(Received for publication, January 22, 1996)
From the
Integrins are heterodimeric (,
) cell adhesion
receptors. We demonstrate that point mutations in the cytoplasmic
domains of both the
and
subunits promote constitutive
signaling by the integrin
. By
generating charge reversal mutations, we show these
``activating'' mutations may act by disrupting a potential
salt bridge between the membrane-proximal portions of the
and
subunit cytoplasmic domains. Thus, the modulation of specific
interactions between the
and
subunit cytoplasmic domains
may regulate transmembrane signaling through integrins. In addition,
these activating mutations induce dominant alterations in cellular
behavior, such as the assembly of the extracellular matrix.
Consequently, somatic mutations in integrin cytoplasmic domains could
have profound effects in vivo on integrin-dependent functions
such as matrix assembly, cell migration, and anchorage-dependent cell
growth and survival.
The integrin family of cell adhesion receptors are heterodimers
of and
transmembrane subunits that play key roles in
important biological processes such as inflammation, wound healing, and
cell growth and survival. Integrins modulate their affinity for ligands
via a process termed ``activation'' or
``inside-out'' signaling(1, 2) .
Furthermore, ligand binding to integrins changes the activities of
cytoplasmic kinases, GTPases, and phospholipases
(``outside-in''
signaling)(2, 3, 4, 5) . Thus,
integrins are bidirectional signaling receptors conducting information
both into and out of the cell.
Inside-out signaling may involve the
propagation of a conformational change from the integrin cytoplasmic
domains to the extracellular domains resulting in high affinity ligand
binding(6, 7) . Integrin and
subunit
cytoplasmic domains share a similar membrane-proximal organization with
apolar and polar sequences following sequentially after the
membrane-cytoplasm interface (Fig. 1A). The conserved
sequences for the
and
subunits are -GFFKR and LLv-iHDR
(highly conserved residues are uppercase, less conserved residues are
lowercase, and dashes represent nonconserved residues). Deletion of
these sequences in either
or
subunit cytoplasmic domain
``activates'' integrins, locking them in the high affinity
state(8, 9, 10, 11) . Consequently,
we termed this region the integrin ``hinge.'' Hence, the
capacity of the conserved membrane-proximal motifs to regulate the
affinity state of integrins may depend on an interaction between them
that constrains integrins to a low affinity state. Furthermore, studies
on integrin assembly suggest that there may an interaction between the
membrane-proximal portions of the
and
subunit cytoplasmic
domains(12) .
Figure 1:
The Ala
substitution of specific residues in the membrane-proximal regions of
the and
cytoplasmic domains
activates
. A, schematic
representation of the topology of the transmembrane and cytoplasmic
domains of
. The conserved
membrane-proximal sequences of the
and
are illustrated (highly conserved residues are uppercase, less
conserved residues are lowercase). Integrin
and
cytoplasmic
domains share a similar membrane-proximal organization with apolar and
polar sequences following on sequentially after the W-K
membrane-cytoplasm interface. The conserved sequences for the
and
subunits are -GFFKR and LLv-iHDR (dashes represent unconserved
residues), respectively. Deletion of these sequences lock integrins in
the high affinity state. The
and
subunits lack these conserved membrane-proximal sequences which
suggests that they may signal via a different mechanism to other
integrins. B, flow cytometry histograms illustrating PAC1
binding to
and
(F992A)
. Depicted are flow
cytometry histograms illustrating PAC1 binding in the presence (open histogram) or absence (filled histogram) of
competitive inhibitor, Ro 43-5054 to CHO cells expressing wild type
and
(F992A)
.The peptidomimetic Ro
43-5054, is a selective inhibitor of ligand binding to
(32) . The
(F992A)
transfectants specifically
bind PAC1, illustrating that this mutation activates
. In contrast, cells expressing
wild-type
, which is in the low
affinity state, can only bind PAC1 in the presence of an activating
antibody (anti-LIBS6) that binds to the extracellular domain of
(13) . C, activation indices of the
mutants. To obtain numerical
estimates of integrin activation, an activation index (AI) was
calculated for each of the
mutants. PAC1 binding was measured in CHO cells expressing
wild-type
,
(G991A)
,
(F992A)
,
(F993A)
,
(K994A)
,
(R995A)
, and
(D723A). Depicted are the mean
activation indices ± S.D. of three independent experiments for
each mutation. For each experiment, wild-type
was included as a control.
Transient transfection of CHO cells with activated forms of
had surface expression levels
similar to wild-type
. In 3
determinations, 5 different activated mutants were expressed at 75
± 16% of wild-type
(not
shown).
In this paper, we describe point mutations in
this hinge region that activate .
Moreover, by generating complementary charge reversal mutations, we
show these activating mutations may act by disrupting a potential salt
bridge between the membrane-proximal portions of the
and
subunit cytoplasmic domains. In addition, we demonstrate that these
active mutations can induce the constitutive outside-in signaling as
assayed by the phosphorylation of pp125
and the ligand
independent recruitment of
to
focal adhesions.
To define those membrane-proximal residues of the
subunit important for affinity modulation, we substituted specific
residues in
with Ala. These variants were then
co-expressed in Chinese hamster ovary (CHO) cells with a wild-type
and the binding of PAC1, an antibody specific for the
active conformation of
was used to
define affinity states (14) (Fig. 1B). The Ala
substitution of
(F992),
(F993),
and
(R995) activated
as determined by high affinity PAC1 binding (Fig. 1, B and C). In contrast, the Ala substitution of
(G991) and
(K994) had minimal
effect (Fig. 1B). Therefore, Ala substitution of
specific residues in the conserved membrane-proximal GFFKR motif of the
subunit can activate an integrin.
The Ala substitution of
(F992A),
(F993A), and
(R995A) may activate
by the disruption of an interaction between the
and
cytoplasmic domains. The
cytoplasmic domain contains a highly conserved Asp residue that
is at a similar displacement from the proposed cytoplasm-membrane
interface as the highly conserved Arg-995 of
. This
raises the possibility that the membrane-proximal regions may interact
via a salt bridge formed between
(R995) and
(D723). To test this idea, we expressed
(D723A); this integrin bound PAC1
with high affinity (Fig. 1C). To further test the
proposed salt bridge we constructed the ``charge-reversal''
mutants,
(R995D) and
(D723R). Both
the single mutations,
(R995D)
and
(D723R), were in the high affinity
state and exhibited spontaneous PAC1 binding (Fig. 2). However,
the double charge reversal mutant
(R995D)
(D723R) complemented the
activating effect of the individual mutations. We suggest that this
double mutation may restore the potential salt bridge between the
and
subunits, reforming the structural constraint which prevents
the activation of the integrin. As a control, we examined the affinity
state of double mutants
(R995A)
(D723A),
(R995A)
(D723R), and
(R995D)
(D723A). All of these
variants bound PAC1 spontaneously (data not shown).
Figure 2:
The
membrane-proximal regions of the and
cytoplasmic domains may interact via a salt bridge between
(R995) and
(D723). The activation
indices of
(R995D)
,
(D723R), and
(R995D)
(D723R) are illustrated. The
activation index of
(R995D)
(D723R)
is significantly less (p
0.001) than
(R995D)
and
(D723R). Depicted are the mean
activation indices ± S.D. of three independent experiments for
each mutation.
Ligand binding
to integrins induces changes in cytoskeletal organization,
intracellular pH, and protein tyrosine phosphorylation (outside-in
signaling)(2, 3, 4, 5) . Above we
described cytoplasmic domain mutations that result in constitutive
inside-out signaling. To determine if these mutations caused
constitutive intracellular signaling, we examined the tyrosine
phosphorylation of focal adhesion kinase
(pp125)(19, 20) . Stable CHO cell lines
expressing the mutants
(F992A)
and
(D723A) exhibited the constitutive
phosphorylation of pp125
when in suspension (Fig. 3a). In contrast, cells expressing wild-type
only phosphorylated pp125
when adherent to a fibrinogen matrix (Fig. 3a).
Figure 3:
Membrane-proximal mutations in both the
and
cytoplasmic domains promote
constitutive intracellular signaling. a, CHO cells expressing
the activating mutations
(F992A)
and
(D723A) constitutively
phosphorylate pp125
. CHO cell lines expressing
,
(F992A)
, and
(D723A) were incubated for 90 min
at 37 °C on plates coated with either fibrinogen or BSA. The cells
were in suspension on BSA-coated plates, but adhered to and spread on
the fibrinogen-coated plates. The cells were then processed for
analysis of pp125
tyrosine phosphorylation(17) .
In contrast to cells expressing wild-type
, those expressing
(F992A)
and
(D723A) exhibited tyrosine
phosphorylation of pp125
when incubated in suspension
over BSA. As expected, cells expressing
phosphorylated pp125
when plated on
fibrinogen-coated plates, as did cells expressing
(F992A)
or
(D723A) (data not shown).
Untransfected CHO cells did not phosphorylate pp125
when
plated on BSA or fibrinogen-coated plates. b, recruitment of
(R995D)
and
(D723R) to focal adhesions in a
ligand independent manner. CHO cells were transiently transfected and,
after 48 h, were cultured on fibronectin for 2 h. The cells were then
stained with a mixture of anti-human
(panels
A, B, C, and D) and anti-hamster
(mouse monoclonal 7E2). In all experiments,
was localized in punctuate structures along the cell
edge and along the ventral surface, characteristic of focal adhesions
(data not shown).
(D119Y) had a
uniform cell surface distribution when plated on fibronectin (panel
A) and was not localized to focal adhesions. In contrast,
(R995D)
(D119Y) (panel B)
and
(D723R)(D119Y) (panel
C) were recruited to the focal adhesions.
(R995D)
(D723R)(D119Y) (panel
D) behaved similarly to
(D119Y) and was not present in
focal adhesions.
The association of integrins with specialized cytoskeletal
structures termed focal adhesions, is regulated by ligand binding to
their extracellular domains(21) . As another assay for
outside-in signaling, we analyzed the effect of the activating
mutations on integrin targeting to focal adhesions. To ensure that the
targeting of the variants to focal
adhesions was independent of ligand binding, each was expressed with a
ligand binding-deficient
mutant,
(D119Y) (22) . When expressed in CHO cells
plated on fibronectin, both
(R995D)
(D119Y) and
(D723R)(D119Y) were spontaneously
recruited to focal adhesions formed by endogenous hamster integrins (Fig. 3b). In contrast, the distribution of
(R995D)
(D723R)(D119Y) was diffuse,
similar to that of
(D119Y), and it
was not recruited to focal adhesions formed by the endogenous integrins (Fig. 3b). Therefore, we conclude that these activating
point mutations allow a spontaneous association of the integrin with
the cytoskeleton in the absence of ligand binding. Thus, activating
membrane-proximal point mutations in both integrin
and
subunits can induce constitutive bidirectional transmembrane signaling.
There presently exists no three-dimensional structure of the native
cytoplasmic domains of .
Furthermore, a high resolution structure of this transmembrane protein
may be difficult to acquire. Consequently, a mutational analysis,
similar to those conducted in bacterial chemoattractant receptors (24) and G protein-coupled receptors(23, 26) ,
can provide a viable alternative to develop a structural hypothesis of
transmembrane signaling. The approach described here has led us to
propose a plausible and testable mechanism for integrin signaling.
Indeed, the present studies may provide insight into a general
mechansim of signaling mediated by a variety of transmembrane
receptors. Point mutations can constitutively activate such
structurally diverse receptors such as G protein-coupled receptors,
growth factor receptors, and bacterial chemoattractant
receptors(23, 24, 25, 26) . However,
only in the
-adrenergic receptor has constitutive
bidirectional signaling been reported(26) . In common with
integrins, the ability of specific mutations to activate these
receptors has been ascribed to the release of a
``constraint'' that maintains the receptor in an off state.
As the topography of integrins is comparatively simple, having two
parallel membrane-spanning subunits, we have been able to identify such
a constraint. Utilizing charge reversal mutations, we provide direct
mutational evidence for a salt bridge constraining integrins into a
nonsignaling state.
As reported here, membrane-proximal point
mutations in both integrin and
subunits can cause
constitutive bidirectional transmembrane signaling. Signals from
integrins can influence cell growth and death, and the assembly of the
extracellular matrix(27, 28, 29) . This
raises the intriguing possibility that activating integrin mutations
may produce dominant phenotypes in vivo. The assembly of a
fibronectin matrix, a process important in wound healing and cell
migration during development, is regulated by integrin affinity
state(29, 30) . Therefore, fibronectin matrix assembly
could be perturbed by activating integrin mutations. To test this idea,
we used CHO B2 cells that are unable to assemble a fibronectin matrix
due to a lack of the appropriate integrins(31) . Transfection
of these cells with the constitutively active mutant
(D723R) enabled them to assemble a
fibronectin matrix (Fig. 4). In contrast, CHO B2 cells
expressing wild-type
failed to
make a fibronectin matrix (Fig. 4). Thus, activating point
mutations in the integrin cytoplasmic domains can influence the
assembly of the extracellular matrix. It will be interesting to
determine if such mutations could account for some of the increased
deposition of extracellular matrix that characterizes certain
pathological states.
Figure 4:
Activating point mutations in
promote fibronectin matrix
assembly.
-deficient CHO B2 cells expressing wild-type
(A) and
(D723R) (B) were cultured
in medium supplemented with human plasma fibronectin (480
nM)(30) . CHO B2 cells expressing wild-type
(A) failed to assemble a
fibronectin matrix. In contrast, cells expressing
(D723R) deposited an abundant
fibronectin matrix (B). The
specific peptide-mimetic Ro 44-883 (1 µM) inhibited
fibronectin matrix assembly by cells expressing
(D723R) (data not shown). Similar
results were obtained with CHO B2 cells expressing the active mutant
(F992A)
(data not
shown).