From the
In this study, we report the use of coiled-coil (leucine zipper)
molecular recognition for the solution assembly of stable, high
affinity, heteromeric interleukin-2 receptor complexes. Co-expression
of interleukin-2 receptor
The hematopoietin receptor family shares several conserved
structural features as well as a common signal transmission mechanism,
that of ligand-induced receptor subunit cross-linking(1) . One
of the first cytokine systems to be classified among this group was
interleukin-2. The high affinity interleukin-2 receptor (IL-2R)
Although the high affinity IL-2R employs three subunits,
heterodimers of IL-2R subunits also play physiologically important
roles. We have previously determined that the (
The ligand recognition properties of the individual ectodomains of
these subunits have been studied. The soluble
In an effort
to better understand the cooperativity of cytokine receptor ectodomains
in ligand binding, we have evaluated the addition of coiled-coil
recognition sequences to the receptor ectodomains as a means of
generating stable solution complexes that mimic cell surface ligand
binding. The coiled-coil interaction was first described 40 years ago
by Crick (14) and has been studied in detail by Hodges as the
structural element present in
tropomyosin(15, 16, 17) . It is now recognized
that the coiled-coil motif or ``leucine zipper'' is a
recognition element common to a variety of macromolecular protein
complexes(18) .
Recently, we have prepared and characterized
an IL-2R
Similarly, the cDNA encoding the
Purification of
IL-2R
After co-infection, preliminary isolation
of IL-2R
The levels of expression of
Since the
tag affinity column was specific only for the
Studies of
competitive neutralization of bioactivity (not shown) determined on Kit
225 cells (13) revealed that the fraction that bound to both
columns was 2-fold more potent (IC
We
compared the ability of this
Coiled-coil
sequences from transcription factors have been employed previously to
help mediate the formation of antibody complexes (for example, see Ref.
32). Unlike these studies, we have chosen multiple copies of idealized
coiled-coil sequences due to their inherently greater stability. In
addition, our goal was to assemble a cooperative system using
noncovalent complexation. Cooperativity in ligand binding by the
Ligand-induced receptor
subunit cross-linking to form both homomeric and heteromeric complexes
is a hallmark of the hematopoietin receptor family(1) .
Refinements in the designs of coiled-coil peptide complexes now make it
possible to prepare stable assemblies containing two, three, or four
subunits (29, 30) as well as to direct the formation of
heteromeric complexes(21, 31) . This is the first study
to demonstrate the feasibility of employing coiled-coil designs as
recognition units to mediate the solution assembly of heteromeric
cytokine receptor complexes. This approach can easily be extended to
other receptor complexes composed of two to four similar or different
subunits and may provide reagents for detailed receptor subunit and
ligand binding characterization, crystallographic analyses, selection
of novel ligands from random libraries, or in vivo therapeutic
evaluation.
Equilibrium
solution binding constants (K
Analytical ultracentrifugation was
carried out as described under ``Materials and Methods.''
Concentration lists cell loading concentrations used in combined
analysis at each rotor speed indicated. M
We thank Kendall Smith, Irwin Chaiken, and Randy
Noelle for helpful comments; Abla Creasy for direction and support; and
Renee Risingsong for technical expertise. We also thank Gwynn Pardee
for help with recombinant baculovirus insect cell expression.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
and
extracellular domains
(ectodomains), each fused to seven coiled-coil heptad repeats, resulted
in the formation of heteromeric complexes that bound interleukin-2 in a
cooperative fashion and with much higher affinity than similar
homomeric complexes. The dissociation constants for these solution
complexes are within the range of values reported for the comparable
cell surface ``pseudo high affinity'' interleukin-2 receptor.
Ligand-induced cross-linking of homomeric or heteromeric receptor
subunits is the common signal transmission mechanism employed by
hematopoietin receptors. Individual receptor ectodomains, however,
often do not bind ligand with measurable affinity. This is the first
study to demonstrate the feasibility of coiled-coil mediated
preassembly of cytokine receptor complexes.
(
)results from the noncovalent interaction of three
cell surface proteins, all of which are capable of independently
binding IL-2 with much lower affinity. The 55-kDa
-subunit binds
IL-2 with a dissociation constant (K
) of
1
10
M(2) . The larger
75-kDa
-subunit was originally reported to have a K
= 1
10
M(3) ; however, the identification of the 64-kDa
-subunit (4) demonstrated that it also participated in the
ligand-induced formation of 1 nM intermediate affinity site.
Cooperative binding of IL-2 by these three proteins ultimately results
in cross-linking of the
- and
-subunits and signaling via
association of their cytoplasmic domains(5) . In addition,
recent reports indicate that the
-subunit is shared by the IL-4
and IL-7 receptors(6) , while both
- and
-subunits
participate in the formation of the IL-15 receptor(7) .
/
)
``pseudo high affinity'' heterodimer exists, preformed, on
the surface of activated T-cells and serves to capture
ligand(8) . This observation was in accordance with theoretical
analyses of ligand binding data(9) . Furthermore, in the
presence of IL-2, a second heterodimer (
/
) is formed. This
signaling complex exists on the majority of natural killer cells in the
absence of the
-subunit(10) . Therefore, the
physiologically functional IL-2R exists in several multimeric forms.
-subunit ectodomain
binds IL-2 with an affinity similar to the cell surface low affinity
site(11) . We have demonstrated that the
-subunit
ectodomain is also capable of binding IL-2 in solution(12) .
Although the
-subunit ectodomain binds IL-2 very weakly in
solution, our studies have demonstrated that it can function
cooperatively with the
-subunit ectodomain to form a stable
/
-ectodomain
IL-2 complex(13) .
ectodomain-coiled-coil fusion protein (19) and
demonstrated the stable formation of a homotrimeric ectodomain complex
(
cc) capable of binding ligand. In this report, we describe
the first solution assembly of a heteromeric IL-2R complex using
idealized coiled-coil recognition sequences. This trimeric
heterocomplex binds IL-2 with an affinity much greater than both the
individual subunit ectodomains or their homomeric complexes and in a
manner that indicates subunit cooperativity. In addition, this complex
binds IL-2 with an affinity similar to that reported for the pseudo
high affinity cell surface site.
Design of the Coiled-coil Sequences
The designs
of the coiled-coil heptads were based on previous studies of the
stability of peptide coiled-coils(16, 20) . Seven
repeats of the sequence LEALKEK were employed as the recognition
sequence for the coiled-coil-modified -subunit ectodomain
(IL-2R
cc), while seven repeats of LKALEKE were chosen
for the
-subunit ectodomain (IL-2R
cc). These sequences (Fig. 1) employed Leu residues at both the a and d positions of the heptad, with like-charged residues at the e and g positions of each helix in order to favor
heteromeric over homomeric association based on electrostatic
interactions(21) . In one version of the
-subunit
coiled-coil construct (IL-2R
cc
), a DNA
sequence encoding the peptide epitope tag EYMPME (13) was added
3` to the heptad repeat sequence to facilitate purification of the
protein.
Figure 1:
Helical wheel representations. A, the heterotrimeric association of the coiled-coil sequences
fused to the IL-2R and
ectodomains in the
cc
complex (arrows indicate potential electrostatic
interactions); B, the homotrimeric association of the
coiled-coil sequences fused to the IL-2R
ectodomain in the
cc complex.
Preparation of the Fusion Proteins
The cDNA for
the -subunit fusion protein possessing the LKALEKE heptad repeat
was constructed in pUC-19. The first double-stranded oligonucleotide
cassette encoding approximately one-half of the seven-heptad repeat
segment, a 5` NheI site and two DsaI sites for
directional cloning of the
-subunit ectodomain, was inserted at
the BamHI site in pUC-19. After verification of sequence and
orientation, the second double-stranded cassette completing the
coiled-coil cDNA was inserted between AflII and BstBI
sites of cassette I. The cDNA encoding the
-subunit ectodomain,
obtained as described previously(19) , was directionally
inserted between the two DsaI sites. The resulting
IL-2R
cc sequence was removed from pUC-19 (NheI and BamHI digestion) and subcloned into the pBlueBac II
baculovirus expression vector (Invitrogen) and verified by sequencing.
-subunit fused to the LEALKEK
coiled-coil repeat was constructed in pUC-8. The first cassette
encoding a 5` XbaI site in addition to the coiled-coil cDNA
was inserted at the BamHI site in pUC-8. The second cassette
with the remaining coiled-coil cDNA was inserted between XhoI
and AflII sites in the first cassette. The cDNA encoding the
-subunit ectodomain was directionally cloned between the BamHI and XbaI sites. The complete fusion protein
cDNA was then removed from pUC-8 (BamHI and BglII)
and subcloned into the BamHI site of the pBlueBac II
baculovirus expression vector (Invitrogen). The sequence and
orientation were verified by sequencing. High titer recombinant virus
was then prepared as described previously(19) . Trichoplusia
ni (High-Five
) insect cells were infected with either a
single virus or simultaneously co-infected with IL-2R
cc and
IL-2R
cc recombinant virus, and the cell-free culture
supernatants were collected 72 h postinfection.
cc was carried out by passing the harvested cell
supernatant over an epitope tag immunoaffinity column. The bound
fraction was eluted from the column with a solution of epitope peptide,
and the protein-containing fractions were pooled and dialyzed against
phosphate-buffered saline.
cc from the cell-free supernatant was performed
in a similar fashion using the tag epitope column. In order to obtain
the heterocomplex containing both receptor ectodomains, the bound
fraction eluted from the epitope immunoaffinity column (
-subunit
specific) was applied to an immunoaffinity column specific for the
-subunit ectodomain (TIC antibody,(12) ). The complex
containing both domains was eluted with 0.2 M HOAc, 0.2 M NaCl and dialyzed against phosphate-buffered saline.
Characterization of the Protein Complexes
The
composition of the purified complexes was analyzed by
SDS-polyacrylamide gel electrophoresis followed by Coomassie Blue
staining. The IL-2Rcc complex was also subjected to
automated N-terminal Edman sequencing. Ultracentrifugation analysis of
the complexes was performed at the centrifugation facility at the
University of New Hampshire on a Beckman XL-A analytical
ultracentrifuge equipped with Rayleigh interference optics (22) using the methods described previously for the
cc complex(19) . The partial specific volume for the
complexes was calculated assuming a protein molecular weight of 31,194
for the
-subunit (
0.73 ml/g) and 30,447 for the
-subunit (
0.74 ml/g) and a carbohydrate molecular weight
(
0.63 ml/g) of 12,200 g/mol and 8,400 g/mol for the
- and
-subunit, respectively (see Ref. 19). Sedimentation velocity was
conducted at 50,000 rpm, and the sedimentation coefficient
distributions (g(s*)) were determined from the time
derivative of the concentration profile as was described for the
cc complex(19) .
Competitive Radioligand Binding
Competitive
displacement of radiolabeled IL-2 from the low affinity IL-2R on the
human leukemic T-cell line MT-1(2) was carried out as described
previously(19) . The assay was performed at 37 °C under
conditions where the depletion of free I-IL-2 is
negligible. Thus, the fraction of maximum bound
I-IL-2 (b = c (1 + K*C*
)/(1 + cK*C*
), where C*
is the total concentration of
I-IL-2 (500
pM), C* is the concentration of
I-IL-2
in the presence of soluble receptor, c = C*/C*
, and K* is the equilibrium constant
for
I-IL-2 binding to the cell surface
-subunit on
MT-1 cells. In the nonlinear least-squares fits to the data, we took
the concentration of
I-IL-2 bound to soluble receptor to
be K
C*R
/(1 + K
C*), where R
is
the total concentration of soluble receptor, and K
is an equilibrium binding constant for the binding of
I-IL-2 to the soluble receptor. Since the total
concentration of
I-IL-2 remains unchanged, C*
= C* + K
C*R
/(1 + K
C*), can be solved for c, and c can then be substituted into the expression for b.
The following equation for total radioactivity bound, cpm, was
used to fit the data cpm = ns + (cpm(0) - ns)b, where ns is
the amount of nonspecifically bound
I-IL-2. In control
experiments where unlabeled IL-2 was used to compete
I-IL-2 (not shown), we determined that the K
for unlabeled binding to the
-subunit on
MT-1 cells was 2.0
10
M
(three separate experiments). When fitting the soluble receptor
data, we took K* equal to this value. Estimates of the
standard errors of K
were obtained using a
bootstrap method (23) where 150 simulations were performed for
each estimate.
Neutralization of IL-2 Bioactivity
The ability of
the complexes to neutralize IL-2 bioactivity was determined as
described previously (8) but in the presence of an EC (10 pM) IL-2. The assay was carried out on normal human
peripheral blood lymphocytes, the IL-2-dependent human T-cell line Kit
225(13) , or the murine IL-2-dependent T-cell line
CTLL-2(24) .
cc and
cc fusion protein complexes in insect cells were
comparable with those previously observed for the expression of the
individual IL-2R ectodomains (12, 13) and for the
cc homomeric complex(19) . The initial attempts at
co-expression of both
and
- receptor subunit fusion proteins
were performed employing IL-2R
cc
virus.
Therefore, preliminary isolation was carried out by passing the
harvested supernatant over an epitope tag immunoaffinity column. The
bound fraction was recovered and tested for its ability to inhibit IL-2
activity in a competitive bioassay(13) . In preliminary
experiments (data not shown), this preparation was 50-100-fold
more potent than a complex of IL-2R
cc
alone
(prepared and isolated in a similar fashion). Although not designed to
be quantitative, these experiments suggested the presence of a mixed
subunit complex with greatly increased binding affinity.
-subunit fusion
protein, it was possible that this fraction also contained homomeric
cc complex. In order to isolate the
cc
heterocomplex from this mixture, the fraction that bound to the tag
affinity column was applied to an immunoaffinity column specific for
the
-subunit ectodomain (TIC antibody(12) ).
SDS-polyacrylamide gel electrophoresis analysis of the fraction bound
to this second column confirmed enrichment of
-subunit fusion
protein when compared with the starting mixture or column flow-through (Fig. 2).
Figure 2:
Gel electrophoresis. SDS-polyacrylamide
gel electrophoresis analysis of immunoaffinity purification of the
cc complex (Coomassie Blue-stained). Lane 1,
the fraction of co-infection cell supernatant bound to the
ectodomain specific tag epitope affinity column; lane 2, the
flow-through fraction obtained when the tag epitope bound sample (lane1) was applied to a
ectodomain-specific
immunoaffinity column; lane 3, the fraction bound to the
ectodomain specific immunoaffinity column, the
cc
complex. Acc and Bcc mark the positions of the
- and
-subunit
coiled-coil fusion proteins, respectively.
Automated N-terminal Edman sequencing of this
product matched the published amino acid sequence of both and
receptor subunits. The results of the first seven cycles for the
-subunit were Glu, 62 pM; Leu, 51 pM; Xaa, 0.7
pM; Asp, 41 pM, Xaa, not quantifiable; Asp, 0.4
pM; Pro, 43 pM, and the results
-subunit were
Ala, 39 pM; Val, 27 pM; Xaa, 0.4 pM; Gly, 20
pM; Thr, 15 pM; Xaa, not quantifiable; Gln, 20
pM. The
/
ratio of cycles 1, 2, 4, and 7 are 1.6,
1.9, 2.1, and 2.2, respectively (cycles 3, 5, and 6 occur at unstable
or glycosylated positions in one or both subunits). Therefore,
N-terminal sequence analysis of the
cc complex revealed
a 2:1 stoichiometry of
-subunit to
-subunit ectodomain. This
stoichiometry was consistent with subsequent characterization of ligand
binding via surface plasmon resonance(35) .
= 0.2 µg/ml)
than the initial tag column-bound fraction (IC
=
0.4 µg/ml) and 3-fold more potent than the TIC column flow-through
fraction (IC
= 0.6 µg/ml). This heteromeric
complex was the most potent ectodomain-related inhibitor ever tested in
the competitive bioassay. Therefore, we carried out a quantitative
ligand binding study to determine the solution affinity. In order to
exclude any potential interference by the tag epitope sequence, we
prepared another
-ectodomain/coiled-coil fusion protein lacking
this tag sequence and isolated the heteromeric complex by sequential
affinity columns employing monoclonal antibodies specific for each
ectodomain (
, TAC(25) ;
, TIC(12) ).
cc heterocomplex, a
complex composed of the
-subunit alone (
cc) and a
mixture of uncomplexed
- and
-ectodomains, to compete with
the low affinity cell surface receptor for IL-2 binding on the MT-1
cell line (2) (Fig. 3). The results of the
competitive binding assay clearly demonstrate that the
cc complex is a much more efficient competitor than the
other two preparations. Since this complex is a multimer of subunits,
each of which is potentially capable of binding ligand independently,
it was important to distinguish cooperative binding from the potential
effects of avidity due to simple multivalency. To quantify the
competition assays and determine if binding was cooperative, we
calculated an effective dissociation constant for the complex (K
) from the equilibrium binding
constants (K
=
1/K
) listed in . If the three
subunits of the complex act independently, then K
= 3/(2K
+ K
), where K
and K
are the equilibrium
constants for the binding of labeled IL-2 to the
and
ectodomains, respectively. If the binding is positively cooperative,
then the K
will be smaller than this
value, while if the binding is negatively cooperative, it will be
greater than this value. We estimate () that for
noncooperative, independent binding, K
76 nM. Nonlinear least square analysis of the
competitive binding data (Fig. 3) yields a K
= 0.32 nM for the heteromeric
cc complex, a K
=
15 nM for the
cc complex, and a K
= 42 nM for the mixture
of individual ectodomains. Previously, we determined that for the
cc trimeric complex, K
= 300 nM per
-ectodomain or K
100 nM for the
complex(19) . Therefore, the results in indicate
that ligand binding for the
cc complex is highly
cooperative. In addition, this complex is an efficient competitor at
concentrations below 1 nM, where occupancy of the third
-ectodomain is insignificant(35) . The data also suggest
that binding of the homomeric
cc complex is consistent with
the three
-ectodomains binding ligand independently and
noncooperatively (as was previously observed for the
cc
complex). For the mixture of
and
ectodomains, there is no
detectable ectodomain association in the absence of the recognition
sequences. This is unlike the ligand-induced association of the
and
ectodomains in solution(13) . Since the full-length
/
pseudo high affinity site exists preformed on the surface
of cells, it is likely that transmembrane or cytoplasmic contact
contributes to the stability of the complex.
Figure 3:
Radioligand binding. Competitive
displacement of I-IL-2 on MT-1 cells. Total bound
radioactivity is plotted versus concentration of soluble
receptor for the
cc complex (
), the
cc
complex (
), and an equimolar mixture of
and
ectodomains (
). Maximum cpm bound in the absence of competitor
(2085 ± 47 cpm) was obtained from the average of six
determinations. The assay was performed at 37 °C under conditions
where the depletion of free
I-IL-2 is negligible. Thus,
the fraction of maximum bound
I-IL-2, b = c (1 + K*C*
)/(1 + cK*C*
), where C*
is the total
concentration of
I-IL-2 (500 pM), C* is
the concentration of
I-IL-2 in the presence of soluble
receptor, c = C*/C*
, and K* is the equilibrium constant for
I-IL-2 binding to
the cell surface
-subunit on MT-1 cells. In the nonlinear
least-squares fits to the data (solidcurves), we
took the concentration of
I-IL-2 bound to soluble
receptor to be K
C* R
/(1 + K
C*), where R
is
the total concentration of soluble receptor (x axis) and K
is an equilibrium binding constant for the
binding of
I-IL-2 to the soluble receptor. Since the
total concentration of
I-IL-2 remains unchanged, C*
= C* +K
C*R
/(1 + K
C*). The following equation for total
radioactivity bound, cpm, was used to fit the data: cpm = ns + (cpm (0) - ns)b, where ns is the amount of
nonspecifically bound
I-IL-2 (410 cpm, visible as
noncompetable cpm in figure). In control experiments where
unlabeled IL-2 was used to compete
I-IL-2 (not shown), we
determined that the K
for unlabeled binding to
the
-subunit on MT-1 cells was 2.0
10
M
(three separate experiments). When
fitting the soluble receptor data, we took K* equal to this
value.
The efficiencies of
these complexes to inhibit IL-2 bioactivity in a human peripheral blood
lymphocyte bioassay (8) were also compared (Fig. 4). The IC value obtained for the
cc complex (
0.9 nM) was slightly greater
than its K
, (0.32 nM) while the
IC
value obtained for the
cc trimer (
100
nM) was significantly greater than its K
(15 nM). The
cc homomeric complex did
not compete in this assay at concentrations in excess of 1 µM (data not shown). Similar assays (not shown) were performed on the
murine CTLL-2 bioassay (24) where the IC
values
were 2 and 500 nM for the
cc and
cc
complexes, respectively. The rapid dissociation rate from the
-
and
-subunits (2, 26) compared with the slower
dissociation rate from the
/
- pseudo high affinity site (26, 35) may account for the poor efficiency of
competition for the homomeric complexes during the 24-h bioassay. The
100-fold increase in potency for the heteromeric complex compared with
the homomeric
cc complex provides further support for the
cooperative nature of ligand binding.
Figure 4:
Competitive bioassays. Neutralization of
IL-2 bioactivity by the cc complex (
) and the
cc complex (
) in the human peripheral blood
lymphocyte bioassay. The assay was performed as described (8) in the
presence of an EC
of IL-2 (10 pM). Each data
point is the mean of triplicate determinations with standard deviation
indicated. Maximum response to 10 pM IL-2 was 6055 cpm with a
background response of 88 cpm in wells where no IL-2 was
added.
In the previous study of the
IL-2Rcc complex, the molecular weight of the trimer complex
was determined by analytical ultracentrifugation(19) .
Therefore, we analyzed the
cc complex using both
equilibrium sedimentation and sedimentation velocity techniques.
Equilibrium sedimentation studies were consistent with a trimeric
complex () with no significant evidence of monomer
at any of the concentrations studied. Comparison with the
cc
complex previously examined (19) indicates that the heteromeric
complex is at least as stable as the IL-2R
-homotrimer.
Sedimentation velocity experiments were consistent with sedimentation
equilibrium results (). At concentrations below
0.25 mg/ml, the major component migrated at s
of 4.6-4.8. At 0.52
mg/ml, the major peak was at 4.3 s, with a shoulder extending
past 5 s (Fig. 5). All curves provide some evidence for
small amounts of both smaller and larger species, but there is no
evidence for either mass action association or dissociation. The
decrease in s with increasing concentration is expected for a
noninteracting asymmetric molecule(27) . The heterocomplex
migrates as an extremely asymmetric molecule; assuming the complex
behaves as a hydrated (0.4 H
O/g of protein) prolate
ellipsoid, the axial ratio is
15. This is consistent with the
expected structural nature of the complex being globular ectodomains
fused to an extended coiled-coil stalk. The observed formation of
trimers using the designs depicted in Fig. 1is in accord
with recent studies confirming that heptads containing Leu residues at
both the a and d positions favor the formation of
trimeric complexes(28, 29, 30) .
Figure 5:
Sedimentation velocity analysis.
Sedimentation coefficient distribution (19) of the cc
complex at a loading concentration of 0.52 mg/ml (--), 0.25
mg/ml (- - -) and 0.08 mg/ml
(
). Aggregated material is noted from
8-12 s at 0.52 mg/ml. Trailing edge from 1-2 s for all
loading concentrations is indicative of some smaller material.
Integration of the major peak at each loading concentration indicates
that the trimeric complex represents 80-95% of migrating
species.
In this
study, we have employed coiled-coil heptad repeats as recognition
elements to mediate the solution assembly of receptor ectodomains that
normally function cooperatively in cell surface ligand
capture(8) . We employed seven coiled-coil heptads in order to
achieve stability at submicromolar monomer concentrations. The results
of the competitive ligand binding assays confirm that the stability of
the cc complex extends into the subnanomolar range
since significant competition is observable at these concentrations.
The effectiveness of the use of like-charged electrostatic residues at
positions e and g of the heptad repeat to favor
heteromeric over homomeric association is uncertain. The
cc
trimeric complex in which each monomer carried like-charged residues at
these positions was also stable. Although electrostatic interactions
can direct heteromeric coiled-coil association(21, 31) ,
the contribution of electrostatic interactions is less significant than
hydrophobic interactions to the overall stability of coiled-coil
complexes. High resolution studies of triple stranded coiled-coil
peptides suggest that like-charged side chains have sufficient
flexibility to avoid close approach(28) .
-
and
-IL-2R subunits in the
cc complex is evident
from the 45- and 300-fold increases in affinity when compared with the
homomeric complexes of
- and
-subunits, respectively. How
closely the
cc complex mimics ligand binding by the
cell surface
-pseudo high affinity site is difficult to
determine. Values ranging between 100 and 600 pM are often
reported for the
-receptor when the individual subunits are
transfected into nonlymphoid cell
lines(26, 33, 34) . The reason for this
variation may be due to cell type or the ratio of subunits expressed,
nevertheless, the K
value for our
cc complex is within the range of these reported
values. Although the stoichiometry of the soluble heterocomplex is not
1:1, neither is the ratio of
to
IL-2 receptor subunits on
the surface of most cells (2, 10, 33). It is highly likely that ligand
binding by the soluble
cc complex resembles, at least
in part, the functional form of the cell surface pseudo high affinity
site. Subsequent study of both equilibrium and kinetic ligand binding
characteristics of this complex employing surface plasmon resonance
support this conclusion (35). (
)
Table: Equilibrium binding constants
) determined from
the competitive radioligand binding data shown in Fig. 3 as described
in ``Materials and Methods.''
Table: Analytical ultracentrifugation
cc IL-2R complex
is the apparent z-averaged molecular weight (19).
Values in parentheses indicate the 95% confidence interval. r.m.s. is
root mean square of variance of fit to a model consisting of a single
ideal thermodynamic component. s
is the sedimentation coefficient determined from the time
derivative of the concentration profile as previously described (19).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.