From the
The ligand binding affinities of the integrins are regulated
through their cytoplasmic domains. To identify specific residues that
are involved in this process, we have generated mutants in the
The regulated adhesion of cells to each other or to elements in
the extracellular environment is crucial in normal physiology. The
integrins, a widely distributed family of
The cytoplasmic
sequences of the integrins appear to be involved in these
bi-directional signaling events. Indeed, several functions have been
associated with the
Mounting evidence implicates a role for both
Little is
known regarding specific
Integrin cDNA constructs were expressed in CHO cells by
liposome-mediated transfection. Twenty-four hours before transfection,
CHO cells were plated at a density of 10
PAC1 binding (FITC staining) was analyzed only on
those gated cells positive for surface expression of
Point mutations were first
generated in the cytoplasmic sequences of
The results described above: 1) identify a distinct set of
The
most significant reductions in constitutive PAC1 binding were achieved
by substitutions of the
In
contrast, amino acid substitutions within the distal NP XY
motif of
The phosphorylation of integrin
tails has been proposed to play a role in affinity modulation. In our
studies, two variants which completely block PAC1 binding,
It is apparent that
mutations at several sites within the
It is also
noteworthy that similar qualitative effects on PAC1 binding were
observed whether the
In summary, we have defined a
subset of
We thank Mark Ginsberg for suggestions and critical
review of the manuscript.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
and
tails and coexpressed them in
Chinese hamster ovary cells with constitutively active
subunits.
These
subunits are chimera of extracellular and transmembrane
joined to the cytoplasmic domains of
,
, or
and confer
an energy-dependent high affinity state when expressed in Chinese
hamster ovary cells. The affinity state of these transfectants was
determined by analyzing the binding of PAC1, an antibody that
specifically recognizes the activated form of the reporter group,
extracellualar
. We have identified
point mutants in several areas of the
tails, which result in a
reduced ability to bind ligand. Complete abolition of PAC1 binding was
obtained with mutants in an NP XY motif found in many integrin
subunits and implicated in the internalization of other cell
surface receptors. Similar effects on PAC1 binding were observed
whether coexpression was with
chimera containing
,
, or
cytoplasmic sequences. These studies identify a novel role for
the NP XY motif in the regulation of integrin binding affinity.
,
heterodimers
(1) , mediate many of these events. Members of this family are
characterized by an ability to dynamically regulate their ligand
binding affinity. In a process termed ``activation'' or
``inside-out signaling,'' integrins change extracellular
conformation and alter ligand binding affinity and cellular adhesion
(1) . Following ligand binding, events are triggered inside the
cell. ``Outside-in signaling'' events include changes in cell
shape, integrin localization, intracellular pH, induction of protein
phosphorylation, and gene transcription
(1) .
subunit cytoplasmic domain. For instance,
these sequences contain sufficient information for specifying
localization to focal adhesions
(2, 3) , are required
for cellular adhesion
(4, 5, 6) , and are
capable of associating with the cytoskeletal proteins
-actinin
(7) and talin
(8) . Independent expression of
tails
results in an inhibition of endogenous integrin function in matrix
assembly, cell spreading, and cell migration
(2, 9) . In
addition,
cytoplasmic domains have copies of an internalization
sequence, NP XY
(10) , and several residues that are
potential phosphorylation sites. The functional properties of
subunit cytoplasmic sequences are less well defined. Nevertheless,
these domains limit the cellular localization of integrins
(11, 12, 13) and play a role in cellular
adhesion, migration, and collagen gel contraction
(14, 15, 16, 17, 18, 19) .
and
cytoplasmic domains in inside-out signaling and the maintenance of high
affinity binding. The identity of the
tail determines the
affinity state of recombinant integrins expressed in heterologous cells
(20, 21) , while deletion of these sequences disrupts
cellular adhesion
(14, 15, 16, 17, 19) . On the
other hand, a point mutation in the
tail has been
associated with an activation defect of
both in vivo (22) and in a recombinant system
(20) . Furthermore, independent coexpression of the
, but
not the
, cytoplasmic domain can abolish ligand binding by
constitutively active, transfected integrins
(23) .
or
cytoplasmic residues required
for high affinity binding or how these relate to other functional areas
in these domains. To map these sequences in the
subunit, we have
mutagenized the
and
tails, joined
them onto extracellular and transmembrane
, and
coexpressed these variants with
cytoplasmic
chimeras that confer constitutive ligand binding properties
(20) . The affinity state of these transfectants was determined
by analyzing their ability to bind PAC1
(24) , a monoclonal
antibody that specifically recognizes the active conformation of the
reporter group,
. Our results have
identified several residues whose substitution reduces constitutive
ligand binding. Complete abolition of PAC1 binding was observed with
variants in an NP XY sequence. This role in affinity modulation
identifies a novel function for this motif. Furthermore, these studies
discount a role for phosphorylation of integrin tails in the regulation
of binding affinity and suggest cytoplasmic splice variants which lack
these sequence elements may be unresponsive to normal inside-out
signaling pathways.
Antibodies and Reagents
The purification and
characterization of several antibodies (D57, anti-LIBS6, PAC1) has been
described elsewhere
(20, 24, 25) , while a
polyclonal antibody against was generated by
previously described methods
(26) . The antibody D57 was
biotinylated with biotin- N-hydroxysuccinimide (Sigma)
according to manufacturer's directions. A peptide-mimetic
compound (Ro43-5054), which specifically blocks binding to
, was a generous gift from Beat
Steiner (F. Hoffman LaRoche, Basel, Switzerland). Oligonucleotides were
synthesized on a model 391 DNA synthesizer (Applied Biosystems Inc.).
Restriction endonucleases, TaqI polymerase, and other enzymes
were from Boehringer Mannheim.
cDNA Constructs
The generation of several wild
type (), chimeric (
,
,
,
), or mutant
(
724,
S752P) integrin constructs
has been described previously
(20, 21, 27) .
Additional
chimera were made by exchanging mutant
cytoplasmic domains for those from wild type
.
Sequences encoding the
mutants S790M, T793V/T794V,
and N797I were recovered by HindIII digestion of the
appropriate pRneo constructs
(28) and ligated into
HindIII-cut pCDM8 (Invitrogen, San Diego, CA). These vectors
were then digested with BspHI and DraIII, and the
fragment containing
cytoplasmic sequences was ligated
to a corresponding BspHI- DraIII fragment from CD3a
(27) containing
transmembrane and
extracellular domains. Cytoplasmic sequences encoding the
variants F771L, E774V, N785I, Y788A, YTRF, S790D, Y800F, and
Y800A were first amplified by the polymerase chain reaction with the 5`
oligonucleotide: GTAAAATCACTGCAGTTTGCCCTA, and the 3` oligonucleotide:
TTGATTTGGAAGCTTCTGATGATC. Amplified fragments were digested with
HindIII and PstI and ligated into HindIII-
and PstI-digested pCDM8. These constructs were then digested
with BspHI and DraIII and ligated as above to a
corresponding BspHI- DraIII fragment from CD3a. The
Y788F variant of
was generated in a two-step
polymerase chain reaction strategy. Overlapping fragments containing
the desired amino acid substitution were first generated in two
amplifications on wild type
sequences using the
oligonucleotide pairs:
GGTGAAAATCCTATTTTTAAGAGTGCCG-GTAAGGTTCCTTCACAAAGAT and
CGGCACTCTTAAAAATAGGATTTTCACC-ATACCTGCAACCGTTACTGCC. The amplified
fragments were combined, denatured at 85 °C for 10 min, and allowed
to cool to room temp. The ends were then filled in with Sequenase
(29) , and double-stranded fragments amplified with the
oligonucleotides: GTAAGGTTCCTTCACAAAGAT and ATACCTGCAACCGTTACTGCC.
Amplified products were digested with AflII and XhoI
and inserted into the AflII- XhoI-digested
chimera, thereby replacing wild type
cytoplasmic sequences with the mutant sequence. The
cytoplasmic variants F727A/F730L/E733V, Y747A, S752A, and Y759A
were generated in a two-step polymerase chain reaction strategy as
above for the
variant Y788F. The final amplified
product was digested with MluI and XhoI and then
ligated into pCDM8. These constructs were then linearized with
MluI and ligated to an MluI fragment from CD3a
containing the remainder of
coding sequences. The
B construct was made by polymerase chain reaction
amplification with the oligonucleotides:
CGAAAAGAATTCGCTAAATTTGAGGAAGAACGCGCCAGAGCAAAATGGGACACCAGTAAGAGAC and
AGAGTCCCCGGGTCAGACCAATGACTTTAGAAAACGCCCAGCCCCGTCTCTTACTGTGC. The
amplified product was digested with EcoRI and SmaI
and subcloned into CD3a cut with EcoRI and SmaI.
Finally, this construct was linearized with EcoRI and ligated
to an EcoRI fragment from
containing its
extracellular and transmembrane sequences. All constructs were
identified by restriction digestion, purified by CsCl centrifugation,
and verified by DNA sequencing before transfection.
Cell Culture and
Transfection
CHO()
cells were obtained
from American Type Culture Collection (ATCC, Rockville, MD) and
maintained in Dulbecco's modified Eagle's medium (DMEM,
BioWhittaker Inc., Walkersville, MD) supplemented with 10% fetal bovine
serum (BioWhittaker Inc.), 1%
L-glutamine (Sigma), 1%
penicillin and streptomycin (Sigma), and 1% nonessential amino acids
(Sigma).
cells/100-cm dish.
A total of 2 µg of each
and
construct and 20 µl of
Lipofectamine (Life Technologies, Inc.) reagent were incubated at room
temperature for 10 min in 180 µl of unsupplemented DMEM.
Unsupplemented DMEM (3.8 ml) was then added and the DNA-liposome
complexes overlaid onto the cells. The cells were incubated for 6 h at
37 °C, washed with phosphate-buffered saline, and then incubated at
37 °C with complete medium. Medium was changed after 24 h and the
cells harvested and analyzed at 48 h.
Immunoprecipitation
Transfectants were
surface-labeled with I by the lactoperoxidase-glucose
oxidase method
(30) and then solubilized in lysis buffer (10
m
M Hepes (pH 7.5), 0.15
M NaCl, 50 m
M octyl
glucoside, 1 m
M CaCl
, 1 m
M MgCl
, 1 m
M phenylmethylsulfonyl fluoride, 0.1
m
M leupeptin, and 10 m
M N-ethylmaleimide).
Cell extracts were immunoprecipitated overnight with preimmune serum or
with a polyclonal antiserum directed against
and then
incubated with protein A-Sepharose CL-4B for 1 h at room temperature.
The Sepharose beads were washed extensively in lysis buffer,
resuspended in sample buffer, and boiled for 5 min. After
centrifugation, immunoreactive proteins were resolved on non-reducing,
7.5% acrylamide gels, the gels dried, and bands visualized by
autoradiography.
Flow Cytometry
Integrin affinity state was
determined by a two color flow cytometry assay. Transient transfectants
were harvested in Tyrode's buffer
(31) containing 0.1
mg/ml
L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated
trypsin (Worthington) and 3.5 m
M EDTA. After a 5-min
incubation at room temperature, the cells were diluted with
Tyrode's buffer containing 0.1% soybean trypsin inhibitor (Sigma)
and 10% bovine serum albumin (Sigma), collected by centrifugation at
1200 rpm for 5 min, and washed once in Tyrode's buffer containing
10% bovine serum albumin. The cells were resuspended in Tyrode's
and 5 10
analyzed for PAC1 binding. Briefly, the
cells were incubated in a final volume of 50 µl with a 1:25
dilution of PAC1 ascites in the presence or absence of 1 µ
M Ro43-5054. After 30 min at room temperature, the cells were
diluted to 0.5 ml with Tyrode's buffer, pelletted, and
resuspended in 50 µl of Tyrode's buffer containing a
biotinylated,
-specific antibody,
D57. After 30 min on ice, the cells were diluted to 0.5 ml with
Tyrode's buffer, pelletted, and resuspended in 50 µl of
Tyrode's buffer containing 4% phycoerythrin-conjugated
strepavidin (Molecular Probes Inc., Junction City, OR) and 10%
FITC-conjugated goat anti-mouse IgM (Tago, Burlingame, CA). After 30
min on ice, the samples were diluted to 0.5 ml with Tyrode's
buffer and analyzed by flow cytometry on a FACScan (Becton Dickinson,
San Jose, CA).
(phycoerythrin staining).
Histograms of FITC staining in the presence or absence of a competitive
inhibitor of PAC1 binding, Ro43-5054, were compared. High
affinity integrins were identified by a rightward shift of the
histogram in the absence of Ro43-5054. PAC1 binding was also
analyzed after incubation with 6 µ
M anti-LIBS6. This
antibody induces the high affinity state of
and reports optimum ligand binding
(25, 32) . As a quantitative measure of affinity state
we have expressed the data as an activation index for each
,
pair. The activation index was defined as: 100
( M - M
/ M
-
M
), where M = median fluorescence
intensity of PAC1 binding, M
= median
fluorescence intensity of PAC1 binding in the presence of
Ro43-5054, M
= median fluorescence
intensity of PAC1 binding in the presence of anti-LIBS6, and
M
= median fluorescence intensity of PAC1
binding in the presence of anti-LIBS6 and Ro43-5054.
To identify residues in Cytoplasmic Mutation Does Not
Disrupt Surface Expression
cytoplasmic domains that are involved in the regulation of integrin
affinity states, we have generated point mutations in these sequences
and coexpressed these variants in CHO cells with
chimeras. These
subunit chimeras consist of the
extracellular and transmembrane domains of
joined
to the cytoplasmic sequences of
,
A,
or
B. In contrast to wild type
,
these
chimeras conferred an energy-dependent, constitutive high
affinity state when coexpressed with constructs encoding the wild type
or
cytoplasmic domains
(20) . Thus, the affinity state of the extracellular reporter
group,
, was determined by the
identity of its cytoplasmic sequences.
(Fig. 1), a
natural partner for the
and
cytoplasmic domains. Areas targeted included several hydroxylated
residues which represent potential phosphorylation sites, the two
NP XY internalization motifs, and residues in the putative
-actinin binding site. Many of these residues have also been
implicated in the recruitment of
to focal adhesions
(28) . These variant cytoplasmic sequences were first joined to
the extracellular and transmembrane domains of
generating
subunit chimeras. To determine if cytoplasmic
mutations affected integrin expression, cells cotransfected with these
chimeras and
(a chimera
consisting of extracellular and transmembrane
joined to cytoplasmic
) were analyzed for
surface expression by flow cytometry. Constructs encoding the wild type
and variant forms of the
cytoplasmic domain
demonstrated comparable levels of surface expression (Fig. 2,
panels A, D, and G) when stained
with an anti-
-specific monoclonal
antibody. Indeed, all of the
variants utilized in
this study were well expressed. In addition, no differences between
wild type or mutant
expression were observed when
cotransfection was with the
A or
B
chimeras. Heterodimer formation and surface expression of
variants was also confirmed by the immunoprecipitation of
iodinated transfectants (data not shown). Thus, the
point mutations we have examined do not disrupt normal subunit
association or cell surface expression.
Mutations in the
To examine the functional
effects of Cytoplasmic Domain
Abolish Inside-out Signaling
cytoplasmic mutation on ligand binding,
transfectants were analyzed for their ability to bind PAC1 by flow
cytometry (Fig. 2). Only those cells positive for surface expression
(denoted by M1 in Fig. 2) were gated and analyzed for
PAC1 binding. Like cells transfected with wild type
( panel B), those expressing the Y788F variant
bound PAC1 constitutively ( panel H). Binding was
specific since it could be blocked with a ligand-mimetic compound,
Ro43-5054. In contrast, cells transfected with the Y788A variant
failed to bind PAC1 ( panel E). Binding in the latter
case was observed only in the presence of an activating antibody,
anti-LIBS 6.
Figure 2:
Representative FACS histograms. Cells
transfected with the and
chimera listed on the left were
stained and subjected to flow cytometry as described under
``Materials and Methods.'' Phycoerythrin staining, indicative
of antibody D57 binding and integrin surface expression levels, is
illustrated in panels A, D, and G.
Note that each heterodimer was expressed at similar levels. Only those
cells positive for surface expression (denoted in each histogram by
M1) were gated and analyzed for FITC staining, indicative of
PAC1 binding ( panels B, C, E,
F, H, and I). Histograms of PAC1 binding in
the absence of inhibitor ( solid line) were
superimposed upon those analyzed in the presence of 1 µ
M Ro43-5054 ( dotted line). A rightward shift
is indicative of an active integrin. Histograms of PAC1 binding done in
the presence of 6 µ
M anti-LIBS6 are shown in the last
column ( panels C, F, and I). All
histograms represent an acquisition of 10
events.
Coexpression with the
and
Y788F constructs result in a
constitutively active integrin ( panels B and
H), while expression of
Y788A results in an inactive integrin ( panel E).
To numerically express PAC1 binding and affinity state,
we have determined an activation index (AI) for each ,
combination as described under ``Materials and Methods.'' The
AI values were determined for each of the
tail
variants when cotransfected with the
chimera and compared to the wild type sequence (Fig.
3 A). When analyzed in this way, the group of
variants fell into three functional categories: those that
conferred near wild type levels (70-100%) of PAC1 binding (Y788F,
Y800F), those that result in reduced (10-70%) binding (F771L,
E774V, S790M, S790D, T793V/T794V, N797I, Y800A), and those that
abolished (<10%) binding (N785I, Y788A, YTRF). To determine if these
effects were specific for the
cytoplasmic tail, the
variants were also transfected with other
constructs. Similar effects on PAC1 binding were observed when
coexpression was with
chimeras containing the
cytoplasmic sequences from the
(Fig. 3 B) and
(data not shown)
subunits. These results suggest that mutations at several sites within
the
cytoplasmic domain reduce energy-dependent ligand
binding.
Figure 3:
Activation indices of
cytoplasmic variants. As outlined under ``Materials and
Methods'' and ``Results,'' we have determined an
activation index for each
variant after
cotransfection with
chimera containing
( A) or
( B) cytoplasmic
sequences. Values for cotransfection with the wild type
chimera
(
) are depicted at the bottom of each
graph. The lowest activation indices (least PAC1 binding) were those
obtained by transfection with the N785I, Y788A, and YTRF variants. A
negative activation index results when the mean fluorescence intensity
of PAC1 binding in the presence of inhibitor is greater than that value
in the absence of inhibitor.
Mutations in the
The Cytoplasmic Domain
Abolish Inside-out Signaling
and
cytoplasmic domains are approximately 60% identical
and contain conservative amino acid substitutions at several other
positions. This high degree of conservation suggests the possibility
that analogous regions in both tails have functional importance. To
look at this possibility, we have generated point mutations in the
tail at sites corresponding to those residues in
which affect its binding function (Fig. 1).
Like those
point mutations, cytoplasmic variants of
do not disrupt normal patterns of
,
subunit
association or cell surface expression (data not shown). With respect
to functional properties, transfectants expressing a
cytoplasmic truncation (
724) or the F727A/F730L/E733V,
Y747A, and S752P, variants demonstrate a greatly reduced (<10%)
ability to bind PAC1 when coexpressed with the
chimera (Fig. 4 A).
Although still reduced relative to wild type
, the
Y759A and S752A variants exhibit a somewhat greater AI. Similar effects
on PAC1 binding were observed when these
variants
were coexpressed with the
(Fig. 4 B) or
A (data not shown) chimeras. Thus analogous residues
in the
and
tails affect ligand
binding affinity. Finally, we have generated a construct
(
B) encoding an alternately spliced form of the
cytoplasmic domain
(33) . This variant
eliminates 8 of 10 hydroxylated residues and the NP XY motifs
in this tail. Coexpression of this construct with the constitutively
active
chimera also resulted in a
low affinity receptor whose binding function was only elicited with
anti-LIBS6 (Fig. 5).
Figure 1:
Wild type and variant
cytoplasmic sequences. Illustrated at top of figure is a schematic of
the integrin chimeras used. The chimeras consist of the
extracellular and transmembrane domains of
joined
to the cytoplasmic domains of
,
, or
. Also represented are the wild type
subunit and a
chimera of extracellular and transmembrane
joined to cytoplasmic
. Listed
below, in single-letter code, are the cytoplasmic sequences of
(beginning with Lys
) and the
chimera.
sequences
begin with the underlined histidine. The position of amino
acid substitutions or a termination codon ( asterisk) in these
domains are illustrated. Dashes represent unchanged amino
acids, while mutant name is listed on the
right.
Figure 4:
Activation indices of
cytoplasmic variants. As outlined under ``Materials and
Methods'' and ``Results,'' we have determined an
activation index for each
variant after
cotransfection with the
( A) or the
( B) chimera. Values for cotransfection with wild type
are depicted at the bottom of each graph. A negative
activation index results when the mean fluorescence intensity of PAC1
binding in the presence of inhibitor is greater than that value in the
absence of inhibitor.
cytoplasmic residues required for inside-out signaling (Fig. 6),
2) define a functional role for the NP XY motif in this is
process, 3) argue against the hypothesis that the phosphorylation of
integrin tails is required, and 4) suggest cytoplasmic splice variants
are not responsive to normal inside-out signaling mechansims.
cytoplasmic residues
Asn
and Tyr
. These residues encompass an
NPIY sequence of this domain, a conserved motif (NP XY) found
in many
subunit cytoplasmic domains. Indeed, the
(34) ,
(35) ,
(36) ,
(37) ,
(38) , and
(39) cytoplasmic
sequences typically contain one of these motifs within 30 residues of
the transmembrane domain (Fig. 6). In addition, the
and
tails also have a more distal
NP XY/F sequence, while other
subunits contain a distal
N XXY/F motif. The NP XY motif has been
identified in the cytoplasmic sequences of several cell surface
receptors
(10) . It functions as an internalization sequence for
the LDL receptor
(10) and is essential for cellular
transformation by the polyomavirus middle T antigen
(40) .
Functional studies suggest that the Asn and Pro residues of this motif
are invariant. Substitution of either in the LDL receptor results in a
significant reduction of coated pit-mediated endocytosis
(10) .
While internalization was maintained when the terminal Tyr of this
motif was substituted with another aromatic residue (Phe, Trp),
substitution with a non-aromatic residue resulted in decreased LDL
receptor internalization
(41) . These functional studies are
similar to ours which demonstrated that substitution of Asn
completely abolished PAC1 binding. Furthermore, we have observed
a complete loss of PAC1 binding with the Y788A substitution but near
wild type levels of binding with the more conservative Y788F variant.
As predicted from these observations, we have determined that a
cytoplasmic splice variant lacking the NP XY
sequence is incapable of supporting constitutive PAC1 binding. Splice
variants of
lacking its NP XY motifs would
also be predicted to be unresponsive to inside-out signaling
mechanisms.
Figure 6:
Alignment of cytoplasmic sequences.
Listed above are the wild type cytoplasmic sequences of the
,
,
,
,
, and
subunits.
Those residues whose substitution abolished constitutive PAC1 binding
are indicated by an asterisk, while those residues whose
substitution results in a reduced ability to bind PAC1 are
boxed.
A second internalization motif, originally identified in
the transferrin receptor, is Y XRF
(42) . Molecular
modeling
(42) and NMR analysis of wild type peptides
(43) suggest that both NP XY and YTRF form a reverse
turn conformation. This conformation is hypothesized to present a
recognition domain to adaptins in clathrin-coated pits thereby
mediating uptake
(43) . Interestingly, peptides derived from
receptors defective in endocytosis do not form the turn conformation.
In our studies, the substitution of NPIY for YTRF in the tail completely abolished PAC1 binding. Since this substitution
might not disrupt overall structure, the abolition of PAC1 binding is
probably related to the loss of specific NPIY residues. It is
conceivable that the NP XY motif could represent a recognition
site for regulatory, intracellular moieties. Interestingly peptides
spanning this region also inhibit talin-integrin interactions in
vitro (44) . Thus, our results suggest a novel role for the
membrane-proximal NP XY motif in the regulation of ligand
binding. Meanwhile, the role of the NP XY motif in integrin
internalization is ambiguous. Expression of a
truncation that lacks its two NP XF motifs or an F-A
substitution variant in the membrane-proximal NP XF both result
in a loss of internalization
(45) . In contrast, substitution of
the tyrosines for serines in the two NP XY motifs of
had no effect on
internalization
(46) . Finally, the NP XY motifs
of
do not mediate internalization of
.(
)
(N797I, Y800A, Y800F) had a lesser effect on
receptor affinity. Similarly, a mutation in
(Y759A)
analogous to
Y800A shows a reduced but variable
ability to bind PAC1. Thus these residues do not seem to be nearly as
crucial for the maintenance of high affinity binding as those in the
first NP XY. Consistent with these results, this motif is not
nearly as well conserved amongst
cytoplasmic sequences. As noted
above, only
and
possess an exact
distal NP XY/F sequence. While these sequences may play a minor
role in the regulation of binding affinity, this motif in
is required for cellular adhesion. Substitution of phenylalanine
in the distal NP XF of this subunit abolished
-intracellular adhesion molecule 1
(ICAM-1) interactions
(5) .
S752P and
Y788A, also represent potential
phosphorylation sites. However, more conservative, yet
phosphorylation-defective, substitutions at these residues (S752A and
Y788F) results in greater or near wild type levels of PAC1 binding
(Figs. 3 and 4). Thus the abolition of binding by S752P and Y788A is
due to an effect other than on phosphorylation. Similarly, other
(S790M, S790D, T793V/T794V, Y800A) and
(Y747A, Y759A) variants, which eliminate potential
phosphorylation sites, retain measurable levels of PAC1 binding. These
results argue strongly against a role of phosphorylation in the
enhancement of binding affinity and are consistent with others that
discount a role for phosphorylation in promoting cellular adhesion
(5, 14, 47, 48) .
tail reduced or abolished
PAC1 binding. In addition to those variants discussed above,
(F771L, E774V) and
(F727A/F730L/E733V) substitutions within the putative
-actinin binding site
(7) also reduced constitutive PAC1
binding. One explanation for these observations is that the dynamic
regulation of ligand binding does involve many sites in the
tail.
Alternatively,
tail folding and conformation may be extremely
sensitive to amino acid substitution and it is these conformational
changes which disrupt normal signaling pathways.
or
point
mutations were coexpressed with chimeras containing the
,
or
tails. It
is possible that high affinity binding specified by these different
tails might involve a single regulatory pathway and thus the same
cytoplasmic sequence elements. Alternatively, each integrin may
be independently regulated yet utilize the same
sequence
elements. In this latter case integrin specific regulation would be
mediated by sequences in the
chain. Distinguishing between these
possibilities and identifying the mechanisms of cell-specific affinity
modulation awaits further study.
cytoplasmic residues required for inside-out signaling.
Substitutions within a membrane-proximal NP XY motif were the
most effective in the abolition of binding, suggesting a novel function
for this sequence. Defining the interaction of intracellular elements
with these sequences will contribute to an understanding of the
mechanisms of affinity modulation.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.