From the Departments of Molecular and Cellular
Biology and ** Biochemistry, University of Arizona, Tucson,
Arizona 85721, the ¶ Friedrich Miescher-Institut,
Postfach 2543, CH-4002 Basel, Switzerland, and the
Department
of Cell and Molecular Biology, Lund University, Box 94, S-22100 Lund,
Sweden
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ABSTRACT |
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Two new potential ligands of the
Drosophila PS2 integrins have been characterized by
functional interaction in cell culture. These potential ligands are a
new Drosophila laminin 2 chain encoded by the wing
blister locus and Ten-m, an extracellular protein known to be
involved in embryonic pattern formation. As with previously identified
PS2 ligands, both contain RGD sequences, and RGD-containing fragments
of these two proteins (DLAM-RGD and TENM-RGD) can support PS2
integrin-mediated cell spreading. In all cases, this spreading is
inhibited specifically by short RGD-containing peptides. As previously
found for the PS2 ligand tiggrin (and the tiggrin fragment TIG-RGD),
TENM-RGD induces maximal spreading of cells expressing integrin
containing the
PS2C splice variant. This is in contrast
to DLAM-RGD, which is the first Drosophila polypeptide
shown to interact preferentially with cells expressing the
PS2
m8 splice variant. The
PS integrin subunit also
varies in the presumed ligand binding region as a result of alternative splicing. For TIG-RGD and TENM-RGD, the
splice variant has little effect, but for DLAM-RGD, maximal cell spreading is supported only by
the
PS4A form of the protein. Thus, the diversity in PS2
integrins due to splicing variations, in combination with diversity of
matrix ligands, can greatly enhance the functional complexity of
PS2-ligand interactions in the developing animal. The data also suggest
that the splice variants may alter regions of the subunits that are
directly involved in ligand interactions, and this is discussed with
respect to models of integrin structure.
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INTRODUCTION |
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The integrins are a family of heterodimeric transmembrane
glycoproteins, consisting of and
subunits, that serve as
receptors for extracellular matrix molecules and cell surface molecules of neighboring cells. Integrins have roles in diverse phenomena, such
as cell adhesion, spreading, migration, and differentiation, as well as
roles in the development and progression of numerous pathological
states, such as cancer and cardiovascular disease (1-4). As might be
expected from these varied requirements, integrins not only provide
mechanical linkages to the matrix and neighboring cells but also
receive and transmit information from the cell exterior to the cell
interior, and vice versa (5). The fruit fly, Drosophila
melanogaster, provides a valuable genetic system in which to
examine these integrin functions in the developing animal (6, 7). As a
complement to these genetic studies, we have utilized cultured cells,
expressing various combinations of Drosophila PS integrin
transgenes, to examine interactions of PS integrins and potential
integrin ligands.
The PS1, PS2, and PS3 integrins of Drosophila consist of a
common PS subunit paired with an
PS1,
PS2, or
PS3 subunit, respectively.
PS,
PS1, and
PS2 were
originally identified as position-specific
(PS)1 antigens in monoclonal
antibody screens of imaginal discs (8, 9). Subsequent biochemical and
molecular analyses of these antigens indicated that they are members of
the integrin family (10-13).
PS3 was identified only
recently, and little is known of its ligand binding properties
(14).
Both the PS and
PS2 subunits may be
alternatively spliced to generate proteins that vary in their
extracellular domains. The
PS subunit mRNA has been
found in alternatively spliced forms to generate proteins referred to
as
PS4A and
PS4B (15, 16). These subunits
differ in the utilization of different fourth exons, which encode 29 amino acids in the ligand binding "head" of the
subunit. The
PS2 subunit exists in splice forms called
PS2C and
PS2m8 (17), referring to the
presence (C, canonical) or absence (m8, missing exon 8) of exon 8 . When present, the eighth exon encodes 25 amino acids, potentially
located in a region that would be expected to influence ligand
associations. Thus, there are at least four possible
/
heterodimer combinations for PS2 integrins:
PS2C
PS4A,
PS2C
PS4B,
PS2m8
PS4A, and
PS2m8
PS4B. These receptors may generate
significant PS2 integrin functional diversity during development.
Ligands that support Drosophila PS2 integrin-mediated cell spreading include mammalian vitronectin and fibronectin (18, 19) and the novel Drosophila extracellular matrix protein, tiggrin (20). A key feature of several vertebrate integrin ligands is the tripeptide sequence, Arg-Gly-Asp (RGD). This same tripeptide is apparently recognized by Drosophila PS2 integrins, as all previously identified PS2 ligands contain an RGD sequence, and PS2 integrin-mediated cell spreading is inhibited by soluble RGD peptides. Moreover, tiggrin polypeptides in which the RGD sequence has been changed to LGA no longer support cell spreading, and the RGD sequence is required for maximal rescue by transgenes of some tiggrin mutant phenotypes in situ (21). In contrast, PS1-expressing cells have been shown to spread on Drosophila heterotrimeric laminin, which does not contain an RGD motif (22), and this spreading is not inhibited by RGD peptides.2
One approach for identifying additional PS2 ligands is to first search
for candidate extracellular matrix molecules based on structure
(e.g. an RGD sequence) or location (e.g. muscle
attachment sites) and ask whether the purified proteins or protein
fragments will support PS2-mediated cell spreading in culture. One such candidate is Ten-m (23), a protein with tenascin-type EGF repeats (Fig.
1). Ten-m contains a C-terminal RGD sequence, and earlier studies had
suggested that it may function as a PS2
ligand.3 Mutants for the
ten-m gene display an early embryonic patterning defect of
the "pair-rule" type (23, 24). Another potential PS2 ligand is
encoded by the wing blister locus, mutations in which can
lead to wing blisters similar to those caused by loss-of-function integrin mutations (25). Recently, this gene was found to encode a new
laminin chain, D-laminin
2, which, in contrast to
the previously characterized Drosophila laminin
chain
(26-28), contains an RGD motif (Fig.
1).4
We have examined the ability of tiggrin and these newly characterized
matrix components to support PS2-mediated cell spreading, utilizing S2
cell lines that express each of the different PS2 integrin /
heterodimer combinations. Our results demonstrate that peptides from
both Ten-m and D-laminin
2 can serve as integrin ligands
in our in vitro assays. Moreover, we find that both
and
splice variants lead to ligand-dependent differences in integrin function.
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MATERIALS AND METHODS |
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Cell Culture, Cell Transfection, and Cell Spreading
Assays--
Cell culture techniques and methods for transfection of
cells have been previously described, as have Schneider's line 2 (S2) Drosophila cells that have been stably transfected with
integrin transgenes under the regulation of the heat shock protein 70 promoter (18, 19, 29). The construct used to express the
PS4B subunit, also under the regulation of the heat
shock protein 70 promoter, is described in Ref. 16. Cell spreading
assays were performed as described previously (19, 20). Briefly, cells
were treated with dispase/collagenase to remove existing matrix
molecules and cell surface proteins. Cells were then heat shocked at
37 °C for 30 min to induce expression of integrin transgenes and
were then plated on TIG-RGD, DLAM-RGD, or TENM-RGD substrates
(described below). 4-6 h following the heat shock, the cells were
fixed and quantified by scoring for spread cells using a Nikon
phase-contrast microscope (Nikon Diaphot-TMD). Three fields of cells
were counted for each well, and the numbers reported represent the
averages (and standard errors) of three separate experiments.
TIG-RGD, TENM-RGD, and DLAM-RGD-- TIG-RGD was a generous gift from Frances Fogerty and has been described (20). It is a polyhistidine-tagged bacterial fusion protein that contains 270 amino acids of tiggrin, residues 1891-2161, with the RGD sequence being residues 1989-1991 (tiggrin has a total of 2186 amino acids).
TENM-RGD is a bacterial fusion protein that contains a polyhistidine tag fused to the final 212 amino acids of Ten-m. Ten-m has a total of 2515 amino acids (23), and the RGD sequence is 72 amino acids from the C terminus. This fusion protein was produced from the expression vector pTrcHisB (Xpress SystemTM, Invitrogen), into which was cloned an XhoI-HindIII fragment of the ten-m cDNA. His-tagged DLAM-RGD was made by cloning a polymerase chain reaction product into pTrcHisA. Genomic DNA from Oregon-R flies was used as template, and sequencing of the wing blister gene showed that there are no introns in this interval.4 The recombinant protein contains 340 amino acids of D-lamininFACS Analysis-- Cells were prepared for flow cytometry following essentially the same procedure as for cell spreading; briefly, cells were protease-treated, heat-shocked, and allowed to recover for 3 h. Cells were then incubated with an anti-PS2 monoclonal antibody (CF.2C7), followed by staining with a fluorescein isothiocyanate-labeled anti-mouse secondary antibody (Jackson ImmunoResearch). Cells were then fixed in 3% formaldehyde. FACS analyses were performed at the Research Flow Cytometry Service Laboratory of the University of Arizona Cancer Center. Data were acquired with a FACScan device (Becton Dickinson), and data were analyzed using FACSvantage and Cell Quest software.
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RESULTS |
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The alternative splicing to produce extracellular variants in
PS2 and
PS have been described previously
(15-17). Recently, models for the structure of the ligand binding
heads of both
and
integrin subunits have been proposed, and the
positions of the variant residues with respect to these models are
detailed under "Discussion."
Cell Spreading on TIG-RGD Is Unaffected by Integrin Subunit
Splice Variants--
Fogerty et al. (20) showed that the
novel Drosophila extracellular matrix protein tiggrin serves
as a ligand in PS2 integrin-mediated cell spreading assays. A 270-amino
acid C-terminal recombinant fragment containing the RGD sequence of
tiggrin (here referred to as TIG-RGD) (Fig.
1) also promoted cell spreading. The
subunit of the integrin receptors used in those assays was
PS4A. We extended the analyses of cell spreading on
TIG-RGD to include integrin heterodimers composed of
PS2
PS4B. As was seen previously (20), PS2C cells spread better on TIG-RGD than PS2 m8 cells (Fig.
2). Additionally, we found that PS2
integrin-expressing cell lines spread equally well on TIG-RGD
regardless of the
subunit splice variant of the integrin. All of
the cell spreading on TIG-RGD was inhibited by soluble RGD peptides
(Fig. 3).
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A Ten-m Fragment Promotes PS2-mediated Cell Spreading--
We
generated a recombinant protein fragment of 212 amino acids of Ten-m,
including the RGD sequence, and used this fragment (TENM-RGD) as a
substrate for cell spreading assays. As shown in Fig.
4, TENM-RGD supported cell spreading for
all tested PS2 integrin-expressing cell lines. Reminiscent of
PS2-mediated cell spreading on TIG-RGD, PS2C cells spread better on
TENM-RGD than did PS2m8 cells. Additionally, the alternative splice
forms of the PS subunit made little or no difference in
the levels of cell spreading on TENM-RGD. As with TIG-RGD, this cell
spreading was inhibited by soluble RGD peptides (Fig. 3).
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A Laminin Fragment Promotes PS2 m8-Mediated Cell
Spreading--
Analysis of the predicted coding sequence of D-laminin
2 indicates that this protein is a member of the laminin
chain
family of extracellular matrix molecules,4 and the putative
protein domain structure may be grouped according to accepted laminin
nomenclature (Fig. 1). Overall, the sequence of D-laminin
2 is similar to murine laminin
2 chains, and it contains 19 laminin EGF-like repeats, 5 laminin G domains, a laminin B motif, and a
characteristic laminin N-terminal domain. D-laminin
2 also possesses
a potential integrin-binding RGD sequence in the N-terminal quarter of
the protein, in the IVb region between two blocks of EGF-like
domains.
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Cell Spreading Is Not Correlated with Integrin Expression
Level--
Although it is formally possible that the differences in
spreading observed between different cell lines are due to differences in integrin expression levels, this does not appear to be the case.
Both FACS analysis (Fig. 6) and
immunofluorescence (see, for example, Ref. 18) indicated that surface
integrin expression on the cells was heterogeneous, but the large
majority (typically 85% or more) of the induced cells expressed
significant integrin for all of the lines. Most importantly, there was
no obvious correlation between expression levels and spreading. For
example, among the four transfected cell lines, the
PS2C
PS4A-expressing and
PS2C
PS4B-expressing cells exhibited the
highest and lowest levels of surface integrin (displaying a difference
of 2-fold or more in mean and median fluorescence values in two FACS
experiments), but showed virtually identical, and very reproducible,
levels of spreading on two peptide ligands. In general, it appears that
once a relatively low level of surface integrin is present, further
increases do not result in large changes in spreading behavior. Indeed,
even uninduced cells (but not untransfected S2 cells), which contain
very small amounts of integrin relative to heat shock-induced cells
(see, for example, Fig. 1 of Ref. 18), will spread in culture if a suitable matrix is present.
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DISCUSSION |
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Integrin Structure and PS2 Isoforms--
Using
structural homology arguments, Springer (30) has generated a model to
describe the organization of the integrin
subunit globular head.
According to this model, seven repeat domains (termed FG-GAP repeats
for the phenylalanyl-glycl and glycyl-alanyl-prolyl consensus
sequences) are folded into a cyclic
-propeller, and each "blade"
of the
-propeller is postulated to be composed of four strands of
anti-parallel
sheet. For
PS2, sequence alignments place the residues encoded by the alternatively spliced exon 8 in the
loop connecting
sheet strands two and three of the third propeller
blade (Fig. 7). Recently, mutagenesis
studies have demonstrated that residues in the corresponding loops of
IIb,
4, and
5 are critical
for ligand binding (33-35), and one possibility is that the extra 25 amino acids extend this loop on the top of the
-propeller, providing
a new surface for integrin-ligand interactions. Polypeptides that
support good spreading of cells expressing PS2C (vitronectin, tiggrin,
and TENM-RGD) also serve as ligands for PS2m8, albeit less well, and so
the exon 8-encoded segment probably does not completely replace the
normal site of ligand interaction on
PS2m8, but it may
provide an additional surface that adds to the stability of binding
(17).
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Integrin Structure and PS Isoforms--
An overall
similarity in hydropathy profiles suggested that the ligand binding
domain of
subunits would fold into a structure similar to the I
domains of some
subunits, with a cation-containing pocket that is
expected to be directly involved in ligand association (37). Recently,
models for
subunit I domain-like structures have been proposed, and
these models differ significantly in the predicted tertiary structure
for the region encoded by
PS exon 4. (A comparison of
the sequences encoded by the alternatively spliced forms of
PS exon 4 (15, 16) is shown in Fig. 7.) In models that
are driven primarily by secondary structure predictions from computer
algorithms (Refs. 38 and 39; see also Ref. 40 for a non-I domain
interpretation of secondary structure profiles), exon 4 encodes
residues that form a loop on the top of the
I domain and then run
via a
sheet to the lower part of the domain, including the
beginning of a motif postulated to be important in integrin regulation
(41). Another model makes adjustments to the secondary structure
predictions in order to more closely copy the structure of
subunit
I domains (42). According to this model, the exon 4-encoded domain
begins low in the structure, and then, via an
helix and loop
structure, moves across the top of the I domain, near the putative
ligand binding region. Thus, in either model exon 4-encoded residues
might be expected to interact directly with ligand, but they are likely
to be different residues in the respective models. It is intriguing
that the exon 4 residues include and connect domains that have been
postulated to interact with ligand and mediate integrin regulation,
based on mutagenesis and antibody binding studies (39, 41). This region
of
PS should prove to be a fruitful location for more extensive site-directed mutagenesis studies.
Ten-m as a PS2 Integrin Ligand-- Ten-m possesses some, but not all, of the features common to most vertebrate tenascins (reviewed in Ref. 43). For example, Ten-m is a secreted glycoprotein with eight tenascin-type EGF-like repeats and putative fibronectin-type III repeats (23). Ten-m lacks a tenascin C-terminal fibrinogen-like domain, and the Ten-m RGD sequence is found 72 amino acids from the C terminus. Recombinant protein fragments containing this RGD sequence promote RGD-dependent, PS2 integrin-mediated cell spreading (Fig. 4), with PS2C cells spreading better than PS2m8 cells.
Levine et al. (24) reported a partial cDNA sequence from the ten-m gene (which they called odd Oz); this partial sequence stops short of the final 325 amino acids and thus does not include the RGD tripeptide near the C terminus, and it also includes 216 N-terminal residues not reported by Baumgartner et al. (23). Levine et al. (24) ascribed properties to the presumed polypeptide that are significantly different from those deduced by Baumgartner et al.; for example, they suggest that Odd Oz is a transmembrane phosphoprotein with tenascin homology in its putative extracellular domain, and they also propose that the polypeptide is cleaved into smaller mature proteins. These apparent discrepancies have yet to be resolved, and it is possible that the protein functions in different forms. In any case, Baumgartner et al. (23) found that a Ten-m polypeptide could be found in conditioned media from Drosophila cells, and so a secreted form is present in at least some instances. Curiously, the ten-m gene is expressed in an embryonic pair-rule pattern, and ten-m mutants display pair-rule patterning defects (23, 24). Since the protein influences expression of downstream genes, it must communicate its presence to the cell nucleus. However, it does not appear that integrin signal transduction is important in early embryonic segmentation. PS integrins are not strongly expressed at this time, and, more importantly, mutations in integrin subunit genes do not cause segmentation phenotypes (6, 44). Ten-m is later localized (among other places) at muscle attachment sites, where integrins are known to accumulate (11, 23, 45). This localization of Ten-m in vivo, as well as the demonstration of TENM-RGD interactions with PS2 integrins in vitro, suggests that Ten-m may function with PS2 integrins in muscle attachment. Interestingly, the heparan sulfate-containing protein D-syndecan also localizes to muscle attachments (46), and Ten-m contains a consensus heparin-binding sequence near the RGD, suggesting the potential of a Ten-m-syndecan-integrin complex. Syndecan proteoglycans recently have been shown to be important in signal transduction in focal adhesions in vertebrate cells (47). The available data, although very suggestive, do not demonstrate unequivocally that Ten-m serves as an integrin ligand at muscle attachment sites. One advantage to using Drosophila is that genetic approaches can often be employed to indicate functional interactions in situ. However, other potential PS2 ligands, such as tiggrin (20), also accumulate at muscle attachment sites, and genetic studies of tiggrin suggest considerable functional redundancy among the extracellular matrix components there (21). Because of this redundancy, a direct genetic demonstration of a role for Ten-m in muscle attachment may require simultaneous disruption of multiple genes encoding matrix proteins, and the early embryonic phenotype of ten-m mutants will further complicate such an analysis. One potential approach might be to demonstrate a dominant genetic effect of ten-m mutations in a background that has been sensitized for loss of function phenotypes by viable mutations in other genes that encode proteins important for muscle attachment or other integrin-dependent processes. Early attempts to do this for Ten-m have been unsuccessful.2D-Laminin 2 as a PS2 Integrin Ligand--
The Drosophila
wing blister locus encodes a new laminin
chain.4
Laminins have long been known to interact with integrins (reviewed in
Ref. 48), and the previously characterized native heterotrimeric Drosophila laminin is a PS1 integrin ligand (22). Our
experiments indicate that a fragment of D-laminin
2 can function as
an RGD-dependent ligand for PS2 integrins.
PS2 Splice Variant Isoforms Affect Ligand
Preference--
Results presented here indicate that
PS2 integrin subunit isoforms differ in their abilities
to mediate cell spreading on fly polypeptides; suggestions that this
might be true had earlier come from studies of PS2 interactions with
vertebrate matrix proteins (19). As was seen previously (20), cells
expressing PS2C integrins spread better on TIG-RGD than PS2m8 cells.
The same is true for cell spreading on TENM-RGD. However, cells
expressing
PS2m8
PS4A integrins spread
better than cells expressing any of the other PS2 subunit combinations
on DLAM-RGD. This is the first Drosophila integrin ligand
that appears to be preferred by a PS2m8 integrin over a PS2C
integrin.
PS Splice Variant Isoforms Affect Ligand
Preference--
S2 cells expressing
PS2m8
PS4A integrins spread more
efficiently on recombinant DLAM-RGD protein fragments than did cells expressing
PS2m8
PS4B. One potential
trivial explanation for the preference for
PS4A is that
the cells might be making more
PS4A than
PS4B. However, we did not see large differences in expression between
PS2m8
PS4A and
PS2m8
PS4B, and as discussed earlier,
spreading does not generally appear to be sensitive to expression
levels above a relatively low threshold. More importantly, there were
no significant
PS-related differences in cell spreading when the same cell lines were plated on TIG-RGD or TENM-RGD; this specificity indicates that the difference in spreading seen with DLAM-RGD reflects a genuine functional difference between the
PS isoforms.
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ACKNOWLEDGEMENTS |
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We thank Frances Fogerty for the gift of TIG-RGD fusion protein and Norma Seaver for help with the FACS analyses.
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FOOTNOTES |
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* This study was supported by Grants T32 CA09213 and R01 GM42474 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Current address: Pediatric Oncology/Hematology, University of Arizona Health Sciences Center, Tucson, AZ 85724.
To whom correspondence should be addressed: Dept. of Molecular
and Cellular Biology, Life Sciences South Bldg., University of Arizona,
Tucson, AZ 85721. Tel.: 520-621-5311; Fax: 520-621-3709; E-mail:
dbrower{at}u.arizona.edu.
1 The abbreviations used are: PS, position-specific; C, canonical; m8, missing exon 8; S2, Schneider's line 2; EGF, epidermal growth factor; FACS, fluorescence-activated cell sorter.
2 T. Bunch, unpublished observations.
3 S. Baumgartner, unpublished observations.
4 D. Martin, S. Zusman, X. Li, E. Williams, R. Chiquet-Ehrismann, and S. Baumgartner, manuscript in preparation.
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REFERENCES |
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