(Received for publication, July 3, 1995; and in revised form, October 13, 1995)
From the
The integrins can be expressed on the surface
of cells in a latent form, which is activated by a variety of stimuli.
As an approach to examining the transition to an active receptor, a
panel of stimulatory antibodies to
were produced and
characterized. These antibodies induced adherence of the T-leukemic
cell line Jurkat to collagen and fibronectin. Competitive antibody
binding assays indicated the existence of at least three distinct
epitope clusters A (B3B11, JB1B, 21C8), B (B44, 13B9), and C(N29)
defined by the indicated antibodies. Two antibodies to the A site, JB1B
and B3B11, were shown to localize to positions 671-703 and
657-670, respectively, of the
. This region is
located in an area encompassing a predicted disulfide bond between
linearly distant cysteines in
(Cys
-Cys
). The homologous region of
the
integrin (490-690 and 602-690) has
been shown to be one of the sites recognized by stimulatory antibodies
to ligand-induced binding sites. The present results indicate the
existence of multiple stimulatory regions and suggest considerable
homology between the locations of
and
regulatory sites.
Members of the integrin family mediate cellular adherence to a variety of extracellular proteins (e.g. collagen, fibronectin, vitronectin, laminin) or to cell surface-associated molecules through homotypic or heterotypic (1, 2, 3) interactions. Several cell types (e.g. platelets, leukocytes) express some of the integrins on their surfaces in latent forms. It is only subsequent to cell activation that these molecules display binding potential for their cognates(4, 5, 6, 7) .
The
mechanisms responsible for the transition to an activated state are
unknown. However, it is apparent that receptor conformational charges
are associated with the generation of a functional
complex(8, 9, 10) . A number of antibodies
that can activate receptors have been described for the
integrins(11, 12, 13, 14, 15, 16, 17, 18) .
In the case of , a number of the
stimulatory antibodies (anti-LIBS1, D3GP3(14) ,
anti-LIBS2(15) , anti-LIBS3, anti-LIBS6(16) , and
AP5(17) ) recognize epitopes that are expressed on ligand
occupancy of the receptor. Some of these anti-LIBS reagents have been
particularly useful in probing aspects of cation requirements for
receptor function and for the localization of possible regulatory
regions of the molecule(10, 17) . However, there is
considerably less information about regulatory sites on other members
of the integrin family.
The integrins play a
critical role in the functional activity of lymphocytes by influencing
cellular distribution patterns and by functioning as costimuli for
differentiation or proliferation induction(20, 21) .
Similar to the
system, the
functional status of the
integrins is stringently
controlled with activation being induced by a variety of stimuli
including antibodies to the
chain (11, 12, 13) .
Two groups have reported on
the locations of some of the regulatory regions of the integrins using monoclonal antibodies to the
chain(22, 23) . The result of one study
suggested that there may be multiple regulatory regions(23) ,
while a second indicated that the majority of stimulatory and
inhibitory antibodies reacted with a very restricted region of the
molecule(22) . Studies from the
integrin system would seem to support the existence of multiple
distinct regions defined by regulatory antibodies.
As an approach to
defining the number and location of potential regulatory sites on the
chain, we have identified and characterized six
stimulatory antibodies. The relative positions of the epitopes
recognized by these antibodies were determined by competitive binding
studies. Furthermore, the location of one set of such epitopes in the
molecule was determined.
CHO cells transfected with human
, provided by Dr. R. Juliano(27) , were
cotransfected by electroporation with pFnR
, a construct containing
the full-length human
gene provided by Dr. E.
Ruoslahti(28) , and pREP4, a vector carrying the hygromycin
reductase marker, provided by Dr. M. Tykocinski(29) . The cells
were cultured for 48 h in RPMI with fetal bovine serum containing G418
(80 µg/ml), after which hygromycin B was added (300 units/ml).
Transfectants reacting with the anti-human
monoclonal, JB1(24) , were selected by fluorescence
activated cell sorter and cultured in the selection media. This cycle
was repeated three times to achieve stable lines. It was found to be
necessary to use the CHO
as the host cell for
expression, as in our hands the wild type CHO cells
did not efficiently express the human
gene product.
Specificity of the transfectants were confirmed using a panel of
monoclonal antibodies to
including JB1A(30) ,
3S3(31) , and JB1(24) .
Blocking assays were
performed by preincubating antibodies (150 ng/ml) with the indicated
peptide concentrations for 2 h. The antibodies were then added to Nunc
plates coated with affinity-purified placental integrin(32) . The color was developed after reaction of
the wells with alkaline phosphatase-conjugated rabbit anti-mouse
immunoglobulin and substrate.
Figure 1:
Specific binding of antibodies to CHO
cells transfected with human integrin. The binding of
antibodies to CHO cells coexpressing human
and
(solid line) but not
to CHO wild type or those expressing only human
(dotted lines) indicates the specificity of these
antibodies for human
.
Previous studies
had demonstrated that Jurkat cells display a low level of adherence to
immobilized collagen(7) . However, following stimulation with
phorbol 12-myristate 13-acetate (7, 12) or some
antibodies to the or
chains(12) , there was a marked increase in adherence.
When a panel of anti-
reagents was analyzed for the
effects on Jurkat adherence, six stimulatory antibodies were identified (Fig. 2). This stimulation was detectable within 30 min of
exposure and lasted for several hours ( (12) and data not
shown). This effect was not a property of all antibodies to
as 3S3 (31) and JB1 (24) did not induce
adherence. In the case of 3S3, there was inhibition of the low level of
spontaneous binding of these cells to collagen.
Figure 2:
The induction of Jurkat cell adherence to
type I collagen. A, Jurkat cells were pretreated with the
indicated anti- monoclonal antibodies for 1 hour,
after which adherence to immobilized collagen was determined. B, a comparison of JB1B, B3B11, and 21C8 induced adherence.
Values represent the mean of sextuplicate assays. All values were
within 10% of the mean.
Fig. 3gives an example of the type of results obtained using
JB1B. There was a dose-dependent inhibition by the unlabeled JB1B.
There was also competition by B3BII and 21C8. In contrast, another
stimulatory antibody(N29) and an inhibitory antibody (3S3) failed to
influence JB1B binding. Similar experiments were performed with each of
the antibodies, and a summary of the results is given in Table 2.
Based on these results, it appears that there are at least three
regions of the integrin that can act as targets for
antibodies which activate integrin function. These are designated group
A (B3BII, JB1B, 21C8), group B (B44, 13B9), and group C(N29).
Figure 3:
Competitive blocking of JB1B binding to
Jurkat cells by monoclonal antibodies to integrin
chain. Blocking studies were performed as described under
``Experimental Procedures.'' The stimulatory antibodies JB1B,
B3B11, and 21C8 inhibit JB1B binding. Another stimulatory
anti-
, N29, and an inhibitory antibody, 3S3, do not
affect JB1B binding. Results are expressed as the percent inhibition of
JB1B binding relative to that of JB1B to untreated
cells.
The library was screened with a
pool of JB1B, B3B11, and 21C8, and five reactive colonies were
identified. Assay of the individual clones with each of the antibodies
indicated that JB1B and B3B11 reacted with all five clones; in
contrast, 21C8 failed to react with these clones. Immunoblot analysis
of SDS-polyacrylamide gel electrophoresis-separated lysates of one of
the clones, B, under reducing conditions indicated that JB1B and B3B11,
reacted with a 46-kDa band corresponding to a fusion protein (Fig. 4B), containing an insert of approximately 65
amino acids. In contrast, an inhibitory anti-, JB1A,
failed to react with this band, thus indicating the specificity of the
reaction. It was noteworthy that 21C8 gave a very weak reaction with
reduced proteins from this clone (Fig. 4B, lane
3) but not with unreduced fusion protein (data not shown).
Figure 4:
Localization of JB1B and B3B11 epitopes on
fusion proteins. Bacterial lysates of clones
expressing
integrin fusion proteins were screened
with 1) JB1B, 2) B3B11, 3) 21C8, 4) JB1A, and 5) no first antibody.
Molecular mass markers indicated by the arrows are 30, 46, 69,
and 98, respectively. A, reactivity of clone B3 with B3B11; B, reactivity of clone B with JB1B and B3B11. Weak reactivity
with 21C8 was also noted under reducing
conditions.
The
screening of a second epitope library containing smaller inserts
(50-150 bp) identified a single colony, which reacted with the
antibody pool. The product of this clone, B3, was found to react with
B3B11 but not 21C8 or JB1B (Fig. 4A). Based on these
results, it is clear that these antibodies recognize distinct epitopes.
Furthermore, as predicted by the competitive binding studies, the JB1B
and B3B11 epitopes are in close proximity to one another on the
chain.
The analysis of the DNA sequences of the
inserts in those clones containing both the B3B11 and JB1B epitopes (A,
B, D, G, E) indicated that these epitopes were located in a peptide
containing a predicted amino acid sequence (33) corresponding
to residues 636-705 of the mature chain (Fig. 5). The B3 insert sequence further served to localize the
B3B11 epitope to amino acids 648-670. Based on the overlap of the
clone sequences, the JB1B epitope would appear to be contained in the
peptide spanning residues 671-703.
Figure 5:
Predicted amino acid location of the
inserts in the mature chain. Fusion proteins from
clones G, A, B, D, and E react with both JB1B and B3B11. The product of
clone B3 reacts with B3B11 but not JB1B. The sequence of positions
636-705 of the
chain is given in the lower
portion of the figure. This corresponds to the region spanned by the
insert in clone G.
The sites of reactivity of
B3B11 were further examined using three overlapping peptides, which
spanned the predicted sequence of the B3 fusion protein. B3B11 reacted
specifically with the peptide P3, which corresponded to residues
657-670 of the sequence (Fig. 6). In
contrast, neither of the other two peptides, P1 and P2, spanning the
remainder of the B3 insert was bound by B3B11. There was also no
reactivity of the other two competing antibodies, 21C8 or JB1B, with
any of the peptides.
Figure 6:
The reaction of anti antibodies with synthetic peptides corresponding to the B3B11
epitope of
. Three overlapping peptides corresponding
to residues 636-649 (P1), 646-659 (P2), and 657-670
(P3) were examined for their reactivities with JB1B, B3B11, and 21C8.
These fragments spanned the entire sequence of the B3 clone isolated
from the epitope fusion library (Fig. 5).
Further support for P3 being the location of
the B3B11 epitope was obtained with the demonstration that only this
peptide inhibited the binding of B3B11 to purified integrin (Fig. 7). Neither P1 or P2 peptides blocked B3B11
binding. Furthermore, the P3 effect appeared to be specific as it did
not influence the binding of an unrelated antibody to
JB1A. These results indicate that the B3B11 epitope is contained
in residues 657-670 and that it lies in close proximity to the
JB1B epitope.
Figure 7:
Competitive inhibition of B3B11 binding to
purified by P3. B3B11 was preincubated with peptides
1 (
), 2 (
), or 3 (
) and assayed for residual binding
to immobilized purified
integrin. The effects of P3
on JB1A (
) were also determined.
The results of the present study provide several new pieces
of information: 1) the characterization of a panel of antibodies to
that induce Jurkat adherence to collagen; 2) the
demonstration of the presence of at least three distinct
sites, which stimulatory antibodies can react with, i.e. group A (B3BII, JB1B, 21C8), group B (B44, 13B9), and group
C(N29); 3) the localization of the epitopes detected by some of group A
antibodies (JB1B, B3B11) to a membrane proximal region (648-705)
of the
integrin. The biological activities of the
antibodies described in this study are not unique as a number of
stimulatory antibodies to both
(11, 12, 13) ,
(18) , and
(14, 15, 16, 17) integrins
have been described. However, it was possible through the use of this
panel to begin to address the location and existence of multiple
regions of the integrin, which could act as targets for stimulatory
antibodies.
In two previous studies relating to this point, there
were somewhat divergent views presented on the question of the number
of regulatory sites(22, 23) . Takada and Puzon (22) observed that the region 207-218 of the appeared to be critical for the generation of epitopes, which
were recognized by a number of inhibitory and stimulatory antibodies.
These results were based on the observations that the expression of
human regulatory epitopes on interspecies
chimeras
required inclusion of this region of the human
integrin. It was also possible to demonstrate that the
introduction of multiple point mutations (S207H, E208K, N210E, K211V)
into the highly conserved homologous region of the chicken integrin
resulted in the generation of epitopes recognized by anti-human
monoclonal antibodies. Thus, it appeared that a
single region of the
was capable of modulating
function in either a positive or a negative fashion.
The authors did, however, note that there might be other regulatory
regions, as it was not possible to localize the epitopes of all
regulatory antibodies.
Shih et al.(23) used a
similar approach but found that the expression of regulatory epitopes
mapped to two distinct regions using chicken mouse chimeric molecules. Epitopes related to the interference with ligand
binding appeared to localize to the first 260 residues of the molecule.
In contrast, those antibodies that altered ligand specificities or
interfered with
-
association were dependent upon
the more membrane proximal parts of the integrin. Their results were
interpreted to indicate the presence of multiple regulatory regions
with the inhibitory and stimulatory epitopes mapping to different sites
on the
chain.
Two antibodies of the group A
cluster, JB1B and B3B11, localize the membrane proximal region of the
(648-705). The third antibody in this cluster,
21C8, reacts with a discontinuous epitope, which is not detectable on
Western blot. The B3B11 (657-670) and JB1B (671-705) flank
Cys
, which is predicted to be involved in a disulfide
bond with Cys
(34) . Such a bond would bring
sequentially distant regions of the
chain into close
spatial proximity. The predicted
disulfide bonding
pattern is based upon those of the
chain, as both
integrins have an identical number of cysteines, and a homologous
pairing pattern has been proposed(33, 35) . However,
the sequences in this region do not show significant homology. Thus,
while the disulfide bond locations may be similar, the intervening
sequences are completely different.
It is noteworthy that several
anti-LIBS to chain, which activate adhesion, have
been suggested to map in the 490-690 using interspecies
chimeras(17) . In the case of anti-LIBS2, the
reduction-sensitive epitope has been localized to
602-690(10) . Thus, a number of stimulatory monoclonal
antibodies appear to bind to the sterically constricted region of
, which is homologous to the A region on
.
The results of our competitive binding assays
support the existence of at least three regions,
which are the targets of stimulatory antibodies. The presence of
multiple sites is consistent with the results of the
chain, where three regions have been identified as sites of
stimulatory anti-LIBS activity (14, 15, 16, 17) . It has been
suggested that these sites may be in close proximity in the intact
molecule as a result of long range disulfide bonds(17) . By
analogy, it would appear that the A region of
corresponds to the LIBS2, LIBS3, LIBS6, P41 site of
(10, 17) . A second site in
(1, 2, 3, 4, 5, 6) recognized by AP5 encompassed a Cys
involved in
a long range disulfide bond(17) . Preliminary data suggest that
N29 reacts with an epitope within the first 100 amino acids of
. (
)It will be of interest to determine the
relationship between the N29 epitope and that of AP5 on
. The group B epitopes have not been localized to
date.
In summary, it would appear that there is homology between the
sites recognized by the group A antibodies and some of the stimulatory epitopes. It remains to be determined if these
antibodies are similar to the LIBS-type reagents of the
system. Preliminary data do indicate differences in expression
levels on adhesion-competent and latent cells. (
)However,
the stringency of expression on the lymphoid cells does not appear to
be as great as for the LIBS.
We have previously demonstrated that several of the antibodies in this panel can have a number of effects on adhesion(12) , intracellular signaling(36) , and the association of cytosolic proteins(37) . It will be of interest to determine whether interactions with the different stimulatory regions will differentially influence these other parameters of integrin activation.