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J. Biol. Chem., Vol. 275, Issue 32, 24540-24546, August 11, 2000
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From the
Received for publication, March 24, 2000, and in revised form, May 22, 2000
Three binary protein-protein interactions,
glycophorin C (GPC)-4.1R, GPC-p55, and p55-4.1R, constitute the
GPC-4.1R-p55 ternary complex in the erythrocyte membrane. Little
is known regarding the molecular basis for the interaction of 4.1R with
either GPC or p55 and regarding the role of 4.1R in regulating the
various protein-protein interactions that constitute the GPC-4.1R-p55 ternary complex. In the present study, we present evidence that sequences in the 30-kDa domain encoded by exon 8 and exon 10 of 4.1R
constitute the binding interfaces for GPC and p55, respectively. We
further show that 4.1R increases the affinity of p55 binding to GPC by
an order of magnitude, implying that 4.1R modulates the interaction
between p55 and GPC. Finally, we document that binding of calmodulin to
4.1R decreases the affinity of 4.1R interactions with both p55 and GPC
in a Ca2+-dependent manner, implying that
the GPC-4.1R-p55 ternary protein complex can undergo dynamic regulation
in the erythrocyte membrane. Taken together, these findings have
enabled us to identify an important role for 4.1R in regulating the
GPC-4.1R-p55 ternary complex in the erythrocyte membrane.
Protein 4.1R (4.1R)1 in
red blood cells has multiple binding sites for transmembrane and
membrane skeletal proteins and plays a critical role in maintaining
cell morphology and membrane mechanical properties (1, 2). The
elasticity and mechanical stability of the erythrocyte membrane is
regulated by 4.1R interaction with the spectrin-actin network and with
membrane proteins such as band 3, glycophorin C (GPC), and p55, a
palmitoylated membrane protein containing a single PDZ domain (3)
belonging to the membrane-associated guanylate kinase
family (for a review, see Ref. 4). Hereditary defects in 4.1R result in
abnormally shaped erythrocytes with decreased membrane mechanical
stability manifested clinically as hemolytic anemia (1, 2, 5).
Three main structural/functional domains have been identified in 4.1R.
A 30-kDa N-terminal membrane binding domain possesses binding sites for
the cytoplasmic tails of integral membrane proteins such as band 3, GPC, and CD44 (1, 6-8). This domain also binds to p55 (9) and
calmodulin (CaM) (10-12). A 10-kDa internal domain contains the
critical spectrin-actin binding activity required for membrane
mechanical stability (1), while the C-terminal 22-24-kDa domain has
recently been reported to bind the immunophilin FKBP13 (13) and NuMA
(14).
Two ternary protein complexes involving 4.1R have been identified in
the erythrocyte membrane: spectrin-actin-4.1R (1) and GPC-4.1R-p55 (4).
The spectrin-actin-4.1R ternary complex plays a critical role in
imparting mechanical stability to the erythrocyte membrane. Binding of
CaM to 4.1R decreases the affinity of its interaction with purified
spectrin and actin in a Ca2+-dependent manner
in vitro (15) and destabilizes erythrocyte membrane
mechanical integrity (16). These data imply an important regulatory
role for 4.1R in the function of the spectrin-actin-4.1R ternary
complex. In the GPC-4.1R-p55 ternary complex, Marfatia et
al. (17, 18) have shown that the PDZ domain of p55 interacts with
the C-terminal YFI tripeptide of the cytoplasmic domain of GPC (GPC-1)
and that the D5 region of p55 interacts with the 30-kDa domain of 4.1R.
In addition to its interaction with p55, the 30-kDa domain of 4.1R also
interacts with a membrane-proximal basic peptide (RHK) in the
cytoplasmic domain of GPC (GPC-3) (7, 17). Thus, three binary
protein-protein interactions, GPC-4.1R, GPC-p55, and 4.1R-p55
constitute the GPC-p55-4.1R ternary complex. At present, little is
known regarding the regulation of the various protein-protein interactions that constitute the GPC-4.1R-p55 ternary complex.
We performed a detailed molecular and functional characterization of
the role of 4.1R in regulating the GPC-4.1R-p55 ternary complex. We
identified that sequences in the 30-kDa domain of 4.1R encoded by exon
8 and exon 10 constitute the binding motifs for GPC and p55,
respectively. We showed that 4.1R binding to p55 increases the affinity
of p55 binding to GPC by an order of magnitude, implying that 4.1R
modulates the interaction between p55 and GPC. Finally, we documented
that binding of CaM to 4.1R decreases the affinity of its interaction
with both p55 and GPC in a Ca2+-dependent
manner, implying that this ternary protein complex can undergo dynamic
regulation in the membrane. These findings demonstrate an important
role for 4.1R in regulating the GPC-4.1R-p55 ternary complex.
Materials
Glutathione-Sepharose 4B was purchased from Amersham Pharmacia
Biotech. Fmoc-amino acids and Fmoc-amino acid-coupled resins were
obtained from PerSeptive Biosystems, Co. Ltd. (Boston, MA). IAsysTM cuvettes coated with aminosilane were supplied by
Affinity Sensors (Cambridge, UK). Bovine serum albumin was purchased
from Sigma. pGEX-KG vector was a gift from Dr. J. E. Dixon (19).
pET30a vector and nickel resin were purchased from Novagen (Madison, WI).
Methods
Preparation of CaM and 4.1R--
Purification of CaM, 4.1R, the
N-terminal 30-kDa domain, and the other chymotryptic fragments of 4.1R
(16-kDa, 10-kDa, and C-terminal domains) were carried out according to
previously published reports (12, 20-22).
Preparation of Recombinant Proteins--
Recombinant proteins
corresponding to the 30-kDa domain of 4.1R (r30kDa) and its mutants
(see Fig. 1) (23, 24), the cytoplasmic domain of GPC (GPCcyt) (9), and
the 4.1R-binding D5 domain of p55 (D5) (17, 25) were produced as
glutathione S-transferase fusion proteins in
Escherichia coli. The minigenes were constructed in modified
pGEX-KG vector (19). The expression, purification, and cleavage of
fusion proteins were previously described (8, 12). The cleaved
glutathione S-transferase was removed using glutathione-Sepharose 4B. Human erythrocyte p55 cDNA was cloned into pET30a vector. His-tagged p55 was purified using a nickel column.
The purity of the recombinant proteins was assessed by analysis with
SDS-polyacrylamide gel electrophoresis (15% gel). The protein sequence
of recombinant GPC cytoplasmic tail was verified by determining the
amino acid sequence of the recombinant protein (amino acid sequencer,
Shimadzu, Kyoto, Japan). The protein concentrations were determined
according to the following equation: protein concentration (mg/ml) = 1.45 A280 Synthesis of Peptides--
Peptide corresponding to the internal
sequence of the 30-kDa domain of protein 4.1R (24), 33 amino acids in
the C-terminal part of exon 10 (214YKDKLRINRFPWPKVLKISYKRSSFFIKIRPGE, pep10), and the
different domains of the cytoplasmic tail of GPC (GPC-1,
112DPALQDAGDSSRKEYFI; GPC-2, 99TEFAESADAALQG;
and GPC-3, 82RYMYRHKGTYHTNDAKG) (see Ref. 7) were
chemically synthesized using the peptide synthesizer (PerSeptive
Biosystems 9050 plus, Boston, MA) according to the Fmoc method (26).
The synthesized peptides were purified by reverse phase high pressure
liquid chromatography. The molecular mass of the different
synthetic peptides was determined by mass spectrometry (matrix-assisted
laser desorption/ionization time-of-flight mass spectrometry; Shimadzu,
Kyoto, Japan) (27, 28) and compared with the theoretically calculated
mass to validate the chemical composition of the synthesized peptides.
The hydrophobicity of the peptides was derived using the Chou and
Fassman algorithm (29, 30) with the software package DNASISTM
(Hitachi, Tokyo, Japan).
Binding Assay by Resonant Mirror Detection--
Protein-protein
interactions and protein-peptide interactions were studied using the
resonant mirror detection method (31-33) of the IAsysTM system
(Affinity Sensors, Cambridge, UK). In the present study, the
immobilized protein or peptide on the cuvette is referred to as the
"ligand," and the protein in the solution added to the cuvette as
the "analyte." The immobilization of proteins or peptides to the
aminosilane cuvette has been previously described (8, 12). All of the
binding assays were carried out in phosphate-buffered saline (0.15 M NaCl, 10 mM
Na2HPO4/NaH2PO4, pH
7.4) at 25 °C with constant stirring. To analyze the binding of the
p55-r30kDa complex to GPCcyt immobilized on the cuvette, equal amounts
of p55 and r30kDa (0.1-1.0 µm) were preincubated at 25 °C for 30 min. To quantitate the effects of Ca2+ and CaM on the
binding of various r30kDa proteins to transmembrane proteins and p55
immobilized on the surface of the cuvette, r30kDa proteins (50 nM to 1 µM) were preincubated with 5 µM CaM in buffer A (20 mM imidazole HCl, pH
7.2, 0.1 M NaCl) and either 0.1 mM EGTA or 1.1 mM CaCl2 and 1.0 mM EGTA at
25 °C for 30 min, and the binding assays were performed using the
same buffers (8, 12).
The procedures employed for kinetic analysis of the binding
interactions and for the quantitation of the stoichiometry of analyte
binding to ligand have been previously described (8, 12). The
dissociation constant from this form of kinetic analysis (termed
"K(D)kin") is calculated
as follows: K(D)kin = kd/ka, where
ka is the association rate constant and
kd is the dissociation rate constant (8, 12). The
stoichiometry of analyte binding to ligand was calculated according to
the following equation: stoichiometry of analyte/ligand = (Bmax of analyte/molecular weight of
analyte)/(amount of immobilized ligand on the cuvette/molecular weight
of ligand) (12). The molecular weight values used in the calculations
for 4.1R, r30kDa, p55, and GPCcyt were 80,000, 35,500, 55,000, and 5000, respectively.
The cuvettes were reused after cleaning with HCl. Original binding
curves could be replicated after HCl washes, implying that the washing
procedure used did not denature the immobilized ligands.
Kinetic Analysis of 4.1R-p55, GPC-4.1R, and GPC-p55 Binary
Protein-Protein Interactions--
Following the addition of native
4.1R to p55 immobilized on the cuvette, the kinetics of binding of 4.1R
to p55 was quantitated by the resonant mirror detection system as an
increase in arc seconds as a function of time (Fig. 2A).
After the binding response reached a plateau value, the addition of
buffer solution without 4.1R resulted in dissociation of bound 4.1R
from p55 and a consequent decrease in the binding response signal. The
new plateau value was significantly higher than the initial base-line
value, implying a specific interaction between 4.1R and p55. A similar
specific binding response was observed following the addition of
r30kDa to p55 immobilized on the cuvette (Fig. 2B).
In marked contrast, neither of the other functional domains of 4.1R
(16-, 10-, and 22/24-kDa domains) nor denatured r30kDa (100 °C at 5 min) exhibited specific binding to p55 (data not shown). These data
imply that 4.1R binds to p55 through its 30-kDa domain, a finding
consistent with earlier studies (9, 18, 34). Analysis of the binding response curves obtained at varying concentrations of analyte provided
association rate constants (ka) of 3.8 × 104 and 4.0 × 104
M
Having characterized the binary interaction between 4.1R and p55, we
then examined binary interactions between GPC and 4.1R and between GPC
and p55. 4.1R, r30kDa, or p55 was incubated with the entire cytoplasmic
tail of GPC (GPCcyt) or different subdomains of the GPC cytoplasmic
tail (synthetic peptides GPC-1, GPC-2, and GPC-3), immobilized on the
cuvette. Analysis of the binding response curves provided association
rate constants (ka) of 2.9 × 105
M Identification of p55 and GPC Binding Sites in the 30-kDa Domain of
4.1R--
To define the binding site for p55 in the 30-kDa domain of
4.1R, we quantitated p55 binding to various deletion mutants of r30kDa
(Fig. 1). Deletion of sequence encoded by
exon 10 completely abolished the binding of p55 to this mutant r30kDa
(r30kDa/
A similar experimental strategy was used to define the binding site for
GPC in the 30-kDa domain of 4.1R. Whereas full-length r30kDa bound
immobilized GPCcyt with a
K(D)kin value of 0.22 µM, deletion of sequence encoded by exon 8 (r30kDa/ 4.1R Modulates p55 Binding to GPC, but p55 Does Not Affect 4.1R
Binding to GPC--
To further define the interactions among the three
protein components of the ternary complex, 4.1R, p55, and GPC, we
determined whether p55-GPC and 4.1R-GPC binary interactions can be
regulated by the third member of the complex, namely, 4.1R and p55,
respectively. The binding affinity of the p55-r30kDa complex to GPC-1
was increased by a factor of 10 compared with that of native p55 to
immobilized GPC-1 (Table III). The
4.1R-induced increase in binding affinity of p55-GPC interaction was in
large part due to an increase in the association rate constant
(ka) from 5.1 × 103 to 1.6 × 105 M Ca2+ and CaM Regulate the Interaction of 4.1R with both
GPC and p55--
Ca2+ and CaM have previously been shown
to bind to 30-kDa domain and regulate 4.1R interaction with band 3 (12). To determine if interactions of 4.1R with GPC and p55 can also be
regulated by Ca2+ and CaM, we quantitated the binding of
r30kDa to GPCcyt and p55 in the presence and absence of CaM and
Ca2+ (Table IV). Either 5 µM CaM alone or 100 µM Ca2+
alone had no effect on the association and dissociation rate constants
of the interactions of r30kDa with either GPCcyt or p55. However, in
the presence of both Ca2+ and CaM, the affinity of these
interactions decreased by a factor of 10 (Table IV). Similar effects of
Ca2+ and CaM were also seen when intact 4.1R was used for
the binding studies (data not shown). These data demonstrate that
Ca2+ and CaM binding to the 30-kDa domain of 4.1R can
down-regulate 30-kDa interaction with both p55 and GPC. The
Ca2+ concentration dependence of CaM-regulated binding of
4.1R to GPC and to p55 was quantitated (Fig.
3). The extent of r30kDa binding to both
GPCcyt and p55 started to decrease at Ca2+ concentrations
greater than 0.1 µM (pCa < 7). Maximal
inhibition of binding was achieved at Ca2+ concentrations
of 100 µM and higher (pCa < 4) for
GPCcyt and at 1 mM and higher (pCa < 3)
for p55. Half-maximal effect was seen at a Ca2+
concentration of 1 µM for r30kDa binding to GPC and at 4 µM for r30kDa binding to p55.
In the present study, we identified the p55 and GPC binding sites
in the N-terminal 30-kDa domain of 4.1R, showed that 4.1R binding to
p55 increases the affinity of interaction between p55 and GPC, and
demonstrated that binding of CaM to 4.1R regulates the affinity of its
interaction with both p55 and GPC in a
Ca2+-dependent manner. These findings imply an
important role for 4.1R in regulating the GPC-4.1R-p55 ternary protein
complex, and they further suggest dynamic regulation of this protein
complex in the membrane by Ca2+ and CaM.
The domain of 30 kDa encoded by exon 10 is responsible for 4.1R binding
to p55, while the domain of 30 kDa encoded by exon 8 is primarily
responsible for 4.1R binding to GPC. In the stretch of 51 amino acids
encoded by exon 10, the 18 amino acids in the N-terminal region are
primarily hydrophobic, while the 33 amino acids in the C terminus are
primarily hydrophilic. We suggest that this C-terminal stretch of
hydrophilic amino acids interacts with the D5 domain of p55, which is
also enriched in hydrophilic amino acids (17). Indeed, a 33-amino acid
synthetic peptide corresponding to the hydrophilic region of exon 10 bound to p55 with similar affinity as native 4.1R and r30kDa. In the
stretch of 73 amino acids encoded by exon 8 (24), the 25 amino acids in
the N-terminal region are primarily hydrophobic, while the 48 amino
acids in the C terminus are primarily hydrophilic. We suggest that the
153ELEE motif in this hydrophilic region is likely to
interact with the 86RHK sequence in GPC. Although it has
been suggested that GPC may bind to the 37LEEDY sequence,
the band 3 binding motif encoded by exon 5 (35), our finding that GPC
bound to r30kDa lacking exon 5-encoded sequences and did not bind to
sequences encoded by exon 5 strongly implies a minimal role for
37LEEDY sequence in GPC binding.
Formation of the ternary complex of 4.1R, p55, and GPC in the
erythrocyte membranes has been previously proposed (for review, see
Ref. 4). However, stoichiometry among these three proteins has not been
well defined. In the present study, we measured stoichiometry of binary
interactions between 4.1R and p55, 4.1R and GPC, and p55 and GPC and
showed that the stoichiometry of each of these three interactions is
approximately 1:1. Measurement of the binding affinities of various
binary interactions showed that 4.1R bound p55 and GPC with a
K(D)kin value on the order
of 0.1 µM, while p55 bound GPC but with a lower affinity
(K(D)kin value on the order
of 1.0 µM). The binding affinities of 4.1R-p55 and
p55-GPC are similar to those previously reported (18, 34). Although
Hemming et al. (34) reported a higher affinity for the
4.1R-p55 interaction (KD of 0.004 µM),
re-examination of their binding data in our hands indicates that the
KD value is actually on the order of 0.1 µM. The measured stoichiometries and the binding
affinities of these three binary interactions lend strong support to
the hypothesis that 4.1R, p55, and GPC exist as a ternary complex in
erythrocyte membranes. Moreover, these in vitro binding
studies are supported by studies of genetically mutant red cells that
exhibit coordinate deficiencies of these proteins (36, 37). We also
measured the binding affinity of the p55-r30kDa complex to immobilized
GPCcyt and obtained an apparent K(D)kin value on the order
of 0.35 µM (Table III). This value is much higher than
the value estimated from the measured affinities of individual binary
interactions. This discrepancy is either due to limitations of the
IAsysTM system or due to conformational change of r30kDa in the
ternary complex.
The observation that 4.1R increases the affinity of p55 binding to GPC
by an order of magnitude demonstrates that 4.1R is an important
modulator of the interaction between a PDZ protein (p55) and an
integral membrane protein (GPC). In contrast, neither p55 binding to
4.1R nor GPC binding to 4.1R affected the interactions of 4.1R with GPC
and p55, respectively. Taken together, these findings imply that 4.1R
alone acts as a critical regulator of the ternary complex of 4.1R, p55,
and GPC. Importantly, this result extends the known function of 4.1R as
a regulator of protein-protein interactions within erythrocyte (2, 3,
6, 8).
CaM is a highly conserved Ca2+-binding protein that
modulates the activities of many Ca2+-dependent
enzymes and also the functional activities of different transport and
structural proteins (for a review, see Ref. 38). In most cases,
Ca2+ is essential for the initial binding of CaM to these
proteins and for subsequent modulation of their function. However, a
small subset of proteins including 4.1R (12, 15) bind CaM in a
Ca2+-independent manner but require Ca2+ for
manifesting changes in function. Previous studies have shown that CaM
in association with Ca2+ at concentrations greater than 2 µM diminishes 4.1R interactions with band 3 as well as
GPC (12, 39). Recently, the 4.1R interaction with CD44 has been
documented to be modulated by Ca2+ and CaM (8). In an
analogous fashion, the present study demonstrated that the 4.1R
interactions with GPC and p55 are also modulated by Ca2+
and CaM. These findings raise the possibility that increases in
intracellular concentration of Ca2+ can modulate binding of
4.1R not only to transmembrane proteins such as band 3, GPC, and CD44
but also to other membrane skeletal proteins such as p55.
Based on the present and previous findings, we propose the following
model for the Ca2+ and CaM modulation of 4.1R interaction
with GPC and p55 in the erythrocyte membranes (Fig.
4). Ca2+ concentration inside
red blood cells is normally maintained at less than 1.0 µM. At this Ca2+ concentration, CaM binds
with high affinity to the Ca2+-independent binding site in
the region encoded by exon 11 of the 30-kDa domain of 4.1R and only
weakly to the Ca2+-dependent binding site in
the region encoded by exon 9 (12). With triggered increases in
Ca2+ concentration, CaM binds to the
Ca2+-dependent site encoded by exon 9 of 4.1R
with high affinity, causing a conformational change in 4.1R that
decreases 4.1R affinity for both GPC and p55. The physiologic
importance of this regulation is not yet known. However, the well
defined ability of 4.1R to interact with spectrin and actin via an
internal 10-kDa spectrin-actin binding domain indicates that 4.1R may
play an important role in linking the spectrin-based skeleton with the
plasma membrane. The current findings suggest that Ca2+ and
CaM can modulate the strength of this interaction in a dynamic fashion.
The red cell model system presented here, in which 4.1R and
Ca2+/CaM can modulate interactions between p55 and GPC, may
serve as a general paradigm for skeletal membrane interactions in other cell types. This proposal is supported by recent studies demonstrating the existence of multiple nonerythroid homologs of both 4.1R and p55,
which share high homology in the appropriate protein-protein interaction domains. For example, three homologues of 4.1R, designated 4.1G, 4.1N, and 4.1B, have been identified and characterized (40-42). All four members of this 4.1 family share high homology in the 30-kDa
domain, particularly in the CaM binding regions (12) and in the p55
binding region (Fig. 5). In contrast,
other 4.1 superfamily members such as ezrin, radixin, and moesin
exhibit much reduced homology in the CaM binding motif and do not bind CaM (43-45). This evolutionary conservation pattern predicts that all
four 4.1 proteins (but not other ERM proteins) can potentially bind to
both CaM and p55 and functionally regulate p55 interactions with
integral proteins in nonerythroid cells in a
Ca2+/CaM-dependent fashion. Moreover, p55 is
also a representative of a larger family of membrane-associated
guanylate kinase proteins that share two conserved domains of
particular relevance to this model. First, these proteins possess PDZ
domains that mediate interaction with the cytoplasmic tails of various
integral membrane proteins. Indeed, some of the target membrane
proteins, such as syndecan, possess a YFI C-terminal peptide similar to
GPC, and the membrane-associated guanylate kinase protein hCASK has
been shown to bind to syndecan (46). A second key functional domain in
membrane-associated guanylate kinase proteins is the protein 4.1 binding region encoded by the HOOK domain. In addition to p55, both
hDlg and hCASK have been demonstrated to bind protein 4.1 (17, 46).
Together these considerations suggest that Ca2+ and CaM
modulation of interactions of 4.1 proteins with transmembrane and
membrane skeletal proteins may play a major role in dynamic cytoskeletal reorganization during cell signaling in erythroid as well
as nonerythroid cells.
We are grateful to Dr. Philippe Gascard of
Lawrence Berkeley National Laboratory for many insightful suggestions.
*
This work was supported in part by Grants-in-Aid for
Scientific Research 12680702, 07305036, and 10877022 from the Ministry of Education of Japan and by the Chiyoda Foundation, the Takeda Scientific Foundation, the Memorial fund for Hiroto Yoshioka, and
National Institutes of Health Grants HL31579, DK 26263 and DK 32094.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Berkeley National
Laboratory, University of California, Mail Stop 74-157, 1 Cyclotron Rd., Berkeley, CA 94720. Tel.: 510-486-7029; Fax: 510-486-6746; E-mail:
mnarla@lbl.gov.
Published, JBC Papers in Press, May 30, 2000, DOI 10.1074/jbc.M002492200
The abbreviations used are:
4.1R, human
erythrocyte protein 4.1;
GPC, glycophorin C;
CaM, calmodulin;
GPCcyt, cytoplasmic domain of GPC;
r30kDa, recombinant 30-kDa domain of 4.1R;
r30kDa/
Regulation of Protein 4.1R, p55, and Glycophorin C Ternary
Complex in Human Erythrocyte Membrane*
§,, Department of Biochemistry, School of
Medicine, Tokyo Women's Medical University, Shinjuku, Tokyo 162-8666, Japan and § Life Sciences Division, Lawrence Berkeley
National Laboratory, Berkeley, California 94720
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
0.74 A260.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 s1
and dissociation rate constants (kd) of 3.8 × 103 s1 and 8.0 × 103 s1 for
interaction of 4.1R and r30kDa, respectively, with p55. Thus, both
intact 4.1R and its r30kDa domain bind p55 with a similar K(D)kin value on the order
of 0.1 µM (Table I). The stoichiometry of 4.1R binding to p55 was calculated as described under
"Experimental Procedures." Using the experimentally determined value of Bmax for 4.1R of 264 arc seconds and
the amount of immobilized p55 on the cuvette to be 170 arc seconds, a
molar ratio of 4.1R binding to p55 was derived to be approximately
1:1.
Binding affinities of binary interactions involving 4.1R, p55, and GPC
1 s1
and 1.5 × 105
M1 s1
and dissociation rate constants (kd) of 2.7 × 102 and 3.3 × 102 s1 for
interaction of 4.1R and r30kDa, respectively, with GPCcyt. Thus, both
intact 4.1R and its r30kDa domain bound GPCcyt with a similar
K(D)kin value on the order
of 0.1 µM (Table I). 4.1R and r30kDa also bound GPC-3
with a K(D)kin value on the
order of 0.1 µM (Table I), but no binding could be
documented between r30kDa and either GPC-1 or GPC-2 (Table I). These
data imply a specific binary interaction between r30kDa and the
membrane-proximal peptide GPC-3. p55 also exhibited specific binding to
GPCcyt but with a much lower affinity
(K(D)kin value on the order
of 1.0 µM) (Table I). However, in contrast to the
findings with 4.1R, p55 bound to GPC-1 (Table I) but not to either
GPC-2 or GPC-3 (Table I). These results are consistent with previous
reports (7, 17, 18), which described distinct binding sites on the
cytoplasmic domain of GPC for 4.1R and p55. Scatchard analysis of
r30kDa and of p55 binding to GPCcyt revealed that the stoichiometry of
both of these interactions is approximately 1:1. The finding that the
stoichiometries of all three binary interactions (4.1R and p55, 4.1R
and GPC, and p55 and GPC) is 1:1 suggests that these three proteins
form a ternary complex in the erythrocyte membrane.
Ex.10) (Fig. 2C and
Table II). In contrast, mutant r30kDa
with deletions of exon 5 or 8 or 11 (r30kDa/Ex.5, r30kDa/Ex.8,
and r30kDa/Ex.11) bound p55 normally (data not shown), consistent
with the idea that the p55 binding site is encoded by exon 10. The
assignment of this region of 30 kDa as the p55 binding domain is
further reinforced by the finding that p55 bound to the recombinant
polypeptide encoded by exons 9-11 (r30kDa/Ex.9+10+11) with a
K(D)kin value on the order
of 0.1 µM (a value very similar to that seen for r30kDa)
but did not bind to a derivative polypeptide from which exon 10 sequences were deleted (r30kDa/Ex.9+11) (Table II). These results
mapped the p55 binding site to a stretch of 51 amino acids within the
30-kDa domain encoded by exon 10. This assignment was confirmed and
further refined using a synthetic peptide. A 33-amino acid peptide
(214YKDKLRINRFPWPKVLKISYKRSSFFIKIRPGE) also bound p55 with
an affinity on the order of 0.1 µM. Furthermore, as with
intact p55, the recombinant D5 domain of p55 (D5), which has previously
been identified as the 4.1R binding site (17), bound r30kDa with a
K(D)kin value on the order
of 0.1 µM but failed to bind the mutant r30kDa/Ex.10 (data not shown).
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Fig. 1.
Structure of the 30-kDa domain of 4.1R
encoded by exons 4-11 and the various mutant constructs. In some
mutant constructs, sequences encoded by specific exons were deleted
(r30kDa/ Ex.5, r30kDa/Ex.8, r30kDa/Ex.10, r30kDa/Ex.9,11),
while in others, sequences encoded by a single exon or a combination of
exons were included (r30kDa/Ex.9+11, r30kDa/Ex.9+10+11, r30kDa/Ex.5,
and r30kDa/Ex.8)
View larger version (29K):
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Fig. 2.
Typical tracings from the IAsysTM
system to quantitate the interactions between p55 and 4.1R
(A); p55 and r30kDa (B); p55 and
r30kDa/ Ex.10 (C); and GPC and
r30kDa, r30kDa/Ex.5, or r30kDa/Ex.8 (D). The
specific binding response monitored as an increase in arc seconds
reached a plateau value at 3.5 ~ 5 min. After the equilibrium
state was reached, the cuvette was washed with protein-free binding
buffer (wash). Following this washing step, the response signal
decreased and reached a new lower plateau value. The cuvette is
subsequently washed with 20 mM HCl to remove all bound
proteins (HCl) and regenerated with phosphate-buffered
saline buffer (Buffer). While a specific association was
noted between p55 and both 4.1R and r30kDa protein, no specific
association was noted between p55 and r30kDa/Ex.10. A specific
association was also documented between r30kDa and r30kDa/Ex.8 with GPC
but not between r30kDa/Ex.5 and GPC.
Mapping of binding sites in 4.1R for p55 and GPC
Ex.10), recombinant
polypeptide encoded by exons 9, 10, and 11 (r30kDa/Ex.9+10+11), and
recombinant polypeptide encoded by exons 9 and 11 (r30kDa/Ex.9+11).
K(Dkin) values, for the interaction
of GPC and r30kDa (wild type and various deletion mutants) are also
shown. Each analyte (50 nM to 1 µM) was
incubated with the identified ligand immobilized on the aminosilane
cuvette as described under "Experimental Procedures." From the
binding curves obtained by the resonant mirror detection method,
K(Dkin) values were determined using
the software package FAST-FitTM.
K(Dkin) represents mean ± S.D.
(n = 3-5).
Ex.8) markedly decreased the affinity of interaction between
mutant r30kDa and the cytoplasmic domain of GPC (Table II). Other
deletion mutants lacking sequences encoded by exons 5, 10, or 9 and 11 (r30kDa/Ex.5, Ex.10, Ex.9,11) bound with normal affinity
(Table II). These findings suggest that the primary binding site for GPC is encoded by exon 8. The assignment of this region of 30 kDa as
the GPC binding domain is further reinforced by the finding that GPC
bound to the recombinant polypeptide encoded by exon 8 but not to the
recombinant polypeptide encoded by exon 5 (Fig. 2D). Taken
together, these findings enable us to assign the sequence encoded by
exon 8 (residues Tyr94 to Arg166) to be the
primary binding motif for GPC and the sequence encoded by exon 10 (residues Tyr214 to Glu246) to be the binding
motif for p55 in 4.1R.
1
s1 with little or no effect on the
dissociation rate constant (kd). This effect of 4.1R
on p55-GPC interaction could also be mimicked using only the synthetic
peptide, pep10, which contains the p55 binding domain of 4.1R (Table
III). Importantly, r30kDa/Ex.10, which can bind to GPCcyt but lacks
the p55 binding domain, had no effect on the affinity of the p55-GPC
interaction (data not shown). These findings imply that the observed
increase in binding affinity is the direct result of 4.1R binding to
p55. In marked contrast, p55 had no effect on the interaction between
r30kDa and GPC (Table III); the affinity of binding of p55-r30kDa
complex to GPC-3 was very similar to that determined for binding of
r30kDa alone to GPC-3. This lack of regulatory effect of p55 was
further confirmed by the finding that D5, the 4.1R binding domain of
p55, did not alter the affinity of interaction between r30kDa and GPC-3 (Table III). We then examined if GPC binding to either 4.1R or p55
could regulate the 4.1R-p55 interaction and were unable to document any
effect of either GPCcyt or GPC peptides on 4.1R-p55 interaction (data
not shown). Taken together, these results imply that 4.1R alone plays a
key regulatory role in modulating the effects of p55 in the ternary
complex.
Modulation of p55-GPC interaction by 30-kDa domain of 4.1R
Ca2+/CaM down-regulation of r30kDa interaction with GPC and p55
View larger version (13K):
[in a new window]
Fig. 3.
Ca2+ concentration dependence of
r30kDa binding to cytoplasmic domain of GPCcyt (A) and
p55 (B). r30kDa binding to cytoplasmic domain of
GPC and p55 was measured at various concentrations of Ca2+
in the presence ( 
) and absence (
) of 5 µM CaM.
Ca2+ concentrations were maintained by a
Ca2+/EGTA buffer system. The maximal extent of binding
under different experimental conditions was quantitated as described
under "Experimental Procedures." Maximal binding in the presence of
EGTA was used to normalize the extent of binding in the presence of
varying concentrations of Ca2+. pCa represents
ionized Ca2+ concentration. The extent of r30kDa binding to
GPCcyt and p55 is plotted as a function of Ca2+
concentration.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (40K):
[in a new window]
Fig. 4.
Model proposed for Ca2+/CaM
modulation of 4.1R interactions with GPC and p55 in the erythrocyte
membrane. The proposed model is based on the binary
protein-protein interaction data obtained in the present study. When
Ca2+ concentration inside the erythrocyte is less than 1.0 µM, CaM is constitutively bound to the
Ca2+-independent CaM binding site of the 30-kDa domain of
4.1R. In this state, CaM-bound 4.1R binds simultaneously to the D5
domain of p55 and to the GPC-3, while p55 binds to GPC-1 through its
PDZ domain. All three of these interactions exhibit binding affinities
on the order of 0.1 µM. Following increased intracellular
Ca2+ concentration, CaM binds with high affinity to the
Ca2+-dependent CaM binding site in the 30-kDa
domain, causing conformational change in binding sites for GPC and p55,
resulting in marked decreases in the affinity of 4.1R for both GPC and
p55 (binding affinities on the order of 1.0 µM). We
suggest that this large decrease in the binding affinities of all three
binary interactions that constitute the GPC-4.1R-p55 ternary complex
will markedly destabilize this complex.
View larger version (35K):
[in a new window]
Fig. 5.
Sequence conservation of p55 binding site
among members of the protein 4.1 family. A stretch of 33 hydrophilic amino acids in the C-terminal part of exon 10-encoded
sequence in 4.1R is highly conserved in the homologues, 4.1G, 4.1N, and
4.1B. Shaded regions indicate identical amino
acids.
ACKNOWLEDGEMENT
FOOTNOTES
ABBREVIATIONS
Ex.5, r30kDa/Ex.8, r30kDa/Ex.10, and r30kDa/Ex.9,11,
r30kDa from which sequence encoded by exon 5, 8, 10, and 9 and 11, were deleted, respectively;
r30kDa/Ex.5, r30kDa/Ex.8, r30kDa/Ex.9+11, and
r30kDa/Ex.9+10+11, recombinant proteins containing sequences encoded by
exon 5, 8, 9 and 10, and 9-11, respectively;
K(D)kin, dissociation
constant from kinetic analysis;
Fmoc, N-(9-fluorenyl)methyloxycarbonyl.
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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