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Prepublished online as a Blood First Edition Paper on October 10, 2002; DOI 10.1182/blood-2002-06-1903.
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Blood, 15 February 2003, Vol. 101, No. 4, pp. 1308-1315
HEMATOPOIESIS
Molecular assembly of the ternary granulocyte-macrophage
colony-stimulating factor receptor complex
Barbara J. McClure,
Timothy
R. Hercus,
Bronwyn A. Cambareri,
Joanna M. Woodcock,
Christopher J. Bagley,
Geoff J. Howlett, and
Angel F. Lopez
From the Cytokine Receptor Laboratory and Protein
Laboratory, Division of Human Immunology, Institute of Medical and
Veterinary Science (IMVS), Adelaide, South
Australia; and Department of Biochemistry and Molecular Biology,
University of Melbourne, Parkville, Australia.
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Abstract |
Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a
hematopoietic cytokine that stimulates the production and functional activity of granulocytes and macrophages, properties that have encouraged its clinical use in bone marrow transplantation and in
certain infectious diseases. Despite the importance of GM-CSF in
regulating myeloid cell numbers and function, little is known about the
exact composition and mechanism of assembly of the GM-CSF receptor
complex. We have now produced soluble forms of the GM-CSF receptor  chain (sGMR) and  chain (sc) and utilized GM-CSF, the GM-CSF
antagonist E21R (Glu21Arg), and the c-blocking monoclonal antibody BION-1 to define the molecular assembly of the GM-CSF receptor
complex. We found that GM-CSF and E21R were able to form low-affinity,
binary complexes with sGMR, each having a stoichiometry of 1:1.
Importantly, GM-CSF but not E21R formed a ternary complex with sGMR
and sc, and this complex could be disrupted by E21R. Significantly,
size-exclusion chromatography, analytical ultracentrifugation, and
radioactive tracer experiments indicated that the ternary complex is
composed of one sc dimer with a single molecule each of
sGMR and of GM-CSF. In addition, a hitherto unrecognized direct interaction between c and GM-CSF was detected that was absent with
E21R and was abolished by BION-1. These results demonstrate a novel
mechanism of cytokine receptor assembly likely to apply also to
interleukin-3 (IL-3) and IL-5 and have implications for our molecular
understanding and potential manipulation of GM-CSF activation of its receptor.
(Blood. 2003;101:1308-1315)
© 2003 by The American Society of Hematology.
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Introduction |
Granulocyte-macrophage colony-stimulating factor
(GM-CSF) is a cytokine produced by many cells in the body that
regulates the production, effector cell function, and survival of
myeloid cells.1-4 Macrophages and granulocytes rise in
numbers and exhibit a prolonged life span and enhanced effector
function in response to GM-CSF,5,6 properties that have
encouraged its use in bone marrow transplantation7 and
infectious diseases such as those associated with AIDS.8
In addition, GM-CSF controls dendritic cell production,
differentiation, and function and potentiates responses of
CD4+ T cells in vivo.9,10 This dual action of
GM-CSF has encouraged its utilization in different vaccination
strategies.11 On the other hand, these same properties
have implicated GM-CSF in myeloid leukemia and several inflammatory
conditions such as asthma12 and rheumatoid
arthritis.13
The actions of GM-CSF are mediated by specific receptors composed of 2 different subunits, a receptor chain (GMR),14 which
provides specificity and the major binding contact, and a chain
(c),15 which is common with the interleukin-3 (IL-3) and IL-5 receptors, promotes affinity conversion, and acts as the major
signal transducer. For this complex to be assembled and to signal,
there exist structural and dimerization requirements, some of which
have been defined. Extensive structure-function analysis has identified
several residues involved in GM-CSF, GMR, and c protein
interaction and biologic activity. For example, the binding of GM-CSF
to GMR involves an electrostatic interaction between Asp112 in the
fourth helix of GM-CSF and Arg280 in the F-G loop of
GMR.16,17 The biologic activities and high-affinity binding of GM-CSF are exquisitely dependent on Glu21 in the first helix of GM-CSF, although direct contact with c has not been demonstrated. Substitution of this amino acid with arginine generates a
GM-CSF analog, E21R (Glu21Arg), which exhibits only
low-affinity binding and is unable to stimulate cellular proliferation
and mature cell functions.18 Importantly, E21R is able to
antagonize GM-CSF binding and function19; however, the
molecular basis of this antagonism is not fully understood. In c,
residues in the B-C loop (Tyr365, His367, Ile368) and F-G loop (Tyr421)
of domain 4 are involved in GM-CSF high-affinity binding and
function.20-23 The monoclonal antibody (mAb) BION-1, which
binds an area in c encompassing these loops, blocks GM-CSF binding
and biologic activities.24
Dimerization of the and c subunits of this family of cytokine
receptors is recognized as a crucial step for their activation; however, the exact composition of the assembled complex remains unclear. A number of studies suggest that simple heterodimerization is
sufficient to activate the GM-CSF receptor,25 whereas both cross-linking and dominant-negative studies using
surface-expressed receptors suggest that the formation of higher-order
GM-CSF receptor complexes is required for receptor
activation.26,27 Dimerization of c in particular has
also been shown to be an important and necessary step for receptor
activation,28,29 probably reflecting the need to bring
into close proximity the cytoplasmic domains of 2 c molecules
associated with Janus kinase-2 (JAK-2), resulting in JAK
transphosphorylation and receptor phosphorylation. Interestingly, c
has been shown to crystallize as a dimer30 and to
exist as a preformed homodimer on the cell surface.26,28
Despite these findings, little is known about the full assembly of this
family of receptors, the intermediate steps in their formation, and how receptor assembly may be selectively modulated.
In this paper we show for the first time the full assembly of the human
GM-CSF receptor in solution. This shows a novel mode of cytokine
receptor assembly in which 1 molecule of GM-CSF associates with 1 molecule of GMR and 2 molecules of c. In addition, these studies
reveal an essential, direct interaction between GM-CSF and c and
provide a molecular understanding of GM-CSF antagonism by E21R or
BION-1. This novel mode of receptor assembly may also apply to the IL-5
and IL-3 receptors.
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Materials and methods |
Human GM-CSF and GM-CSF analogs
Soluble wild-type human GM-CSF was produced in Escherichia
coli and recovered from the periplasmic space by osmotic shock as
described previously.19 Crude periplasmic extracts were
adjusted to 25 mM N-ethylmorpholine HCl (NEM), pH 7.0, loaded onto Q
Sepharose Fast Flow (Amersham Biosciences, Sydney,
Australia) equilibrated in 25 mM NEM, pH 7.0, and a linear
gradient of 0 to 600 mM NaCl in 25 mM NEM, pH 7.0, used to elute the
bound proteins. GM-CSF purified by anion exchange was further purified
by reversed phase high-performance liquid chromatography (HPLC),
lyophilized, dissolved in phosphate-buffered saline (PBS) as
previously described,19 and sterile-filtered (0.45 µm).
The E21R analog of GM-CSF (BresaGen, Adelaide, South Australia)
contains a glutamate to arginine substitution at residue 21 and a
modified 12-amino acid leader peptide, MFATSSSTGNDG, to facilitate
expression in E coli.31
Radiolabeling of human GM-CSF
To enable phosphorylation of GM-CSF under mild conditions, we
made the GM-CSF analog, SGMKIN, in which the amino acids from alanine
at position 3 to proline at position 6 were replaced by the peptide
sequence RRASV, which is recognized by the catalytic subunit of cyclic
adenosine monophosphate (cAMP)-dependent protein kinase from
heart muscle.32 Complementary oligonucleotides were used
to create a HindIII/NcoI fragment encoding the
N-terminal 12 amino acids of SGMKIN. This fragment was ligated with
an NcoI/BamHI fragment encoding the
C-terminal 116 amino acids of human GM-CSF (hGM-CSF)
into HindIII/BamHI-digested pIN-III-OmpH3
expression vector19 to create the plasmid, pSGMKIN.
Soluble SGMKIN was expressed in E coli and purified as
described for wild-type GM-CSF. The final product was at more than 95%
purity by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE), and the SGMKIN analog displayed
biologic activity indistinguishable from the wild-type GM-CSF (data not
shown). Labeling of SGMKIN with 32P used a protocol adapted
from Kaelin et al.32 Fifty micrograms of purified SGMKIN
was incubated in a 200-µL reaction mix containing 20 mM Tris
(tris(hydroxymethyl)aminomethane) HCl, pH 7.5; 100 mM NaCl; 12 mM MgCl2; 10 mM -mercaptoethanol; 1 µCi/µL (0.037 MBq/µL) [ -32P]adenosine
triphosphate ([-32P]ATP) (3000 Ci/mmol
[111 000 GBq/mmol]; Geneworks, Adelaide, South
Australia), and 1 U/µL of the protein kinase catalytic subunit (Sigma, Castle Hill, Australia). The reaction proceeded at 4°C for 30 minutes, was terminated by the addition of 200 µL of 100 mM
EDTA (ethylenediaminetetraacetic acid), adjusted to 0.1%
(vol/vol) trifluoroacetic acid (TFA; Auspep, Parkville,
Australia), 1% (vol/vol) acetic acid, and loaded onto a
Sep-Pak C18 reversed phase cartridge (Waters, Rydalmere,
Australia) equilibrated in 0.01% TFA. The cartridge was
washed with 0.01% TFA and bound SGMKIN eluted using 10 mL of 50%
(vol/vol) acetonitrile in the presence of 0.01% TFA. Ten equal
fractions were collected, and those containing the peak of eluted
radioactivity were pooled and concentrated using a Speed-vac (Savant
Instruments, Farmingdale, NY) to a final volume of approximately 100 µL.
Production of recombinant soluble GM-CSF receptor subunits
DNA fragments encoding the soluble extracellular domains of
GMR (sGMR) or c (sc) were generated by PCR using the
primers 5'-CTGACCGGATCCATGCTTCTCCTGGTGACAAGCC-3' and
5'-GTACACGGATCCGAATTCTTACCCGTCGTCAGAACCAAATTC-3' for
sGMR and 5'-CTGACCGGATCCATGGTGCTGGCCCAGGGGCTGC-3' and
5'-CAGCACGGATCCGAATTCTTACGACTCGGTGTCCCAGGAGCG-3' for
sc, with EcoRI and BamHI restriction sites
underlined. Stop codons were inserted immediately prior to the
transmembrane domain for each receptor molecule, following Gly at
position 320 for GMR and Ser at position 438 for c. The PCR
products were digested with BamHI and EcoRI and
cloned into the baculovirus transfer vector BacPAK9 (Clontech, Palo
Alto, CA) and the sequence of the cloned inserts were verified by cycle
sequencing with BigDye chemistry (Applied Biosystems, Foster City, CA).
The cDNA encoding sGMR and sc was introduced into the genome of
Bsu36I-digested BacPAK6 viral DNA (Clontech) by homologous
recombination following the manufacturer's instructions. Expression of
recombinant protein is under the control of the strong polyhedrin
promoter. Large-scale expression of sGMR or sc was performed by
infection of Sf21 cells, grown in serum-free Ex-Cell 420 medium (JRH
Biosciences, Brooklyn, Australia), with recombinant baculovirus at a
multiplicity of infection of 0.3. Supernatant containing soluble
receptor was harvested following incubation at 27°C for 5 to 7 days.
Purification of soluble GMR and sc
Conditioned media containing sGMR (20 L) or sc (9 L) were
concentrated to less than 1 L using tangential flow filtration cartridges (10 000 molecular weight cutoff, 0.23 m2)
(Millipore, Northryde, Australia) operated at 80 kPa and 4°C. Insoluble material in the concentrate was pelleted at 3000g
for 30 minutes and the resulting supernatant filtered (3 µm) prior to
affinity chromatography. Affinity matrices were prepared by coupling
E21R or the anti-c mAb, BION-1,24 to cyanogen
bromide (CNBr)-activated Sepharose 4B (Amersham Biosciences)
following the manufacturer's instructions. Recombinant soluble
receptor was bound to the affinity matrix, washed extensively in PBS
containing 0.01% (vol/vol) polyoxyethylene 20 sorbitan monolaurate
(Tween 20), and bound proteins eluted with 100 mM NaCl, 100 mM sodium acetate (pH 4.0). The eluate fractions were immediately neutralized using 2 M Tris and analyzed for the presence of soluble receptor by
SDS-PAGE. Fractions containing purified soluble receptor were pooled
and concentrated using a stirred-cell device with a 10 000 molecular
weight cutoff, low protein-binding membrane (YM10; Millipore) operated
at 300 kPa and 4°C. Concentrated soluble receptor was dialyzed
extensively into PBS, sterile-filtered (0.2 µm), and stored at
4°C.
SDS-PAGE
Samples were analyzed on 10% or 12.5% polyacrylamide gels
containing 38:1 acrylamide/bisacrylamide under reducing or nonreducing conditions as specified. Bands were visualized by staining with either
Coomassie brilliant blue R-250 or silver.33
Mass spectrometry
Electrospray ionization mass spectrometry was performed using a
PE/Sciex API100 mass spectrometer (Perkin-Elmer Sciex Instruments, Ontario, Canada). Protein samples were desalted in-line using a
1 × 10 mm reversed phase column eluted with 60% (vol/vol)
acetonitrile in the presence of 0.04% (vol/vol) TFA and the primary
mass spectrum transformed to give a true-mass profile using
instrument software.
Protein analyses by size-exclusion chromatography
Size-exclusion chromatography was initially used to quantify
purified soluble receptors and their ligands. Samples were
chromatographed on a SMART system with a Superdex 200PC 3.2/30 (3.2 mm × 300 mm) column (Amersham Biosciences) operated at 40 µL/min at 25°C using 150 mM NaCl, 50 mM sodium phosphate, pH 7.0, as running buffer. The area under the protein peak was integrated using
the extinction coefficient (absorbance
units × mL 1 × mg1)
calculated for each protein: GM-CSF, 0.95; E21R, 0.88; sGMR, 1.17;
sc, 1.95.
To analyze protein-protein interactions, individual proteins and
protein complexes were prepared in a final volume of 50 µL, adjusted
with PBS as required, and incubated at 25°C for at least 1 hour.
Samples were analyzed by size-exclusion chromatography using the SMART
system as described above with data presented from representative
experiments (n = 5). The dependence of elution time on the
log10 (MW) of protein standards was used to calibrate the
column and to generate a trend line for each set of standards. External
standards included myoglobin, MW 17 kDa; ovalbumin, MW 44 kDa;
-globulin, MW 158 kDa; and thyroglobulin, MW 670 kDa (Biorad
Laboratories, Hercules, CA). Internal standards were GM-CSF, MW 14.5 kDa; E21R, MW 15.7 kDa; sGMR, MW 43 kDa; and sc, MW 101 kDa as
determined by mass spectrometry and SDS-PAGE. Soluble c was found to
be a dimer by size-exclusion chromatography consistent with previous
reports.30 Calibration curves constructed from the
external and internal standards were essentially parallel (see
Figure 2A). The calibration curve for the internal standards was extrapolated to higher mass (670 kDa) because this was found to be
the limit of the linear range for the external standards.
Analytical ultracentrifugation
The molecular weights of GM-CSF, E21R, sGMR, sc, and the
binary and ternary complexes were determined by sedimentation
equilibrium. Individual proteins and protein complexes were isolated by
size-exclusion chromatography using a fast protein liquid
chromatography (FPLC) system with a Superdex 200 10/30 (10 mm × 300 mm) column (Amersham Biosciences) operated at 0.5 mL/min
at 25°C using 150 mM NaCl, 50 mM sodium phosphate, pH 7.0, as running
buffer. Pooled fractions were concentrated using Centricon 10 microconcentrators (Amicon, Beverly, MA). Sedimentation equilibrium
experiments were performed using a Beckman XL-A analytical
ultracentrifuge equipped with a Ti60 rotor (Beckman, Palo Alto, CA) and
filled epon centerpieces (12-mm path length). Sedimentation
equilibrium profiles were obtained at 20°C using the rotor speeds
indicated. Equilibrium distributions were fitted by nonlinear
regression analysis to obtain best-fit values for the M (1-  ),
where M is the molecular weight and the partial specific
volume of the sedimenting species and the solution density. The
compositional molecular weights of the proteins and the partial
specific volumes of GM-CSF and E21R were calculated from their amino
acid sequences. Partial specific volumes for the glycosylated forms of
sGMR and sc were calculated assuming these proteins were monomer
and dimmer, respectively. A value of 0.622 mL/g was assumed for the
partial specific volume of carbohydrate. The experimental value of M
(1-) and the molecular weight (Mp) and partial
specific volume of the protein component were then used to solve for
the weight fraction of bound carbohydrate and hence the partial
specific volume of the carbohydrate-bound protein. Values for the
partial specific volumes of the GM-CSF/sGMR and E21R/sGMR
complexes were calculated assuming a 1:1 complex and no volume change
on association. A value of 0.72 mL/g was assumed for the
GM-CSF/sGMR/sc complex.
Cross-linking experiments
Stable cross-linking of sc or soluble complexes of ligand
with sc was performed by incubation of 2.6 µg sc with either 2.4 µg GM-CSF or E21R for 1 hour at 25°C followed by addition of
BS3 cross-linker (Pierce, Rockford, IL) at a final
concentration of 0.1 mg/mL for 10 minutes. The reaction was then
stopped by addition of ethanolamine HCl, pH 8.0, to a final
concentration of 100 mM. Cross-linked proteins were subjected to
reducing SDS-PAGE and compared with non-cross-linked material.
Antibody Fab fragments of BION-1 (anti-c fourth domain blocking mAb)
and 2H1 (anti-c fourth domain control mAb) used in cross-linking
experiments were generated by digestion with ficin using the Immunopure
IgG1 Fab Preparation Kit (Pierce) following the
manufacturer's instructions. Fab fragment (18 µg) was preincubated
with 2.6 µg sc for 30 minutes at 25°C prior to the addition of
2.4 µg GM-CSF in a final volume of 20 µL for a further hour.
Cross-linking was then performed as above followed by SDS-PAGE analysis.
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Results |
Production, purification, and analysis of GM-CSF soluble
receptor components
Complementary DNA fragments encoding the extracellular domains of
GMR and c (sGMR and sc) were generated by PCR and cloned into a baculovirus transfer vector. Following introduction into a
baculovirus expression system by homologous recombination, the soluble
receptor components were generated by infection of Sf21 cells.
Purification of the soluble receptors was achieved by affinity chromatography using immobilized ligand for sGMR and immobilized mAb
BION-124 for sc. Purified soluble receptors were
recovered at more than 95% purity as assessed by silver-stained
SDS-PAGE under reducing conditions (Figure
1A) with an apparent molecular weight
(MW) of approximately 43 kDa for sGMR and 55 kDa for sc. Importantly, these MWs determined for sGMR and for sc did not alter when analyzed under nonreducing conditions (Figure 1B), indicating the absence of disulfide-linked dimers. A small amount of
disulfide-aggregated sc was visible by nonreducing SDS-PAGE (Figure
1B) and as an early, minor peak during size-exclusion chromatography
(Figure 2C). The absence of detectable
disulfide-linked dimers in sc was confirmed by ion-spray mass
spectrometry, which demonstrated that the protein preparation had a
major species of 50.623 kDa with several minor species representing
glycosylation variants.
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| Figure 1.
SDS-PAGE analysis of purified sGMR and sc.
Soluble GMR and sc were produced by Sf21 cells infected with
recombinant baculovirus encoding appropriate cDNA and affinity purified
from the supernatant as described in "Materials and
methods." Soluble GMR (1 µg) and sc (0.5 µg) were
fractionated by 10% SDS-PAGE under reducing (A) and nonreducing (B)
conditions and silver stained. The positions of molecular weight
markers are shown in kilodaltons.
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| Figure 2.
GM-CSF but not the GM-CSF analog E21R induces the
assembly of the ternary GM-CSF receptor complex in solution.
The presence and molecular weight of individual proteins and protein
complexes were determined using size-exclusion chromatography as
described in "Materials and methods." (A) Linear regression
of log10 (MW × 103) versus elution times
using external ( ) and internal ( ) standards for calibration
of the column. (B-I) Individual proteins sGMR (B), sc (C), GM-CSF
(D), and E21R (G) were applied separately. Mixtures of sGMR
(6 µM) and GM-CSF (12 µM) (E); sc (3 µM), sGMR (6 µM),
and GM-CSF (12 µM) (F); sGMR (6 µM) and E21R (12 µM) (H);
sc (3 µM), sGMR (6 µM), and E21R (12 µM) (I) were incubated
for 1 hour before being applied to the column. The number above each
peak represents elution time. Peaks containing binary (BC) or ternary
(TC) complexes are indicated.
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The physical properties of sGMR and sc were further characterized
by size-exclusion chromatography. We initially determined the retention
times of sGMR (Figure 2B), sc (Figure 2C), GM-CSF (Figure 2D),
and E21R (Figure 2G). The individual proteins eluted at 39.17 minutes
for sGMR, 34.75 minutes for sc, 44.56 minutes for GM-CSF, and
44.11 minutes for E21R. External MW standards for calibration of the
size-exclusion chromatography (Figure 2A) indicated that sGMR,
GM-CSF, and E21R were monomeric but that sc was dimeric. The dimeric
nature of sc was confirmed by cross-linking experiments with
purified sc, which produced a covalent dimer with a MW of 100 kDa as
determined by SDS-PAGE (see Figure 8B). The observation that
sc exists as a dimer is consistent with a recent report describing
the structure of the extracellular domain of c expressed in insect
cells.30,34 We observed that both the ligands and the
receptor components eluted from size-exclusion chromatography earlier
than expected from the elution times of the external MW standards
(Figure 2A). We chose to use the proteins of interest as internal MW
standards and constructed a calibration curve for the internal MW
standards that is parallel to that constructed from the external MW
standards (Figure 2A). This is expected to provide a superior estimate
of the masses of the receptor complexes.
Soluble GMR interactions with GM-CSF and E21R
Purified sGMR (6 µM) was incubated with GM-CSF (12 µM) and
fractionated on a Superdex 200 column, producing a modest shift (from
39.17 minutes to 38.51 minutes) in the elution time of sGMR (Figure
2E). The shifted peak, with an apparent MW of 48 kDa, contained both
GM-CSF and sGMR as determined by SDS-PAGE analysis of fractions
(data not shown). The MW of the GM-CSF/sGMR binary complex is
consistent with a stoichiometry of 1:1 as has previously been
described.35 The complete peak shifts observed when
sGMR binds GM-CSF suggest that all of this soluble receptor is
competent to bind ligand. Saturation binding experiments revealed that
GM-CSF bound to sGMR with a dissociation constant
(Kd) of 1.5 to 9 nM, similar to that seen with
cell surface-expressed GMR (data not shown).
Purified sGMR (6 µM) was incubated with E21R (12 µM) and
fractionated on a Superdex 200 column, producing a modest shift (from 39.17 minutes to 38.40 minutes) in the elution time of
sGMR (Figure 2H). The shifted peak, with an apparent MW of 49 kDa, contained both E21R and sGMR as determined by SDS-PAGE analysis of
fractions (data not shown). The MW of the E21R:sGMR binary complex
is consistent with a stoichiometry of 1:1.
The sc induces the formation of a GM-CSF ternary
complex
Purified sc (3 µM) was incubated with sGMR (6 µM) plus
GM-CSF (12 µM) and fractionated on a Superdex 200 column, producing a
complete shift in the elution time of sc (from 34.75 minutes to
32.67 minutes) as well as peaks corresponding to the binary complex at
38.40 minutes and free ligand at 44.61 minutes (Figure 2F). The peak
eluting at 32.67 minutes had an apparent MW of 155 kDa and contained
GM-CSF, sGMR, and sc as determined by SDS-PAGE analysis of
fractions (Figure 3A). The MW of this
ternary GM-CSF receptor complex is consistent with a stoichiometry of 1 GM-CSF:1 sGMR:2 sc.
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| Figure 3.
SDS-PAGE analysis of the ternary GM-CSF receptor
complex.
Mixtures of sc, sGMR, and either GM-CSF (A) or E21R (B) were
analyzed by size-exclusion chromatography as described for Figure 2F
and I. Fractions were collected at 1-minute intervals, fractionated by
12.5% SDS-PAGE under reducing conditions, and silver
stained.33 The positions of individual components are
indicated.
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In contrast, no ternary complex was observed when purified sc (3 µM) was incubated with sGMR (6 µM) plus E21R (12 µM) and fractionated on a Superdex 200 column (Figure 2I). Whereas the binary
complex eluting at 38.32 minutes contained E21R and sGMR, the peak
at 34.80 minutes contained sc but no sGMR or E21R as determined
by SDS-PAGE analysis of fractions (Figure 3B).
E21R disrupts the formation of the ternary GM-CSF receptor
complex
To investigate whether the formation of a binary complex was an
intermediate step in the formation of the ternary GM-CSF receptor complex, we tested the effect of E21R in this process. Purified sc
(3 µM) was incubated with sGMR (6 µM) and GM-CSF (12 µM) for 1 hour. A 100-fold molar excess of E21R was then added, and after a
further 1-hour incubation the mixture was fractionated on a Superdex
200 column. In the absence of E21R the ternary GM-CSF receptor complex
eluted at 33.12 minutes (Figure 4).
Significantly, in the presence of a 100-fold molar excess of E21R
(Figure 4) there was a reduction in the amount of ternary GM-CSF
receptor complex and an increase in its elution time (34.10 minutes),
more comparable with the elution time of free sc (34.75 minutes). The reduction in the amount of ternary complex along with an increased amount of binary complex (38.40 minutes) and free ligand (44.00 minutes) is consistent with sGMR preferentially forming a binary complex with E21R, which is unable to recruit sc into a ternary complex.
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| Figure 4.
E21R prevents formation of the ternary GM-CSF receptor
complex.
Following formation of the GM-CSF/sGMR/sc ternary complex using a
1:2:4 molar ratio, a 100-fold molar excess of E21R over GM-CSF was
added for a further hour at 25°C before size-exclusion
chromatography. The chromatogram shows the A280 profile of
a GM-CSF/sGMR/sc mixture in the absence (thick line) or
presence of E21R (thin line) or in sc alone (dashed
line).
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Stoichiometry of the ternary GM-CSF receptor complex
To confirm the 1 GM-CSF:1 sGMR:2 sc stoichiometry of the
ternary GM-CSF receptor complex obtained by size-exclusion
chromatography, we utilized 2 other complementary and independent
methods. In one of these the molecular weights of the individual
proteins and of the binary and ternary complexes were determined by
sedimentation equilibrium. The results showed (Figure
5; Table
1) values similar to those obtained by
gel filtration. The estimates of the molecular weight of the binary
complexes GM-CSF/sGMR, 52.7 kDa (Figure 5A; Table 1), and
E21R/sGMR, 54.8 kDa, (Figure 5B; Table 1), are consistent with a 1:1
stoichiometry. The molecular weight of the ternary GM-CSF/sGMR/sc
complex was determined to be 135 kDa (Figure 5C and Table 1). This
value is consistent with a model where one sc dimer (97.4 kDa)
associates with one GM-CSF/sGMR binary complex (52.7 kDa) (Table 1)
with a theoretical molecular weight of 150.1 kDa.
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| Figure 5.
Analyses of the ternary GM-CSF receptor complex by
sedimentation equilibrium.
The individual proteins or protein complexes in 150 mM NaCl/50
mM sodium phosphate, pH 7.0, were centrifuged at 20°C at
angular velocity, W rpm, for 16 hours. The equilibrium
profiles are presented as (W/W1)2 Ln(c/co)
versus the square of the radial distance, where c/co is the
optical density at 280 nm divided by the initial optical density and W1
is 20 000 rpm. For a single species, this plot is linear with a slope
proportional to the molecular weight of the sedimenting species. The
initial concentrations were in the range 0.40 to 0.47 mg/mL,
and samples were centrifuged at 20 000 rpm except for E21R, where the
initial concentration was 0.2 mg/mL and the angular velocity 15 000
rpm. Panel A samples: GM-CSF (), sGMR ( ),
GM-CSF/sGMR complex (). Panel B samples: E21R (), E21R/sGMR
complex (). Panel C samples: purified sc () was centrifuged at
15 000 rpm with an initial concentration of 0.45 mg/mL, whereas the
GM-CSF/sGMR/sc ternary complex at an initial concentration of
0.47 mg/mL was centrifuged at either 8000 rpm ( ) or 15 000 rpm
() for 16 hours at 20°C.
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Table 1.
Sedimentation equilibrium analysis of the molecular
weights of GM-CSF, E21R, sGMR, sc, and their complexes
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In a separate approach, we used radiolabeled GM-CSF as a tracer
molecule. Purified sc (0 to 7 µM) was titrated against a mixture
of sGMR (3.2 µM) and cold GM-CSF (7.3 µM) spiked with the GM-CSF
analog 32P-SGMKIN and subjected to size-exclusion
chromatography as above. Addition of sc to the GM-CSF/sGMR
mixture led to the dose-dependent formation of the ternary complex and
depletion of the binary complex (Figure
6A). Once the concentration of sc
saturated the available binary complex, a shoulder appeared on the
trailing edge of the ternary complex peak, presumably reflecting the
presence of free sc. Formation of the ternary complex was associated
with a dose-dependent accumulation of radioactivity at the appropriate
elution time of the ternary complex and was accompanied by a reduction
of radioactivity at the elution time of the binary complex (Figure 6B).
Titration of sc did not lead to a reduction of radioactivity at the
elution time of free ligand, although a modest shift at the leading
edge of the free ligand peak was observed. We then determined the
distribution of 32P-SGMKIN into the ternary complex and
expressed it as a percentage of total label in the ternary and binary
complexes versus proportion of sc present (Figure 6C). When compared
with the theoretical distribution predicted for a ternary complex
with a GM-CSF/sGMR/sc ratio of 1:1:2 or 2:2:2, the observed
distribution was consistent with a 1:1:2 stoichiometry. The observed
distribution only departed from the modeled linear distribution as the
concentration of binary complex became limiting.
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| Figure 6.
Radiolabeled GM-CSF differentially partitions to the
ternary GM-CSF receptor complex.
(A-C) A titration of purified sc (0 to 7.03 µM) against a mixture
of 3.2 µM sGMR and 7.3 µM GM-CSF spiked with
32P-labeled SGMKIN. Reaction mixes were set up with 0 (dashed gray), 0.88 µM (dashed black), 1.76 µM (thin gray), 3.52 µM (thin black), 5.27 µM (thick gray), or 7.03 µM (thick black)
sc and incubated at 25°C for 1 hour before size-exclusion
chromatography. Fractions were collected at 1-minute intervals. A
control reaction was also prepared with 5.27 µM sc and 3.2 µM
sGMR but no GM-CSF (dashed black). (A) Chromatogram of
A280 profiles for each sample with the location of the
ternary complex (TC), binary complex (BC), and free ligand (GM)
indicated. (B) Distribution of radioactivity among the ternary complex,
binary complex, and free ligand for the reactions described in panel A. (C) Radioactive GM-CSF distributed into the ternary complex,
expressed as a percentage of the total radioactive GM-CSF in ternary
and binary complexes; comparing experimentally observed values for the
reactions described in panel A () with a theoretical
distribution based on 1GM:1:2 () and 2GM:2:2 ()
models. (D) Titration of GM-CSF (0 to 7 µM) spiked with
32P-labeled SGMKIN against a mixture of 3.5 µM sGMR
and 3.5 µM sc. Reaction mixes were allowed to reach equilibrium at
25°C for at least 2 hours before being fractionated by size-exclusion
chromatography. The distribution of radioactivity among ternary ()
and binary () complexes was determined and the radioactivity in each
complex was expressed as a percentage of total bound counts where
counts in TC plus counts in BC is 100%.
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The use of radiolabeled GM-CSF also allowed us to investigate whether
the presence of sc in the ternary complex led to affinity conversion. GM-CSF spiked with the GM-CSF analog 32P-SGMKIN
was titrated against an equimolar mixture of sGMR and sc, allowed
to equilibrate, and fractionated by size-exclusion chromatography. For
each GM-CSF concentration point, radioactivity bound in the
binary and ternary complexes was determined and the proportion in each
complex was expressed as a percentage of total bound counts. We found
(Figure 6D) a 4-fold preferential distribution of
32P-SGMKIN into ternary complexes at subsaturating
concentrations of ligand, indicating that the presence of sc in the
ternary complex induces a measurable degree of affinity conversion.
GM-CSF binds sc in the absence of sGMR
Initial chromatography experiments at approximately
equimolar concentrations indicated that GM-CSF was unable to form a
complex with sc in the absence of sGMR. However, close inspection
of the elution profile of radiolabeled GM-CSF in the presence of free
sc (Figure 6B) revealed a modest decrease in the elution time of
GM-CSF suggestive of a weak interaction between sc and GM-CSF. To
investigate this further and to determine the specificity of this
interaction, we titrated sc against GM-CSF or the E21R analog
(Figure 7). Titration of sc against
GM-CSF had a dose-dependent effect on GM-CSF peak height with a
concomitant spreading of the GM-CSF profile to earlier elution times
(Figure 7A). Titration of sc against E21R had no effect on E21R
elution time or profile (Figure 7B). These results show that GM-CSF
directly interacts with sc through the functionally important Glu21
residue and that substitution of this residue makes a qualitative
difference to the GMR-independent recognition of c by
GM-CSF.
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| Figure 7.
GM-CSF binds directly to c.
Purified sc was titrated (0 to 20 µM) against 5 µM GM-CSF (A) or
5 µM E21R (B). Reaction mixes were set up with 0 (dashed black), 1 µM (thin black), 2.5 µM (medium gray), 5 µM (medium black), 10 µM (thick gray), or 20 µM (thick black) sc, incubated at 25°C
for 2 hours, and fractionated by size-exclusion chromatography.
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A second approach confirmed the direct interaction of GM-CSF with
c and extended these findings to the identification of the
reciprocal region in c. We incubated purified sc with a 3-fold
molar excess of GM-CSF or E21R, treated with the BS3 cross-linker and analyzed the mixture by SDS-PAGE under reducing conditions (Figure 8). No covalent
interactions between sc and GM-CSF or E21R were observed in the
absence of cross-linker (Figure 8A). GM-CSF and E21R were not dimerized
by cross-linker under the conditions used, whereas the sc dimer was
partially cross-linked, yielding a band of MW 100 kDa (Figure 8B).
Significantly, when GM-CSF was incubated with sc and cross-linked, a
unique band of MW 70 kDa was observed (Figure 8B). Western blotting
with anti-c or anti-GM-CSF antibodies showed that this band
contains both sc and GM-CSF (data not shown). E21R could not be
cross-linked directly to sc as seen by the absence of the 70 kDa
band (Figure 8B), thus confirming that Glu21 of GM-CSF is necessary for
direct contact with c. Neither the structurally related cytokine
human growth hormone nor sGMR was able to be cross-linked to sc
under these conditions (data not shown).
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| Figure 8.
Discrete regions in GM-CSF and c mediate their direct
interaction.
Purified sc was incubated at 25°C for 1 hour alone or in the
presence of either GM-CSF or E21R. Samples were left untreated (A) or
were treated for 10 minutes with BS3 cross-linker (B). To
determine if GM-CSF was interacting with the cytokine-binding site in
the fourth domain of c, purified sc was preincubated with a Fab
fragment of the neutralizing anti-c mAb, BION-1, or the
nonneutralizing control anti-c mAb, 2H1. GM-CSF was then allowed to
bind and the mixture and individual mAb treated with BS3
cross-linker (C). Samples were analyzed on 12.5% (A) or 10% (B-C)
SDS-PAGE gels and stained with Coomassie. The positions of molecular
weight markers are shown in kilodaltons, and the position of sc
cross-linked to GM-CSF is indicated by  .
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To determine if the interaction between sc and GM-CSF was occurring
through a functionally relevant region of c, we used BION-1, a mAb
that blocks GM-CSF, IL-3, and IL-5 binding and signaling through
c.24 BION-1 recognizes a discrete region in the fourth domain of c associated with high-affinity GM-CSF binding and function.20,22,36 Preincubation of sc with BION-1 Fab
fragment prevented GM-CSF from being cross-linked to sc, as seen by
the absence of the 70-kDa band (Figure 8C). The Fab fragment of a mAb
that binds to the fourth domain of c but does not block cytokine binding was unable to perturb the cross-linking of sc to GM-CSF (Figure 8C).
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Discussion |
We report here the first demonstration of a fully assembled
GM-CSF/GM-CSF receptor ternary complex in solution and describe the
molecular interactions required for its formation. It is shown that the
ternary complex exhibits a novel mode of cytokine receptor assembly
that comprises 1 molecule of GM-CSF and 1 molecule of GM-CSF receptor
chain interacting monovalently with a noncovalently linked dimer of
c. In addition, a direct interaction between GM-CSF and c in the
absence of the receptor chain could be demonstrated. The
recruitment of c as a preformed dimer may facilitate receptor
activation and may also represent a mechanism utilized by the related
IL-3 and IL-5 receptors. The GM-CSF ternary complex was demonstrated by
gel filtration and sedimentation equilibrium analyses to have a
molecular weight of between 135 kDa and 156 kDa, consistent with a
GM-CSF/sGMR/sc stoichiometry of 1:1:2. In addition, the relative
distribution of radiolabeled GM-CSF fitted a ternary complex with a
1:1:2 stoichiometry. The preferential distribution of radiolabeled
GM-CSF into the ternary complex is indicative of sc-mediated,
affinity conversion. No disulfide linkages between receptor subunits
were observed; there were no differences seen when the ternary complex
was analyzed by SDS-PAGE under either reducing or nonreducing
conditions or when the free cysteine groups in sc were blocked with
iodoacetamide (data not shown). These results are consistent with
previous reports suggesting that GM-CS |
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Abstract
Introduction