, which may be linked to changes in CR3 activity.

scholarly journals Identification of the ligand-binding regions in the macrophage colony-stimulating factor receptor extracellular domain.

1993 ◽  
Vol 13 (9) ◽  
pp. 5348-5359 ◽  
Author(s):  
Z E Wang ◽  
G M Myles ◽  
C S Brandt ◽  
M N Lioubin ◽  
L Rohrschneider
Keyword(s):  
Ligand Binding ◽  
Extracellular Domain ◽  
Colony Stimulating Factor ◽  
High Affinity ◽  
Baculovirus Expression ◽  
Baculovirus Expression Vector ◽  
Macrophage Colony Stimulating Factor ◽  
Polymerase Chain ◽  
Macrophage Colony ◽  
Stimulating Factor

The c-fms gene encodes the receptor for the macrophage colony-stimulating factor (M-CSF), and its extracellular domain consists of five immunoglobulin-like subdomains. To identify which of the five immunoglobulin-like regions are involved in ligand binding, we polymerase chain reaction-cloned five segments of the extracellular domain of the murine c-fms gene, each starting with the normal initiation codon and containing successive additions of the immunoglobulin-like subdomains. These protein segments are designated A, B, C, D, and E and contain, from the N-terminal end, either one, two, three, four, or all five immunoglobulin-like subdomains, respectively. Each segment was expressed as a secreted soluble protein from a baculovirus expression vector in Sf9 insect cells. In addition, segments A, B, C, and E were produced as soluble alkaline phosphatase fusion proteins, as was a segment containing only the fourth and fifth immunoglobulin domains. These segments of the Fms extracellular domain were used to assess M-CSF binding by competition radioimmunoassays, plate binding immunoassays, and immunoprecipitation analyses. The results indicated that the first two N-terminal immunoglobulin-like domains did not interact with M-CSF but, in combination with the third immunoglobulin-like domain, provided high-affinity M-CSF binding. The fourth and fifth immunoglobulin-like domains near the cell membrane did not exhibit M-CSF binding and may inhibit interaction of M-CSF with the first three immunoglobulin domains. These results suggest that the three N-terminal immunoglobulin-like domains constitute the high-affinity M-CSF binding region and that the fourth and fifth immunoglobulin-like domains may perform functions other than ligand binding.

scholarly journals Human granulocyte-macrophage colony-stimulating factor receptor signal transduction requires the proximal cytoplasmic domains of the alpha and beta subunits

Blood ◽  
1993 ◽  
Vol 82 (11) ◽  
pp. 3298-3306 ◽  
Author(s):  
M Weiss ◽  
C Yokoyama ◽  
Y Shikama ◽  
C Naugle ◽  
B Druker ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Beta Subunits ◽  
Colony Stimulating Factor ◽  
Wild Type ◽  
Gm Csf ◽  
High Affinity ◽  
Alpha Subunits ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) controls the production, maturation, and function of cells in multiple hematopoietic lineages. These effects are mediated by a cell-surface receptor (GM-R) composed of alpha and beta subunits, each containing 378 and 881 amino acids, respectively. Whereas the alpha subunit exists as several isoforms that bind GM-CSF with low affinity, the beta common subunit (beta c) does not bind GM-CSF itself, but acts as a high- affinity converter for GM-CSF, interleukin-3 (IL-3), and IL-5 receptor alpha subunits. The cytoplasmic region of GM-R alpha consists of a membrane-proximal conserved region shared by the alpha 1 and alpha 2 isoforms and a C-terminal variable region that is divergent between alpha 1 and alpha 2. The cytoplasmic region of beta c contains membrane proximal serine and acidic domains. To investigate the amino acid sequences that influence signal transduction by this receptor complex, we constructed a series of cytoplasmic truncation mutants of the alpha 2 and beta subunits. To study these truncations, we stably transfected the IL-3-dependent murine cell line Ba/F3 with wild-type or mutant cDNAs. We found that the wild-type and mutant alpha subunits conferred similar low-affinity binding sites for human GM-CSF to Ba/F3, and the wild-type or mutant beta subunit converted some of these sites to high- affinity; the cytoplasmic domain of beta was unnecessary for this high- affinity conversion. Proliferation assays showed that the membrane- proximal conserved region of GM-R alpha and the serine-acidic domain of beta c are required for both cell proliferation and ligand-dependent phosphorylation of a 93-kD cytoplasmic protein. We suggest that these regions may represent an important signal transduction motif present in several cytokine receptors.

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