(Received for publication, August 28, 1995; and in revised form, November 20, 1995)
From the
Peptides corresponding to the cytoplasmic tails of the
(
(985-1008)) and
(
(713-762)) subunits of the
integrin receptor
(glycoprotein
IIb-IIIa) were synthesized and used to characterize their interaction
with cations and with one another.
(985-1008)
was found to contain a functional cation binding site as assessed by
both terbium luminescence and electrospray ionization mass
spectroscopy. The binding of Tb
to
(985-1008) was of high affinity (K
= 8.8 ± 5.2
nM), occurred with a 1:1 stoichiometry, and was mediated by
its acidic carboxyl terminus (
(999-1008),
PLEEDDEEGE). The affinity of this site for divalent cations was in the
micromolar range, suggesting that this site would be constitutively
occupied in the intracellular environment. Incubation of
(999-1008) with
(713-762) resulted in the formation of a
complex, both in the presence and absence of cations. The interactive
site for
(999-1008) in
was
mapped to
(721-740), and complex formation was
associated with a stabilization of secondary structure as assessed by
circular dichroism. Both a binary
(
(985-1008)
(721-740))
and a ternary
(Tb
(999-1008)
(721-740))
complex were detected by mass spectroscopy, but the distribution and
intensity of the mass/charge peaks were distinct. These difference may
reflect the involvement of distinct cation coordination sites and the
formation of salt bridges in stabilizing the ternary complex. These
data demonstrate the formation of a novel entity composed of the
cytoplasmic tails of
and
and a
cation which may constitute a functional intracellular domain.
The integrin family of receptors mediate many of the cellcell
and cell-substratum interactions that are central to cell adhesion,
migration, growth, and differentiation. Integrins are noncovalent
/
heterodimers; each subunit contains a large extracellular
region of several hundred amino acids, a transmembrane domain, and a
single, short (usually less than 50 amino acids) cytoplasmic
tail(1, 2, 3, 4, 5, 6, 7) .
The extracellular regions of the subunits interact with each other to
form a binding site for a wide variety of ligands, including
extracellular matrix proteins, counter-receptors on other cells, and
circulating plasma proteins(8, 9) . Numerous studies
support a model in which amino acid sequences in both subunits
coordinate ligand and cations within close proximity to form a
``reactive center'' for ligand
binding(10, 11, 12, 13) .
The
affinity of integrins for their ligands is tightly regulated through a
process termed inside-out
signaling(5, 14, 15) . Accordingly,
intracellular signals initiate conformational changes, and the
extracellular domains can be transformed from a low to a high affinity
ligand binding state. The cytoplasmic tails of integrins must be
centrally involved in initiating and propagating the conformational
changes that mediate such inside-out signaling. At the same time,
binding of ligands to the extracellular domain elicits intracellular
responses (outside-in signaling), and activation of such signaling
pathways must be dependent upon integrin cytoplasmic tails and their
conformation(16) . In addition, the cytoplasmic tails serve as
binding sites for intracellular ligands, including cytoskeletal
proteins such as talin and -actinin(17, 18) . The
transmission of conformational change to the cytoplasmic tails must
also be centrally involved in such signaling. Thus, the cytoplasmic
tails of integrin
and
subunits play a pivotal role in
regulating integrin function.
The platelet integrin
(glycoprotein IIb-IIIa) provides
evidence for both inside-out and outside-in signaling and for the
importance of its cytoplasmic tails in these
processes(19, 20, 21, 22, 23) .
On resting platelets
is in a
``latent'' and/or noncompetent state, as it does not bind its
abundant blood-borne ligands, such as
fibrinogen(24, 25, 26) . Platelet stimulation
with agonists, such as thrombin and ADP, induces inside-out signaling
which leads to activation of
to a
competent receptor. Outside-in signaling is manifest by numerous
intracellular changes, including the initiation of cytoskeletal
reorganization, activation of kinases and phosphatases, and
translocation of intracellular
constituents(23, 27, 28) . Several lines of
evidence directly support the involvement of the cytoplasmic tails of
and
in these events. A single
point mutation within the
cytoplasmic tail, serine
752 to proline, prevents
from
becoming a competent receptor(21) . More recently, it has been
reported that transfected cells expressing this
substitution in
have
impaired
-mediated cell spreading,
focal adhesion, and fibrin clot retraction, suggesting that this
mutation also affected outside-in signaling(29) . Truncation of
the cytoplasmic tail of
results in a constitutively
active
(20) , direct
evidence of inside-out signaling.
The central features of integrin
extracellular domains and their ligand binding sites extend to
: the subunits interact with one
another and bind divalent cations, and the ligand binding function is
conformationally regulated. In a sense, the cytoplasmic tails of
and other integrins can be viewed
as a second ligand binding domain by virtue of their interactions with
cytoskeletal proteins and intracellular signaling molecules and by
their predicted conformational sensitivity. In the present study, we
have put this analogy to a direct test. Interaction of the cytoplasmic
tails with one another and with cations are demonstrated; and, in turn,
these interactions are shown to effect conformation. These findings may
have substantial bearing on integrin structure and function.
Crude peptides were dissolved in 10% aqueous acetic acid, diluted 1:20 with 5% aqueous acetonitrile (containing 0.1% trifluoroacetic acid), and finally purified by either semipreparative or preparative HPLC. Following lyophilization, the purity of each peptide was confirmed to be >98% as assessed by both analytical HPLC and ESIM spectroscopy. The molecular mass of each peptide, obtained from its ESIM spectrum, was identical to its expected mass, determined using MacProMass (PESciex, Thornhill, ON).
In
preliminary studies, the peptides tended to
precipitate within 2 h when dissolved in buffers containing
20
mM KCl or NaCl. Therefore, all experiments reported here were
performed in buffers containing 10 mM KCl, unless otherwise
specified.
For peptide
nomenclature, the subunit identification ( or
) is followed
by the sequence position, with residue 1 being the NH
terminus of the mature subunit(31, 32) .
Peptides that contained the natural COOH terminus of the subunit was
synthesized with a terminal carboxylate; otherwise the
carboxyl-terminal residue was in an amide form. The peptides used in
this study are as follows:
(985-1008),
LAMWKVGFFKRNRPPLEEDDE-EGE;
(985-998),
LAMWKVGFFKRNRP(amide);
(999-1008), PLEEDDEEGE;
(1008E
A), PLEEDDEEGA(amide);
(1003DD
AA), PLEEAAEEGE(amide);
(713-762),
LIWKLLITIHDRKEFAKFEEERARAKWDTANNPLYKEATSTFTNITYRGT;
(715-737), WKLLITIHDRKEFAKFEEERARA(amide);
(721-740), IHDRKEFAKFEEERARAKWD(amide); and
(117-131), LMDLSYSMKDDLWSI(amide).
Tb binding
constant for
(985-1008) was determined by
nonlinear least squares fit of fluorescence intensities to (34) .
In this study, the peptides were dissolved in 5 mM KCl,
5 mM PIPES, pH 6.8. The fluorescence intensities at zero
receptor concentration (F) and at receptor
saturation (F
) and the dissociation constant (K
) were all optimized to achieve the best fit of
the data as described previously(34) . In addition, a K
was also calculated using experimental values
for F
and F
. The
Tb
concentration, [L]
,
was kept constant at 300 nM, while the concentration of
(985-1008),
[R]
, was varied from 50 nM to
10 µM.
The binding of other cations to the peptides
under analysis was assessed by their capacity to displace
Tb complexed with a peptide, measured as a decrease
in the luminescent signal at 545 nm (33) . The K
values for these other cations were derived from their
Tb
displacement curves using the calculated K
for Tb
(see above). For these
calculations, the K
was the concentration that
decreased the fluorescence intensity by 50%, assuming that
[cation]
= [cation]
- 0.5[R].
Distance measurements using
fluorescence energy transfer from tryptophan residues within the
peptides to bound Tb were calculated with the
following equations(35, 36) : E = 1
- F
/F
and r = [E
-
1]
R
, where E is the
efficiency of energy transfer from donor to acceptor; F
and F
are the fluorescence intensity of the
donor in the presence and absence of the acceptor, respectively; R
is the Förster critical
distance at which E = 0.5 and r is the
distance between donor and acceptor. F
and F
were measured at 350 nm, the emission maximum
for tryptophan within the Tb
-binding peptide,
(985-1008).
The program solved the equation using four secondary structural
component spectra ( helical,
sheet,
turn, and
random/extended coil). These spectra were based on either proteins,
different peptides sets, and a combination of peptide and protein
components(37) . Validation of the program was established in
two ways. First, the program always produced correct results when
combinations of the four secondary structural component spectra were
mathematically added and entered into the program. Second, the program
gave results almost identical to those obtained by Yang et al.(37) and with a deconvolution CD program provided with the
J600 instrument for four protein CD spectra (sperm whale myoglobin, egg
white lysozyme, bovine pancreas ribonuclease A, and bovine pancreas
-chymotrypsin).
Figure 1:
Binding of Tb to the
cytoplasmic tail of
as monitored by terbium
luminescence spectroscopy. Peptides (25 µM) encompassing
the cytoplasmic tails of
(
(985-1008)) and
(
(713-762)), the amino terminus of
(
(985-998)), and the
extracellular calcium-binding segment of
(
(117-131)) were dissolved in 5 mM MOPS, pH 6.8, containing 50 µM Tb
.
After a 5-min incubation, luminescence of Tb
bound to
the tryptophan containing peptides was induced by excitation at 289 nm,
and Tb
emission was quantitated at 545 nm. Each data
point is a mean of three separate experiments with an average of five
measurements of each point in each
experiment.
The stoichiometry of Tb binding to
(985-1008) was evaluated by adding increasing
amounts of Tb
to a fixed concentration of peptide (10
µM). The results of three separate experiments are shown
in Fig. 2A. In the absence of Tb
,
(985-1008) displayed no Tb
luminescence at 545 nm. Increasing amounts of
Tb
, in the range of 0.25:1 to 1:1
Tb
/peptide ratio, resulted in a linear increase in
Tb
luminescence (r = 0.998).
Increasing the amount of Tb
added above the 1:1 ratio
did not further increase the luminescent signal. Assuming that all the
Tb
luminescence was directly related to the amount of
complex formed, the stoichiometry of Tb
binding to
(985-1008) was 1:1. Using the data points from
ratios of
1:1, the K
of Tb
binding to
(985-1008) was estimated to
be below 10 nM. Accurate measurement of K
from titrations, such as shown in Fig. 2A,
required using peptide concentrations below 50 nM, where the
signal-to-noise ratio was unacceptable. Therefore, the K
for Tb
binding to
(985-1008) was determined as described by
Kuzmic et al.(34) . In these experiments, the
concentration of Tb
was fixed at 300 nM and
the concentration of
(985-1008) varied (50
nM to 10 µM). The experimental data points are
shown in Fig. 2B. The curve represents the theoretical
fit of the data to (see ``Experimental
Procedures''), after optimizing F
, F
, and K
(34) . The
optimized values obtained were: F
= 0.399
± 0.377, F
= 8.231 ± 2.167,
and K
= 8.8 ± 5.2 nM. Using
experimentally obtained values for F
and F
(0.380 ± 0.512 and 8.492 ± 1.15,
respectively), the K
was calculated to be 14.6
± 6.3 nM. Thus, the two approaches yielded very similar
values for the various parameters. For comparison, the K
of five cation binding peptides from the extracellular region of
, including that of
(118-131), fell within the 1-15 µM range(33) .
Figure 2:
Characterization of
(985-1008)
Tb
complex. A, stoichiometry of the
(985-1008)
Tb
complex.
(985-1008) (
) or
(117-131) (
), each at 25 µM,
were incubated with increasing amounts of Tb
and
terbium luminescence measured as in Fig. 1. B,
determination of the dissociation constant of Tb
for
(985-1008). Increasing amounts of
(985-1008) were added to a fixed amount of
Tb
(300 nM). After 5 min, Tb
luminescence was measured. Each data point (
) is an average
of five measurements. The displayed curve (-) represents
the theoretical fit of the data (see under
``Experimental Procedures''). C, displacement of
Tb
from the
-(985-1008)
Tb
complex
by cations.
(985-1008) (30 µM)
was incubated for 5 min with Tb
(25 µM)
and then various concentrations of divalent cations were added
(Mn
,
; Ba
,
;
Ca
,
; Mg
,
). After an
additional 5-min incubation, the amount of Tb
remaining complex to
(985-1008) was
assessed by Tb
luminescence spectroscopy. As all
three monovalent cations (K
, Na
, and
Li
), used only at a final displacement concentration
of 50 mM, were marked with the same symbols (
), as they
all gave similarly low levels of
displacement.
To determine the cation specificity of
(985-1008), the ability of divalent
(Ba
, Ca
, Mn
, and
Mg
) and monovalent (K
,
Na
, and Li
) cations to displace
Tb
from
(985-1008) was
measured (Fig. 2C). In these displacement experiments,
the amount of Tb
was nonsaturating (25 µM Tb
with 30 µM
(985-1008)), such that the decrease in
Tb
luminescence was proportional to the amount of
Tb
displaced by the competing ion. A 2000-fold excess
(50 mM) of any of the monovalent cations relative to
Tb
resulted in <20% decrease in Tb
luminescence. In contrast, the divalent cations did displace
bound Tb
from
(985-1008).
Mn
was 100-fold more effective than either
Ca
or Mg
in competing with
Tb
. The displacement curves shown in Fig. 2C were analyzed to calculate the dissociation
constants for the divalent cations for
(985-1008) using the K
for Tb
calculated above. These values are
summarized in Table 1and indicate that the cation preference of
(985-1008) is Mn
Ba
> Ca
Mg
monovalents.
The absence of a luminescent signal with
(985-998) suggests that the carboxylate-rich
carboxyl-terminal aspects (
-(999-1008)) must
be required for the cation binding properties of
.
However,
(999-1008) does not contain a natural
aromatic residue, precluding the use of Tb
luminescence to directly test this hypothesis. Therefore, the
cation binding properties of
(999-1008) were
directly measured by ESIM spectroscopy. The results of the ESIM
spectroscopy experiments are summarized in Fig. 3, A and B.
(999-1008) (10
µM) was dissolved in water containing ± 20
µM Tb
. In the absence of
Tb
, the expected ESIM spectrum for the peptide was
obtained (Fig. 3A). Only two major electrospray
responses (m/z peaks) were observed. Both these peaks
corresponded to those expected for
(999-1008) (m/z = 1161.4 and 581.2 at charge states 1+ and
2+, respectively). Addition of Tb
to
(999-1008) (Fig. 3B) resulted
in almost complete loss of the two m/z peaks in the
(999-1008) spectrum with the concurrent
appearance of a new ESIM spectrum. This spectrum corresponded to that
predicted for the
(999-1008)
Tb
complex,
with the loss of three protons for each Tb
bound (m/z = 659.2 and 439.8). Similar results were obtained
with
(985-1008), the peptide encompassing the
entire cytoplasmic tail of
(data not shown). In
contrast,
(721-740), IHDRKEFAKFEEERARAKWD, which
contained three sequential glutamic acid residues showed no
Tb
binding by ESIM spectroscopy (Fig. 3C
versus three-dimensional). Similarly,
(985-998),
(715-737),
and
(713-762) failed to bind
Tb
, as did a number of control peptides (RGDW,
YQAAIDYIN, and YGSTGVFSSWVDRIEEA), demonstrating the specificity of
ESIM spectroscopic methodology in determining the capacity of peptides
to bind cations. Thus, we conclude that the cation binding activity of
the cytoplasmic tail of
resides in its
carboxyl-terminal aspects.
Figure 3:
Electrospray ionization mass spectroscopic
analysis of terbium binding to the and
cytoplasmic tails. Peptides (10 µM) were dissolved
in water containing ±20 µM Tb
and
infused into the mass spectrometer at a flow rate of 3 µl/min using
a Hamilton syringe pump. Spectrum for
(999-1008) (A, B) and
(721-740) (C, D), in the absence (A, C) and presence (B, D) of Tb
,
are displayed. The predicted electrospray response peaks corresponding
to uncomplexed(-) and Tb
complexed (+)
peptides are identified.
(715-737) yields
similar results similar to those obtained for
(721-740) (data not shown). Full-scan spectra
(400-1600 M/Z) were acquired at a 0.2 m/z accuracy, and the spectra shown represent an average of six to ten
scans.
Having established that the cation
binding properties of (985-1008) resided
within
(999-1008) (PLEEDDEEGE, containing a
free carboxyl terminus), two mutant peptides were synthesized in an
attempt to determine which oxygenated residues were involved in cation
coordination. The carboxyl terminus of
(999-1008) was mutated to PLEEDDEEGA-amide
(
(1008E
A)), and
(1003-1004) was mutated from DD to AA
(
(1003DD
AA), PLEEAAEEGE-amide). Terbium
luminescence was used to evaluate the cation binding properties of
these mutant peptides. Specifically, since these peptides lacked a
tryptophan, we evaluated their cation binding capacity as competitors
of Tb
binding to
(985-1008).
Incubation of an equimolar amount of
(985-1008) (20 µM) with either
(999-1008),
(1008E
A),
or
- (1003DD
AA), in the presence of 17.5
µM Tb
, resulted in a 55 ± 4%, 55
± 5%, and 1.3 ± 12.5% decrease in Tb
luminescence, respectively. Thus,
(1008E
A) competed as effectively as the
control native peptide for Tb
binding, but
(1003DD
AA) bound Tb
poorly.
These data suggest that neither the carboxylate terminus of
nor the carboxylate side chain of glutamate
(1008) is required for cation binding, while at
least one of the carboxylate side chains of the aspartates at
(1003) and/or
(1004) is essential
for cation binding.
Finally, we attempted to estimate the distance
from the tryptophan at (986) to the bound
Tb
ion. The efficiency of energy transfer from donor
to acceptor, E, was determined to be 8.3 ± 0.4% (n = 9). Assuming a Förster critical
distance (R
) range of 10-22.7 Å for
donor-Tb
(35, 36, 40) , the
range in distance between donor (
(988W)) and
acceptor (Tb
) was calculated to be 15-34
Å.
Figure 4:
Evidence for interaction of the
cytoplasmic tails of and
by
fluorescence quenching.
(985-1008) and
(713-762) peptides (10 µM) were
dissolved together in 5 mM phosphate, pH 7.2, in the absence
or presence of 1 M GuHCl. The peptides were co-incubated for
60 min on ice and then placed in a 4 °C thermally regulated quartz
cuvette. The samples were then excited at 289 nm (bandwidth = 3
nm) and the emission spectra recorded from 300 to 450 nm using 0.5-nm
steps and a bandwidth of 3 nm. Background contribution was then
subtracted from each spectra. The average spectra from five scans of
the peptides obtained in the absence (-) and presence
(- - - -) of GuHCl are displayed. Similar
results to those obtained in the presence of GuHCl were obtained by
mathematical addition of the individual spectra of
(985-1008) and
(713-762) (data not
shown).
Terbium luminescence provided a
more incisive approach to demonstrate interaction between the
and
cytoplasmic tails. In these
analyses, we exploited the fact that
(999-1008) contains the high affinity
cation-binding site (see above), but lacks an aromatic residue for the
excitation of bound Tb
. However, if the
(999-1008)
Tb
binary
complex formed a stable ternary complex with a tryptophan-containing
peptide, then fluorescence energy transfer could occur, provided that
the tryptophan and Tb
were in close proximity. Thus,
if the tryptophan-containing peptide itself was incapable of binding
Tb
, then all Tb
luminescence would
depend upon the formation of the ternary complex. This was found to be
the case. When
(985-998) or
(721-740) peptides were incubated with a 1.5
molar excess of Tb
, no Tb
luminescence signal was detected (Fig. 5A),
confirming our ESIM data (Fig. 3) that these peptides do not
bind Tb
. As expected,
(999-1008) did not generate a luminescent
signal as it lacks a tryptophan donor (Fig. 5A).
However, when
(999-1008) was co-incubated with
(721-740) and Tb
, a large
luminescence signal was generated (Fig. 5A). These data
provide direct evidence for the formation of an
(999)
(721-740)
Tb
ternary complex. Similar results were also obtained when
(721-740) was replaced with either
(713-762) or
(715-737)
(data not shown), suggesting that
(721-737)
encompasses a majority of the
Tb
binding domain.
Figure 5:
Evidence for interaction of the
cytoplasmic tails of and
by
terbium luminescence. A, mapping of the interactive sites in
cytoplasmic tails of
and
.
Individual peptides (25 µM) corresponding to either
(985-1008),
(985-998),
(999-1008), and
(721-740), or
(999-1008) co-incubated with
(985-998) (
/
) and
(721-740) (
/
), were incubated with
37.5 µM Tb
for 30 min before
luminescence was measured. B, stoichiometry of
Tb
complex as measured by Tb
luminescence.
(999-1008) (10 µM) was incubated
with a 1.5 molar excess of Tb
and incubated with
increasing amounts of either the
(721-740)
peptide (
) or with a control
peptide,
(985-998) (
). The resulting terbium
luminescence was measured as outlined in the legend to Fig. 1.
The stoichiometry of this ternary complex with
respect to its peptide constituents was determined to be 1:1 (Fig. 5B). Varying amounts of
(721-740) were added to a constant concentration
of
(999-1008) (10 µM) in the
presence of a molar excess of Tb
. As shown in Fig. 5B, the maximum Tb
luminescence
was observed at a 1:1 molar ratio of the two peptides and did not
increase as excess
(721-740) was added. In
addition, the interaction of
(999-1008) with
(721-740) was not merely a nonspecific
charge-charge interaction between the two peptides, as
(999-1008) failed to complex with
(985-998) (Fig. 5, A and B), a peptide containing four positively charged residues
(three side chains and one free amino terminus).
Figure 6:
Stabilization of secondary structure by
formation of the cytoplasmic
tail complex as assessed by circular dichroism.
(985-1008) and
(713-762) peptides (30 µM) were
dissolved either individually or together in 5 mM phosphate,
buffered to either pH 7.2 (A) or pH 3.2 (B) with 1 M KOH. The peptides were incubated for 30 min on ice and then
placed in a 4 °C thermally regulated quartz cuvette, and their CD
spectra obtained (an average of five scans). Background contribution
was then subtracted from each spectrum, and the spectra obtained for
the co-incubated peptides are displayed (-). The spectra
obtained from the individual
and
peptides were
mathematically added together, and this calculated combined spectrum is
displayed(- - - -).
Despite the fact that ESIM measurements are performed in
the gaseous phase, ESIM spectrometry has been proven to be a powerful
technique for obtaining information about three dimensional protein
structure and ligand binding and to be in good agreement with solution
phase
techniques(11, 42, 43, 44, 45, 46, 47, 48) .
This was also the case in our study, the interaction of the cytoplasmic
tails with cation and each other as detected by ESIM spectroscopy, were
in agreement with Tb luminescence, CD spectroscopy,
and fluorescence quenching. Therefore, further information pertaining
to the conformation of the binary peptide-peptide and the ternary
cation-peptide-peptide complexes were obtained by ESIM spectrometry.
When 10 µM
(999-1008) and
(721-740) peptides were co-incubated, a series
of electrospray response peaks were generated (Fig. 7A). This series corresponded to the expected m/z peaks for the
(999-1008)
(721-740)
complex (-). In agreement with our terbium luminescence data,
both
(999-1008)
(713-762)
and
(999-1008)
(715-737)
complexes could also be generated. The relative abundance of molecular
charged ion components of the complex can be determined by comparing
the height of each m/z peak. The ``envelope'' of the m/z peaks of the
(999-1008)
(721-740)
complex had one maximum (m/z 941.4, charge state of 4+),
and all charge states from 3+ to 8+ were represented. These
data demonstrate that none of the charge states were suppressed, which,
in turn, indicates that none of the positively charged groups in the
complex were masked by the molecular associations. The addition of 20
µM Tb
to the complex resulted in the
formation of an additional series of peaks (+) corresponding to
the
Tb
(999-1008)
(721-740)
ternary complex (Fig. 7B). In comparing the intensities
of the two series (- versus +), it was evident that
the relative abundance of the binary complex(-) was at least
2-fold greater than that of the ternary complex (+), if one
assumes that each complex yields a electrospray series of equal
intensity. The envelope of the binary complex(-) in the absence versus the presence of Tb
(Fig. 7, A versus B, and Table 2) exhibited changes in the
overall intensity of m/z peaks, but the shape and the location
of the peaks were not altered. Thus, the presence of Tb
did not distort the pattern of the binary
cytoplasmic tail complex.
However, when the envelope of the ternary complex (+) (Fig. 7B and Table 2) was examined, the pattern
was markedly different. Two maxima were present at m/z 980.4
and 490.7 were observed, corresponding to the 4+ and 8+
charge states, respectively. Furthermore, all charge states were not
present. Missing were the expected peaks at m/z 1306.9
(3+) and m/z 560.7 (7+) (the 6+ charge state of
the ternary complex (m/z 654) was masked by the 2+ charge
state of
(999)
Tb
(m/z 659.2)). These distortions indicate alterations in the charges
and/or their distribution in the ternary as compared with the binary
complexes. Finally, the positions of the m/z peaks shown in Fig. 7, A and B, are consistent with binary
and ternary complexes at stoichiometries of 1:1 and 1:1:1. Evidence for
higher order complexes was not detected.
Figure 7:
Observation of an
binary and an
Tb
ternary complex by electrospray ionization mass spectroscopy.
(999-1008) and
(721-740) peptides (10 µM) were
mixed in the absence (A) and presence (B) of 20
µM Tb
. After a 5-min incubation, the
samples were infused into the mass spectrometer and spectra collected
as outlined in the legend to Fig. 3. Spectra corresponding to
expected series for the
(999-1008)
(721-740)(-)
and for the
(999-1008)
(721-740)
Tb
(+) complexes are highlighted. For clarity, all off-scale
electrospray response peaks corresponding to either
(999-1008),
(721-740),
or
(999-1008)
Tb
were
deleted from the displayed spectra.
In this study, we have examined the interactions of the
cytoplasmic tails of and
with
each other and with cations. The data support the following
conclusions. First, the carboxyl-terminal aspect of
contains a high affinity cation binding site. Second, the two
cytoplasmic tails interact with each other to form a binary
(peptide-peptide) or a ternary (peptide-peptide-cation) complex. Third,
complex formation between the
and
cytoplasmic tails involves their carboxyl- and amino-terminal
aspects, respectively. Fourth, as a consequence of cytoplasmic tail
interaction, a complex-specific conformation is stabilized. Taken
together, these data support a model in which the cytoplasmic portions
of the
and
subunits interact with
each other and a cation to form the intracellular domain of the
receptor.
The negatively charged carboxyl terminus of the
cytoplasmic tail was found to be a high affinity
cation binding site. This stretch of 10 amino acids,
(999-1008), bound Tb
with a
1:1 stoichiometry as determined by terbium luminescence and ESIM
spectroscopy (Fig. 1Fig. 2Fig. 3). From
Tb
displacement experiments, we demonstrated that the
cation binding properties of this segment was divalent ion specific
with a preference for Mn
Mg
Ca
. The K
for
Mn
was calculated to be 110 nM and
15
µM for both Mg
and Ca
.
This cation specificity is different from that of the peptides
corresponding to the extracellular cation binding segments of
(33) , which bind these
cations with much lower affinity (K
mM) and little preference. As the intracellular concentrations
of both Mn
and Mg
(49) are
at least 3-100-fold greater than their K
for
, it is predicted that this site should be
constitutively occupied. Constitutive occupancy and/or extremely high
affinity of this site for Tb
are reasonable
explanations for the failure to detect this site in previous analyses
of the cation binding properties of intact
(33, 50) . Using
the Förster theory and equations(51) , we
calculated that the bound Tb
within
(985-1008) would be located 15-34
Å from the tryptophan at
(988). Recently, we
have constructed a molecular model of
(985-1008) using the Biosym Insight II
software. (
)Docking of Tb
within the
carboxyl terminus of the peptide was possible, and the distance to
(988W) was 18-26 Å. In our molecular
model, the carboxyl terminus of
is directed toward
the plasma membrane, due to a predicted turn motif at the Pro-Pro
sequence (
(998-999)). Thus, the Pro-Pro turn
motif in
brings the bound cation close to the
plasma membrane.
Do other integrin subunits contain a cation
binding domain? The clustering of charged residues at the carboxyl
terminus is unique to
. In addition, we have
tentatively identified the carboxylate side chains of
(1003-1004) as providing at least one key
coordination site for cation binding. The
cytoplasmic
tail also contains a highly charged sequence, QEEQEREQLQPHENGE, but not
at its carboxyl terminus. Other integrin
subunits appear to lack
such sequences, but definitive conclusions cannot be drawn until all
the residues that are directly involved in cation coordination in
(999-1008) are precisely defined. The
cytoplasmic cation binding site in
has no homology
to the integrin extracellular ``DXSXS''
cation-binding segments(52) , nor to other known cation binding
domains(53, 54, 55, 56) . However,
the sequence of
-(999-1008), EEDDEEGE, is
identical to the carboxyl-terminal acidic tail of xUBF Xenopus transcription factor(57) , and it is predicted that this
factor also has cation binding properties. Finally, with respect to
cytoplasmic tail of
, our combined terbium luminescent
and ESIM spectroscopic data indicate that this segment does not bind
cation with high affinity in the absence of
. Given
that the
subunits are highly homologous, we expect that none of
the
cytoplasmic tails have strong cation binding properties.
Four independent sets of observations, fluorescence quenching,
terbium luminescence, ESIM spectroscopy, and CD spectroscopy, indicate
that the cytoplasmic tails of and
interact with each other. In addition to our preliminary report
of this observation(58) , two subsequent studies have suggested
that the cytoplasmic tails of
and
can interact(41, 59) . In a modeling study,
Rocco et al. (59) proposed that the two cytoplasmic
tails might interact. Muir et al.(41) designed and
synthesized a model protein (MP-1) containing the cytoplasmic tails of
both
and
covalently-linked
through a constrained helical coiled-coil motif. The coiled-coil motif
tertiary structure was incorporated to mimic the transmembrane domains
of an integrin receptor, with both cytoplasmic domains aligned in
parallel arrangement. In MP-1, the tryptophan emission from
(739) was protected from heavy metal quenching, which
suggested that this residue may be buried in a complex. In our study,
using unconstrained free peptides, complex formation was clearly
demonstrable. It should be noted that in the linked construct of Muir et al.(41) , the proline-proline (Pro-Pro) amino acid
sequence at
(998-999) was mutated to
histidine-threonine(41) . Although these substitutions may very
well alter the conformation of the
cytoplasmic tail
(in
, the Pro-Pro sequence is predicted to be a turn
motif (60) ), complex formation still occurred, suggesting that
a highly constrained conformation for the
subunit
may not be essential for complexation. Furthermore, as MP-1 contained
model transmembrane regions, the combination of data from our study and
from that of Muir et al.(41) provide strong evidence
that this
/
cytoplasmic interaction occurs in a biological
setting, in which
is inserted into
the plasma membrane.
The contact sites in the and
cytoplasmic tails were mapped to the
cation-binding domain of
, its carboxyl terminus,
and to
(721-740) ( Fig. 5and Fig. 7). Furthermore, as substitution of
(721-740) with either
(713-762) or
(715-737)
yielded similar terbium luminescence and ESIM results, we further
concluded that the majority, and most likely all, of sites of contact
of
(999-1008) for
must lie
between
(721) and
(737). This region
is highly conserved among the integrin
subunits. In
, this region encompasses the ``cyto 1''
region, which has been implicated as an
-actinin binding
site(18, 62) . O'Toole et al.(63) also have implicated this region of
as regulating integrin inside-out signaling. These observations
raise the possibility of a role for cytoskeletal elements in modulating
the integrin intercytoplasmic tail interactions, which, in turn, could
influence inside-out signaling. At first glance, this possibility
appears to be contradicted by the data of Kassner et
al.(64) , who demonstrated that a short, nonspecific
stretch of amino acids after the highly conserved
region, the
GFFKR motif, was the only requirement to maintain integrin function in
both
and
. However, it should be
noted that neither
nor
contains a
potential turn motif in its cytoplasmic tail and are unlikely to bind
cation. Furthermore, truncation of
cytoplasmic tail
before the GFFKR motif resulted in the expression of a constitutively
active
receptor(20) ,
which was not reversed with either
or
chimeras, in which the
recognition sequence in
,
(999-1008), was
destroyed(20, 22) . Thus, the regulation of
and
integrins may differ from that
of
, and cation binding to
subunits may individualize integrin functions.
That the cytoplasmic
tail complex of and
forms a
conformational entity, distinct from its free constituents, is
supported by our fluorescence quenching, CD and ESIM spectroscopic
data. In the fluorescence analysis (Fig. 4), we observed
quenching, but no shift in the tryptophan emission spectrum from the
350 nm of each individual subunit. The maximum emission for tryptophan
in an exposed hydrophilic environment is near 350 nm and is shifted
toward the 320-340-nm range in a hydrophobic
environment(65) . Thus, it appears that the three tryptophans
(
(988),
(715), and
(739)) remain in a hydrophilic environment and are
still partially exposed to solvent in the formed complex. One factor
which can quench the intensity of the tryptophan emission spectrum is
the proximity of the tryptophan to a carboxylate side chain of either
aspartate or glutamate(65, 66) . Accordingly,
interaction of the negative carboxyl terminus of
with or near one of the tryptophans in
, most
likely near
(739), could account for the fluorescence
quenching. From the CD analyses, a small increase in helical content of
the
and
cytoplasmic tail complex
was evident (Fig. 6). It is most likely that the increase in
helical content involves the
rather than the
subunit, based upon our observations that the
helical content of
(713-762), but not that of
(985-1008), could be markedly increased by
trifluoroethanol, a helix promoting agent; and predictions of the
helical content of the
versus the
cytoplasmic tails by molecular modelling.
Most interesting was the large increase in
turn
conformation which occurred upon complexation. The
and
cytoplasmic tails are predicted to contain a single turn motif, located
at
(998-999) (Pro-Pro motif) and
(744-747) (NPXY motif). The
NPXY motif is highly conserved in all
integrins and has
been implicated as an
-actinin binding site in
integrins(18, 62) . Thus, our data raises the
possibility that
/
subunit complexation may regulate
cytoskeletal attachment and/or vice versa. A functional role
for the
subunit Pro-Pro turn motif is suggested in our study in
establishing subunit interaction. Although this motif is present in
only a few
subunits, it is present in
, the
other
subunit.
In addition to the binary
peptide-peptide complex, the formation of a
cation ternary complex
was demonstrable by ESIM spectroscopy and terbium luminescence. The
stoichiometry of the complexes was 1:1:1 (Fig. 5B and
7B). We did not detect any major differences in the
conformation of binary versus the ternary complex by CD. Such
was not the case with ESIM spectroscopy, which indicated major
differences between the binary and ternary complexes ( Fig. 7and Table 2). The ESIM spectra of the
(999)
(721-740) binary
complex contained all the expected charge states from 2+ to
8+, with a typical ESIM spectrum envelope, indicating that all
positive charge groups were exposed. In contrast, the ESIM spectrum
envelope of the cation
ternary complex was clearly not typical, in that it contained two
maxima and diminished or missing charge states. These data could arise
if positively charged groups were either buried in a hydrophobic core
of the complex or were involved in hydrogen bonding and in salt
bridges(44, 48) . As the peptides involved are small
(
20 residues) and hydrophilic, the first possibility seems remote.
More likely is the possibility that one or more of the charged residues
is involved in an interaction, such as forming a salt bridge.
The
role of cations in stabilizing protein-protein interactions has become
increasingly apparent from structural analyses. The crystal structure
of two cation binding domains, the A domain of the subunit of
(52) and the epidermal
growth factor-like domain of human clotting factor IX (54) provide examples in which cations participate directly in
and stabilize protein interactions. The distortion of the m/z peaks in the ternary complex could be due to such a rearrangement
in cation coordination sites in
upon complexation
with
. To speculate, complexation of the two
cytoplasmic tails would allow one of
's
cation-coordinating carboxylate side chains to be provided by a residue
in
instead (either by a main chain carbonyl oxygen or
by a carboxylate/carboxyamide side chain). The freed carboxylate side
chain in
could then form the proposed salt bridge
with a lysine or arginine side chain in
. This
arrangement in cation coordination sites would explain the loss of ESIM
peaks and provide a mechanism driving the conformation difference
between the ternary and binary complexes. In support of this
hypothesis, our molecular models
of the
and
cytoplasmic tails do permit interactions of
the carboxyl-terminal aspects of
with the
amino-terminal aspects of
. Such an arrangement is
possible because of the predicted
turns in each cytoplasmic tail.
Nevertheless, the same regions of
also have been
implicated in binding multiple intracellular constituents, including
cytoskeletal proteins (18) and endonexin(61) . It may
be that the different conformational states of the cytoplasmic domain
will dictate which interactions are favored. Studies to test this model
and to assess the role of complex stabilization and destabilization in
initiating and propagating the conformational changes associated with
outside-in and inside-out signaling are in progress.