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Direct observation of ligand-induced receptor
dimerization with a bioresponsive hydrogel†
Jongseong Kim,*
a
Yongdoo Park,
b
Ashley C. Brown
c
and L. Andrew Lyon
d
Multimerization of biomolecules is essential for biological function
and thus there is a need for sensitive biochemical assays that deter-
mine whether a molecule associates with one or more other mole-
cules in the context of biological function. In this contribution we
demonstrate a simple yet versatile method for the identification of
physiologically important receptor dimerization events induced by a
ligand. Bioresponsive hydrogel microparticles (microgels) conjugated
with a receptor, Glycoprotein Iba(GPIba), display large changes in
optical (microscopic) appearance under conditions known for to
promote thrombin-induced GPIbadimerization. In support of X-ray
crystal structures, we identify that one thrombin molecule associates
with two GPIbamoieties, which may play a role in efficient hemostatic
function by increasing local concentration of GPIbaon platelet
surfaces. This microgel assay could provide a new way of studying
important physiological and pathological mechanisms related to
receptor dimerization and/or clustering.
Characterization of how receptors interact with each other and
with their native ligands is a long-standing goal of biochemical
assay development.
1–4
Receptor multimerization (or clustering)
that occurs independent of or is induced by ligand binding acts
as a crucial step not only for normal physiological function,
5
but
also in the progression of pathological infections such as those
caused by human immunodeciency virus (HIV),
6,7
and inu-
enza virus.
8,9
However, technology for directly identifying such
clustering is limited by the capability of distinguishing multi-
valent (two or more receptors with one ligand) interactions from
monovalent (one receptor with one ligand) interactions. One
common technique is isothermal titration calorimetry (ITC)
that quanties thermodynamic parameters of molecular inter-
actions in solution. However, this specialized instrumental
approach can be technically challenging and consumes rela-
tively large amounts of puried protein. Here we show a simple
but unique microgel-based assay for the characterization of
ligand-induced receptor dimerization in real time.
Thrombin is an enzyme that mediates the cleavage of bri-
nopeptides from brinogen, thereby initiating the assembly of
brinogen into brin. This is an integral component of both
hemostasis and thrombosis. In addition, thrombin binds to
platelet glycoprotein Iba(GPIba) with both high and interme-
diate affinity sites.
12
X-ray crystal structures of the thrombin–
GPIbacomplex showed that both molecules have two distinct
binding sites for its counterpart, which suggests multivalent
binding of the two proteins simultaneously (Fig. 1a).
10,11
GPIba
is a membrane protein of platelets and has an important role in
blood coagulation at the site of vascular injury through its
Fig. 1 Schematic of ligand-induced receptor dimerization assay using
microgels. (a) X-ray structure of a-thrombin–GPIbacomplex showing
multivalent binding.
10,11
(b) Conjugation of GPIbato hydrogel particle.
(c) Thrombin-induced GPIbadimerization.
a
Department of Chemistry, Eberly College of Science, Pennsylvania State University,
University Park, Pennsylvania, 16802, USA. E-mail: jxk90@psu.edu
b
Department of Biomedical Engineering, College of Medicine, Korea University, Seoul,
136-705, Korea
c
School of Chemistry and Biochemistry and the Petit Institute for Bioengineering and
Bioscience, Georgia Institute of Technology, Atlanta, 30332-0400, Georgia
d
Schmid College of Science and Technology, Chemistry, Chapman University, Orange,
California, 92866, USA
†Electronic supplementary information (ESI) available: Details of microgel
synthesis, protein expression and purication, fabrication of microgel assay.
See DOI: 10.1039/c4ra13251c
Cite this: RSC Adv.,2014,4,65173
Received 27th October 2014
Accepted 20th November 2014
DOI: 10.1039/c4ra13251c
www.rsc.org/advances
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binding to von Willebrand factor (vWF) A1 domain. For the last
few decades, the functional signicance of thrombin binding to
GPIbahas remained controversial, with some question as to
whether it is prothrombotic or antithrombotic. Nonetheless,
both hypotheses suggest that a-thrombin induces dimerization
of platelet GPIbaby cooperating with the exocite I and II.
10,11
To interrogate thrombin-induced GPIbadimerization, we
utilized bioresponsive microgels. Microgels are hydrogel
microparticles, which are water swellable polymeric networks
with high water content.
13,14
Microgels with the composition
poly(N-isopropylacrylamide-co-acrylic acid) (pNIPAm-co-AAc)
were prepared through aqueous free-radical precipitation
polymerization (details in ESI†). Receptor, GPIba, was tran-
siently expressed in mammalian cells (HEK293T) and then
puried using Ni–NTA affinity chromatography followed by
size-exclusion chromatography (see ESI†). Functionalization of
microgels with GPIbaor thrombin allows for direct observation
of the multivalent interaction between thrombin and GPIba
(Fig. 1b). The microgels were then attached on an amino-
prophytrimethoxysilane (APTMS)-functionalized glass substrate
via Coulombic interactions.
14
The optical microscopy image of
the assembled microgels is tuned by multivalent protein
binding, which is characterized by formation of a dark ring
(Fig. 1c). This phenomenon has been observed previously for
simple model systems, which is the response of biotin-
functionalized microgels to avidin and anti-biotin.
13,14
Despite
the lackage of the physiological signicance, we were successful
in demonstrating that the increase of local crosslink density in
microgels due to multivalent binding causes change in the
refractive index of the microgel rim followed by dark ring
formation in the microscopy image.
The behavior of the microgels in different concentrations of
a-thrombin and GPIbais shown in Fig. 2. Under these condi-
tions, both the GPIba- and the thrombin-functionalized
microgels showed dark ring formation above a critical concen-
tration of the thrombin (Fig. 2a) and GPIba(Fig. 2b), respec-
tively. The critical concentrations of two proteins indicate that
overall multivalent binding energy between thrombin and
GPIbais sufficient to cause deswelling of the microgel
periphery. Thus, the elastic restoring force of microgel network
is overwhelmed by the strength of the multivalent thrombin–
GPIbainteractions. These results indicate that thrombin can
induce GPIbaclustering, which may have relevance for the
receptor's behavior on platelet membrane surfaces. In light of
the results, we hypothesize that such clustering increases a local
concentration of GPIba, which would be favorable for interac-
tions with vWF in the hemostatic function of platelets.
The dimerization assay was further tested by using two kinds
of specic antibodies for GPIbathat are 6D1 IgG for the N-
terminus region (Fig. 3a) and anti-His IgG for the C-terminus
6His-tag (Fig. 3b). The GPIba-functionalized microgels dis-
played dark ring formation upon incubation with the both
antibodies above the critical concentrations required for
microgel shidue to multivalent binding.
13,14
However, the
microgels are insensitive to monovalent binding of vWF A1
domain to GPIbaup to 5 mM(K
d
!30 nM (ref. 15)). Under this
condition, monovalent interactions are incapable of changing
the optical properties of the microgel. Note that the local
deswelling of the water-swollen hydrogel is efficiently caused by
the formation of cross-links at the gel surface due to dimer-
ization, but in monovalent binding, the elastic restoring force of
the gel-network exceeds the free energy change accompanied by
the complex formation. Thus, dark ring formation is exclusively
induced by increasing the crosslink density and concomitant
deswelling of the microgel periphery.
To develop a biochemical assay system, the capability of
responding only in positive signals and operating in rigorous
and physiological condition is a fundamental requirement for
general application. Therefore, we examined the response of
GPIba-functionalized hydrogel particles under various concen-
trations of bovine serum albumin (BSA) (Fig. 4a) and sodium
chloride (NaCl) (Fig. 4b). BSA is used not only for preserving
many kinds of puried proteins but also for decreasing
nonspecic interaction in most biochemical assays. Our results
show that the dimerization signal was not observed until 10 mg
ml
"1
of BSA in PBS buffer (!10 times higher than a normal dose
for preserving proteins). Thus, the nonspecic interaction
between BSA and the microgel network is incapable of causing
noticeable dimerization signal under our experimental
Fig. 2 Response of bioresponsive microgels to ligand-induced
receptor dimerization. DIC microscopy images of a GPIba-function-
alized microgel (a) and an a-thrombin-functionalized microgel (b) at
the indicated concentrations of a-thrombin and GPIba, respectively.
Above a critical concentration, the microgels show modulation of the
images through dark ring formations. The scale bar is 2 mm.
Fig. 3 Response of bioresponsive microgels to the presence of anti-
body and vWF A1 domain. DIC images of a GPIba-functionalized
hydrogel at the indicated concentrations of mAb 6D1 for GPIba(a),
mAb for His-tag in GPIba(b) and vWF A1 domain (c) in PBS buffer.
Only the antibodies which bind to GPIbacause the change in
optical properties of microgels above a critical concentration, and not
vWF A1 domain that binds to GPIbain monovalent manner. The scale
bar is 2 mm.
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conditions. We hypothesize that the BSA either interacts with
the hydrogel network in monovalent manner or the bond
strength, in case of multivalent binding, is weaker than the
restoring force of hydrogel network.
We have also observed the reliability of the microgel assay in
physiological salt concentration (!150 mM including our
dimerization assay in PBS buffer) and even in more rigorous
condition (300 mM of NaCl). Indeed, freshly puried recombi-
nant proteins oen include !300 mM salts, requiring dialysis.
Thus, our microgel assay could be used for such proteins even
without desalting step. It is also worthwhile to note that all the
assays have been done in incubation with only 6 ml of protein
solution, which makes the method highly desired for most in
vitro biochemical assay. We note that the formation of a dark
ring for GPIba-functionalized microgels was observed in 0.6 M
and 1 M NaCl. These data suggest that extremely high salt
conditions are required to induce deswelling of the microgel,
which could be explained by both charge shielding effect of AAc
group in the microgel followed by decreasing the Columbic
repulsion and osmotic pressure due to higher solute
concentration.
16
In conclusion, we show the development of a new
biochemical assay that enables the observation of ligand-
induced receptor dimerization. The utility of the microgel
assay was highlighted by its ability to respond only to multiva-
lent binding, not for monovalent and nonspecic binding,
under physiological salt concentrations. This novel but simple
methodology, in which the responsive microgels are conjugated
with biomolecules, will allow for unique and powerful
biochemical assay in real-time measurement of protein multi-
merization. Furthermore, the method described here could be
improved by combination with uorescence microscopy, which
provides uorescence signals for both monovalent and multi-
valent bindings, allowing for detection of both but discrimi-
nation between the two conditions. All of these features make
the microgel assay attractive for future applications in identi-
fying biological pathway that is coupled to protein assembly,
multimerization, and disassembly.
Acknowledgements
JK and YP acknowledge support from National Research
Foundation of Korea (NRF-2013S1A2A2035518). LAL acknowl-
edges support from Georgia Tech. ACB acknowledges support
from American Heart Association postdoctoral fellowship.
Notes and references
1 A. Brown, C. J. Robinson, J. T. Gallagher and T. L. Blundell,
Biophys. J., 2013, 104, 1720.
2 Y. Wu, B. Honig and A. Ben-Shaul, Biophys. J., 2013, 104,
1221.
3 C. Matsushita, H. Tamagaki, Y. Miyazawa, S. Aimoto,
S. O. Smith and T. Sato, Proc. Natl. Acad. Sci. U. S. A., 2013,
110, 1646.
4 A. Arachiche, M. M. Mumaw, M. de la Fuente and
M. T. Nieman, J. Biol. Chem., 2013, 288, 32553.
5 H. Tanji, U. Ohto, T. Shibata, K. Miyake and T. Shimizu,
Science, 2013, 339, 1426.
6 D. Lyumkis, J. P. Julien, N. de Val, A. Cupo, C. S. Potter,
P. J. Klasse, D. R. Burton, R. W. Sanders, J. P. Moore,
B. Carragher, I. A. Wilson and A. B. Ward, Science, 2013,
342, 1484.
7 A. Bartesaghi, A. Merk, M. J. Borgnia, J. L. Milne and
S. Subramaniam, Nat. Struct. Mol. Biol., 2013, 20, 1352.
8 S. E. Swalley, B. M. Baker, L. J. Calder, S. C. Harrison,
J. J. Skehel and D. C. Wiley, Biochemistry, 2004, 43,
5902.
9 T. Ivanovic, J. L. Choi, S. P. Whelan, A. M. van Oijen and
S. C. Harrison, Elife, 2013, 2, e00333.
10 J. J. Dumas, R. Kumar, J. Seehra, W. S. Somers and
L. Mosyak, Science, 2003, 301, 222.
11 R. Celikel, R. A. McClintock, J. R. Roberts,
G. L. Mendolicchio, J. Ware, K. I. Varughese and
Z. M. Ruggeri, Science, 2003, 301, 218.
12 A. Zarpellon, R. Celikel, J. R. Roberts, R. A. McClintock,
G. L. Mendolicchio, K. L. Moore, H. Jing, K. I. Varughese
and Z. M. Ruggeri, Proc. Natl. Acad. Sci. U. S. A., 2011, 108,
8628.
13 J. Kim, N. Singh and L. A. Lyon, Angew. Chem., Int. Ed. Engl.,
2006, 45, 1446.
14 J. Kim, S. Nayak and L. A. Lyon, J. Am. Chem. Soc., 2005, 127,
9588.
15 E. G. Huizinga, S. Tsuji, R. A. Romijn, M. E. Schiphorst,
P. G. de Groot, J. J. Sixma and P. Gros, Science, 2002, 297,
1176.
16 T. Lopez-Leon and A. Fernandez-Nieves, Phys. Rev. E: Stat.,
Nonlinear, SoMatter Phys., 2007, 75, 011801.
Fig. 4 Response of microgels to nonspecific interaction and salt
concentration. DIC images of a GPIba-functionalized hydrogel at the
indicated concentrations of BSA in PBS (top row) and NaCl in 50 mM
Tris pH 7.5 buffer (bottom row). The scale bar is 2 mm.
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