(Received for publication, January 23, 1995; and in revised form, March 24, 1995 )
From the
Integrin affinities for ligands can change markedly via a
process termed inside-out signaling. We expressed several truncations
of the cytoplasmic domain in conjunction with an
``activating''
subunit chimera,
. Deletion of the 4 C-terminal
residues of the
tail blocked inside-out signaling as
assessed by the binding of an activation-specific antibody, PAC1.
Several additional truncations remained in the low affinity state, but
complete truncation (
717) caused PAC1 binding.
Activation by this truncation mutant did not depend on the
subunit cytoplasmic domain and was resistant to inhibitors of cellular
metabolism and the over-expression of an isolated
cytoplasmic domain. Since deletion of
(Leu
-Asp
) results in a
constitutively activated integrin, this membrane-proximal seven amino
acids of the
cytoplasmic domain is required to
maintain
in a default low affinity
state. The amino acid sequence of this region is conserved among
integrins. Moreover, the conserved membrane-proximal sequence in
subunit tails seems to serve a similar function. Consequently, the
conserved membrane-proximal regions of both integrin cytoplasmic
domains control the ligand binding affinity of the extracellular
domain.
Changes in cell adhesion are often mediated via integrins, a
heterodimeric family of cell adhesion receptors composed of two type I
transmembrane subunits, and
(1) . An important
feature of integrins is their ability to modulate their affinity for
extracellular ligands in response to developmental and environmental
cues, a process termed inside-out signaling
(2) . Affinity
modulation seems to be a general property of integrins, being found in
(3) ,
(4) ,
(5) , and
integrins
(6) . For example, in order for platelets to
aggregate, the integrin
must bind
fibrinogen
(7) , which requires prior platelet
``activation.'' Conformational changes in the extracellular
domain of
seem to be responsible
for regulation of its affinity
(8, 9) . Platelet agonists
increase the affinity of
(activation) via cytoplasmic signaling pathways. These pathways
include heterotrimeric GTP-binding proteins, phospholipid metabolism,
and serine-threonine kinases and may also involve calcium fluxes,
tyrosine kinases, and low molecular weight GTP-binding
proteins
(2, 9, 10, 11, 12, 13) .
How cytoplasmic signals result in changes in the conformation and
ligand binding affinity of the extracellular domain (``inside-out
signal transduction'') of the integrin remains obscure.
Integrin and
cytoplasmic domains play key roles in
inside-out
signaling
(2, 14, 15, 16, 17, 18) .
As summarized in Fig. 1, chimeras composed of the cytoplasmic
domains of various
subunits fused to the transmembrane and
extracellular domains of
established that the
subunit cytoplasmic domains mediate
cell type-specific inside-out signaling (19). Inside-out signaling is
an active cellular process as high affinity ligand binding to these
chimeras and to
was reduced by
inhibitors of oxidative phosphorylation (NaN
) and anaerobic
glycolysis (2-deoxyglucose)
(19) . The
cytoplasmic domain
is important in this process because partial deletion of the
cytoplasmic domain disrupted the high affinity state
of the chimeric receptors
(19) (Fig. 1) and a naturally
occurring point mutation,
S752P, disrupts activation
of
(20, 21) .
Furthermore, overexpression of isolated
or
cytoplasmic domains can inhibit inside-out signaling
(22) (Fig. 1). Thus, physiological inside-out integrin signaling
is an energy-dependent process, with cell type-specific signaling
machinery operating through both
and
integrin cytoplasmic
domains to induce changes in the ligand binding affinity of the
extracellular domain.
Figure 1:
Control of integrin
affinity via the cytoplasmic domains. This schematic summarizes
previous studies (19, 22, 26) analyzing the role of integrin
cytoplasmic domains in inside-out signaling in CHO cells. The nature of
the cytoplasmic domain joined to the transmembrane and
extracellular domain of
is shown schematically with
a shaded box indicating the conserved GFFKR motif. Wild-type
is in the low affinity state
(dash). In contrast, a chimera with the cytoplasmic domain of
is activated as indicated by the star. The
chimera's active state requires the
cytoplasmic
domain because the chimera is not active when co-expressed with
724 (see sequence in Fig. 2). Cellular signaling
events are involved in the activation of this chimera because it is in
the low affinity state in the presence of a combination of inhibitors
of oxidative phosphorylation and anaerobic glycolysis (ATP) or
over-expressed isolated
or
cytoplasmic domains (Free
). Deletion of the GFFKR
motif in
991 or
also
induces the high affinity state. In contrast to the chimeras, high
affinity ligand binding to these constructs is not blocked by
truncation, reduction of ATP, or over-expressed free
cytoplasmic
domains.
The membrane-proximal regions of both
and
cytoplasmic domains are highly conserved across integrin
families
(23) . The putative membrane-cytoplasm interface of both
the integrin
and
subunits is defined by a conserved Lys
residue. In both the
and
subunits this Lys is immediately
followed by a short (4-6 residue) apolar segment and then a
charged region
(23) . The corresponding conserved sequences for
the
and
subunits are XGFFKR and LLviXhDR
(less conserved amino acids are in the lower case, X indicates
a non-conserved amino acid). As summarized in Fig. 1, mutations
that eliminate the highly conserved membrane-proximal GFFKR motif in
the
subunit cytoplasmic domain increase ligand binding affinity,
i.e. ``activate''
(19) . In contrast to the
aforementioned chimeras, the high affinity state of these deletion
mutants is independent of the cell type, cellular metabolism, and the
majority of the
subunit cytoplasmic domain
(19) (Fig. 1). In addition, ligand binding affinity of
these deletion mutants is not reduced by over-expressed isolated
cytoplasmic domains
(22) . Consequently, the activation of these
mutants appears to be independent of cytoplasmic signaling pathways,
and the GFFKR motif is required to maintain a low affinity state in the
absence of physiological activation.
To examine the role of the
cytoplasmic domain in affinity modulation, we have
expressed a series of truncation mutants in Chinese hamster ovary
(CHO)
(
)
cells in conjunction with
or an ``activating''
subunit chimera,
. We found that the deletion of a
conserved membrane-proximal region of the
cytoplasmic
domain results in constitutive high affinity ligand binding that is
independent of the usual cellular signaling pathways and the sequence
of
subunit cytoplasmic domain. Thus, the conserved
membrane-proximal portion of the
subunit, like that of the
subunit, maintains the default low affinity state of
.
In
experiments using metabolic inhibitors, the cells were first incubated
with glucose-free Tyrode's containing 2 mg/ml 2-deoxyglucose and
0.1% sodium azide for 30 min at room temperature prior to the addition
of PAC1. After washing, 5 10
cells were incubated
in a final volume of 50 µl containing 0.1% PAC1 ascites in the
presence or absence of 1 µM Ro43-5054 in
glucose-free Tyrode's containing 2 mg/ml 2-deoxyglucose and 0.1%
sodium azide.
We generated truncation
mutants by placing a series of stop codons at residues
Tyr, Thr
, Tyr
,
Arg
, and Leu
in
. To
assess ligand binding affinity, we examined the capacity of the
transfectants to bind PAC1, a murine IgM
antibody specific for the
high affinity conformation of
(25) . PAC1 mimics the
ligand binding characteristics of the natural ligand,
fibrinogen
(25, 30, 31) and fails to bind to
ligand binding defective mutants of
(32, 33) .
Consequently, the binding of this monoclonal antibody faithfully
reflects the binding of the physiological ligand.
When coexpressed
with
(Fig. 2), a
cytoplasmic domain mutant (
759) with the 4 C-terminal residues deleted, failed to bind
PAC. The lack of PAC1 binding was manifest in all truncations through
724 (Fig. 2), indicating that an almost
complete
tail is required for inside-out signaling.
Surprisingly, high affinity PAC1 binding to
was restored
following the complete truncation (
717) of the
cytoplasmic domain. Moreover, this same truncation,
when co-expressed with wild-type
also bound PAC1
(Fig. 2). Thus, its activating effect did not require the
cytoplasmic domain.
Figure 2:
The
deletion of membrane-proximal residues activates
. CHO cells were co-transfected
with 2 µg of cDNA encoding each of the
constructs
and 2 µg of cDNA encoding wild-type
(
) or
the chimera
(
). After 48
h, the cells were harvested and analyzed for PAC1 binding as described
under ``Materials and Methods.'' To obtain a quantitative
estimate of PAC1 binding, we calculated an activation index, as
described under ``Materials and Methods.'' Depicted are the
mean ± S.E. of three independent experiments for each
construct plotted versus
tail length starting at Leu
= 0. The
cytoplasmic domain sequence is indicated above with
the positions of the stop codons marked as inverted triangles.
Thus, the first stop codon is at Leu
, producing a
-tail length of 0 in
717. Depicted are the
mean ± S.E. of three independent
determinations.
To characterize further the
activating effect of the 717 truncation, we
established a stable cell line expressing
717. This receptor was
expressed as a heterodimer that could be immunoprecipitated with an
anti-
, but not an antibody directed
against the
cytoplasmic domain
(Fig. 3A). Moreover,
717 bound both PAC1 (not
shown) and fibrinogen (Fig. 3B) without prior
activation. Furthermore,
717
was similar to native
in that it
bound a number of conformation-dependent monoclonal antibodies
including A2A9 (34), 4F10
(35) , 2G12
(35) ,
D57
(19) , and mAb15
(36) (data not shown). Since
724 retains the membrane-proximal region and is
not activating, removal of the LLITIHD sequence is responsible for this
effect. To test this idea, we internally deleted residues Lys
to Ile
. When this
construct was
transiently expressed with
, it also bound PAC1
spontaneously (not shown). Consequently, this membrane-proximal seven
amino acids of
Lys
to
Asp
, influences the ligand binding affinity of the
extracellular domain of
.
Figure 3:
A, characterization of
717. Upper panel,
CHO cells stably transfected with
or
717 were
biotinylated, lysed, and immunoprecipitated with an antibody to the
cytoplasmic domain (
tail),
a monoclonal anti-
, or a control
monoclonal antibody (MOPC21). Following SDS-polyacrylamide gel
electrophoresis and electrophoretic transfer, blots were developed with
Avidin-Peroxidase (Vectastain ABC). The luminograms were purposely
overexposed to establish the absence of precipitated
717 when the anti-
tail antibody was used. Lower panel, Western blots of
anti-
or MOPC21 precipitates
described in the upper panel were probed with the
anti-
tail antibody, and the blots were developed with
biotinylated anti-Immunoglobulin and Avidin-Peroxidase. Note the
absence of reactivity in the immunoprecipitate from cells bearing
717. B, fibrinogen
binding to
717.
Fluoresceinated fibrinogen (160 nM) was added to 50 µl of
modified Tyrode's solution containing CHO cells stably
transfected with the indicated integrin in the presence or absence of 2
µM Ro43-5054. After 30 min at 37° C, 450 µl
of Tyrode's solution was added, and fibrinogen binding was
measured by flow cytometry as described under ``Materials and
Methods.'' Depicted are specific fluorescence (mean fluorescence
intensity in the presence of Ro43-5054) for each cell line in the
presence or absence of 2 µM Anti-LIBS6, an activating
monoclonal antibody. Depicted are mean ± S.E. of three
independent determinations.
Figure 4:
High affinity ligand binding induced by
the
717 truncation is independent of
chain
cytoplasmic domain sequences. PAC1 binding was measured in CHO cells
expressing
717 (shaded box) with
996,
, and wild-type
as a control. In
parallel, we co-expressed these
subunit variants with wild-type
(open box). The data depicted are the mean
activation indices ± S.E. of three independent
experiments.
The conserved membrane-proximal GFFKR
motif of the subunit could interact with
(Lys
-Asp
) (23). To determine whether
a GFFKR deletion could reverse the activating effect of the
(Lys
-Asp
) deletion, we co-transfected
717 and the
chimera, in which the GFFKR motif of the
cytoplasmic domain is disrupted
(19) . The transient
expression of
leads to high
affinity PAC1 binding with both
717 and
wild-type
(Fig. 4). Similar data were obtained
with the GFFKR truncation mutant
991
(26) (not shown). Thus, no portion of the
subunit
cytoplasmic domain is required for the activation of
717.
First, we examined the
effect of treatment with the metabolic inhibitors NaN and
2-deoxyglucose on activation of
717. There was no change in
PAC1 binding to CHO cells stably transfected with
717 (Fig. 5). In
contrast, the metabolic inhibitors caused an 8-fold reduction of PAC1
binding to CHO cells expressing a constitutively active
chimera.
Therefore, high affinity PAC1 binding to
717 does not require intact
cellular ATP metabolism.
Figure 5:
717 does not
require cellular metabolism to activate
. Stable CHO cell lines expressing
717 and the constitutively
active chimera
were assayed for PAC1 binding in the presence and absence of the
metabolic inhibitors NaN
and deoxyglucose. Depicted are the
mean activation indices ± S.E of three independent experiments
in the presence (open box) and absence (shaded box)
of the inhibitors.
Next, we examined the effect of
coexpression of an isolated tail on the affinity
state of
717. The free
tail was transiently expressed in the form of a
chimera of the cytoplasmic domain of integrin
subunit
fused to the extracellular and transmembrane domains of the Tac subunit
of the interleukin-2 receptor
(44) . The high affinity state of
717 was resistant to
Tac-
coexpression (Fig. 6). However, high
affinity PAC1 binding was inhibited in the constitutively active
chimera,
,
demonstrating the potency of Tac-
as an inhibitor of
inside-out signal transduction. This result, together with the
observation that the high affinity state of
717 does not require cellular
metabolism suggests that the activation of this receptor is independent
of the usual cellular signaling machinery.
Figure 6:
Overexpression of an isolated
cytoplasmic domain fails to inhibit PAC1 binding to
717. Depicted are flow
cytometry histograms in which fluorescence intensity is plotted on the
abscissa and cell number on the ordinate. PAC1
binding in the presence (open histogram) and absence
(filled histogram) of competitive inhibitor, 1 µM
Ro43-5054, is depicted in panels A-D.
717 specifically binds PAC1
(panel A); this binding is not effected by co-expression of
Tac-
(panel B). The integrin chimera
binds PAC1 with
high affinity (panel C); coexpression of Tac-
blocks this high affinity PAC1 binding (panel D). High
affinity PAC1 binding was restored following the addition of the
activating antibody anti-LIBS6. Tac-
expression levels
were similar in both transfections (data not
shown).
The most important findings presented here are: 1) the
removal of the 7-residue highly conserved inner membrane-proximal
region of the subunit (
717)
increases the ligand binding affinity, i.e. activates the
integrin
. 2) The high affinity
state of
717 is independent of
the cellular signaling machinery as: (a) high affinity ligand
binding is preserved in the absence of the distal portion of the
cytoplasmic domain, a region required for
``physiological'' inside-out signal transduction.
(b) High affinity ligand binding was not reduced by the
addition of inhibitors of oxidative phosphorylation and anaerobic
glycolysis. (c) The overexpression of an isolated
cytoplasmic domain, a potent inhibitor of inside-out signaling,
failed to inhibit the activation of
717. 3) The high affinity
ligand binding conferred by the
717 truncation
is independent of the
chain cytoplasmic domain sequences. Thus,
the highly conserved inner membrane proximal portion of the integrin
subunit influences the affinity state of the extracellular domain
of
.
A complete truncation of
the cytoplasmic domain,
717,
promotes high affinity ligand binding. In contrast,
724, a truncation that spares
(Leu
-Asp
), is not
activating when co-expressed with either
or
. Lys
is proposed to
be the first cytoplasmic domain residue in
(23) , consequently the
Leu
-Asp
would be the most membrane proximal
portion of the
cytoplasmic domain. Disruption of this
sequence seems to be the cause of the increased ligand binding affinity
because a similar result was observed with a 6-residue
``loop-out'' mutation of this sequence that spared the distal
cytoplasmic domain. Moreover,
717 was expressed as a heterodimer and bound several
conformation-sensitive monoclonal antibodies. Since integrin surface
expression is sensitive to mutations and
deletions
(45, 46, 47, 48, 49, 50) ,
these results suggest that
717 is not grossly misfolded. Moreover, this integrin also
bound the physiological ligand, fibrinogen, suggesting that its high
affinity ligand binding state is similar to that of the physiologically
activated receptor.
(Leu
-Asp
) is a conserved
sequence in other integrin
cytoplasmic domains (Fig. 7).
Moreover, mutations that disrupt this sequence in
stimulate the binding of iC3b-coated particles
(51) and in
(5) stimulate VCAM-1 binding. Thus, this
region appears to be important in controlling the affinity state of
multiple integrin families.
Figure 7:
Alignment of subunit cytoplasmic
domains. The cytoplasmic domains of human
subunits, with the
exception of
, were aligned using PILEUP and displayed
using PRETTY programs of the UWGCG package (61). Consensus residues
were defined by their presence in at least four of the sequences and
are displayed in capital letters. Note the conservation of the
sequence in
(Leu
-Asp
)
(dark gray box) in all
subunits. The
actinin-binding decapeptide (56) of
is surrounded by
the hatched box. A light gray box surrounds the
conserved NPXY that has been implicated in particle phagocytosis (51)
in
. Ser
is circled;
substitution of a Pro at this position disrupts bidirectional integrin
signaling (21). The TTT sequence involved in the adhesive function of
is boxed (62).
The high affinity state of
717 is independent of the
usual cellular signaling machinery. Previous studies established that
C-terminal sequences of the
cytoplasmic domain are required for
the active high affinity state of several
integrins
(5, 19, 21) as well as for normal
adhesive function
(52, 53) . We found that the high
affinity state of the chimera,
, was abolished
by removal of as little as 4 C-terminal residues. Yet, the
717 constructs were ``active'' in the
complete absence of these distal sequences. Conversely, the distal
portions of the
cytoplasmic domain are also required for both the
active high affinity state
(19) and for normal cell
adhesion
(14, 37, 38, 39, 40, 41, 42, 43) .
Nevertheless, these sequences were not required for high affinity
ligand binding to
717 because
996
717, a mutant in
which the
cytoplasmic domain has been truncated
following the GFFKR sequence, bound PAC1. In addition, the
physiological integrin high affinity state is sensitive to depletion of
cellular ATP
(19, 54) or overexpression of isolated
cytoplasmic domains (22). The binding of PAC1 to
717 was unaffected by either
of these treatments. Consequently, the high affinity state of
717 does not appear to require
the signaling mechanisms involved in integrin activation.
The
capacity of the membrane-proximal portion of the cytoplasmic
domain to regulate integrin affinity may be due to its interaction with
an intracellular partner. The cytoskeletal proteins
actinin
(55, 56) and talin
(57) bind to
subunit cytoplasmic domain peptides, but the relevant binding sites do
not include
(Leu
-Asp
).
pp125
, a tyrosine kinase, was recently reported to bind
to a
peptide that contains this region
(58) .
Consequently, the role of pp125
in integrin affinity
modulation may bear further study. Finally, the
subunit
cytoplasmic domain may contain a binding site for
(Leu
-Asp
). As already
noted, the membrane proximal
cytoplasmic domain is highly
conserved, and deletions here change ligand binding
affinity
(19) . The proximal portions of the
and
cytoplasmic tails must be parallel to each other and contiguous with
presumptively helical transmembrane regions
(23) . Further, there
appear to be interactions between the
and
tails in
synthetic neo-protein mimics of the integrin cytoplasmic
face
(59) . Thus, the conserved membrane-proximal region of the
integrin cytoplasmic domains may serve to constrain these receptors
into a default low affinity conformation. In addition, it could act as
a conduit for the proposed long range allosteric rearrangements
(60) involved in inside-out integrin signaling.