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J Biol Chem, Vol. 273, Issue 29, 18235-18241, July 17, 1998


Splice Variants of the Drosophila PS2 Integrins Differentially Interact with RGD-containing Fragments of the Extracellular Proteins Tiggrin, Ten-m, and D-Laminin alpha 2*

Michael W. GranerDagger §, Thomas A. BunchDagger , Stefan Baumgartnerparallel , Arthur KerschenDagger , and Danny L. BrowerDagger **Dagger Dagger

From the Departments of Dagger  Molecular and Cellular Biology and ** Biochemistry, University of Arizona, Tucson, Arizona 85721, the  Friedrich Miescher-Institut, Postfach 2543, CH-4002 Basel, Switzerland, and the parallel  Department of Cell and Molecular Biology, Lund University, Box 94, S-22100 Lund, Sweden

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 alpha 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 alpha PS2C splice variant. This is in contrast to DLAM-RGD, which is the first Drosophila polypeptide shown to interact preferentially with cells expressing the alpha PS2 m8 splice variant. The beta PS integrin subunit also varies in the presumed ligand binding region as a result of alternative splicing. For TIG-RGD and TENM-RGD, the beta  splice variant has little effect, but for DLAM-RGD, maximal cell spreading is supported only by the beta 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.

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The integrins are a family of heterodimeric transmembrane glycoproteins, consisting of alpha  and beta  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 beta PS subunit paired with an alpha PS1, alpha PS2, or alpha PS3 subunit, respectively. beta PS, alpha PS1, and alpha 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). alpha PS3 was identified only recently, and little is known of its ligand binding properties (14).

Both the beta PS and alpha PS2 subunits may be alternatively spliced to generate proteins that vary in their extracellular domains. The beta PS subunit mRNA has been found in alternatively spliced forms to generate proteins referred to as beta PS4A and beta 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 beta  subunit. The alpha PS2 subunit exists in splice forms called alpha PS2C and alpha 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 alpha /beta heterodimer combinations for PS2 integrins: alpha PS2Cbeta PS4A, alpha PS2Cbeta PS4B, alpha PS2m8beta PS4A, and alpha PS2m8beta 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 alpha  chain, D-laminin alpha 2, which, in contrast to the previously characterized Drosophila laminin alpha  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 alpha /beta heterodimer combinations. Our results demonstrate that peptides from both Ten-m and D-laminin alpha 2 can serve as integrin ligands in our in vitro assays. Moreover, we find that both alpha and beta  splice variants lead to ligand-dependent differences in integrin function.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 beta 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-laminin alpha 2. These are residues 492-832 (RGD is found at 689-691) of a total of 3325 residues.

Protein induction was performed according to the manufacturer's protocols, and recombinant peptide was affinity purified using a nickel resin (Ni-NTA, Qiagen). Purified fusion proteins were dialyzed from a buffer containing 8 M urea stepwise into 50 mM Tris, 100 mM NaCl, pH 7.5. Protein concentrations were determined by SDS-polyacrylamide gel electrophoresis and comparison of fusion protein staining with that of protein molecular weight standards (Bio-Rad). Gels were stained with Coomassie Brilliant Blue. Total protein concentrations were determined by using a BCA protein assay (Pierce) with bovine serum albumin as a standard.

FACS 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.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The alternative splicing to produce extracellular variants in alpha PS2 and beta PS have been described previously (15-17). Recently, models for the structure of the ligand binding heads of both alpha and beta  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 beta  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 beta  subunit of the integrin receptors used in those assays was beta PS4A. We extended the analyses of cell spreading on TIG-RGD to include integrin heterodimers composed of alpha PS2beta 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 beta  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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  		Fig. 1.   A, the ligands and histidine-tagged recombinant protein fragments used in cell spreading assays. The size and location of each fragment relative to the entire protein molecule is shown beneath each ligand. Laminin domains are indicated under the D-laminin alpha 2. B, region of homology surrounding the RGD motifs in D-laminin alpha 2 (region IVb) and murine laminin alpha 5 (region IVa).


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  		Fig. 2.   Spreading of integrin-expressing cells on recombinant TIG-RGD fragment. Drosophila S2 cells expressing the indicated integrins (CA, alpha PS2Cbeta PS4A; CB, alpha PS2Cbeta PS4B; m8A, alpha PS2m8beta PS4A; m8B, alpha PS2m8beta PS4B) were plated on microtiter wells coated with TIG-RGD at the indicated concentrations. Spread cells were counted using phase contrast microscopy; see under "Materials and Methods" for details. Spreading levels recorded are the average of at least three experiments; error bars represent S.E.


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  		Fig. 3.   Inhibition of cell spreading by RGD peptides. Cell spreading was performed as before in the presence of increasing concentrations of the peptides GRGDSP and GRGESP. For simplicity, data are presented only for three cell lines (CA, CB, and m8A), representing various combinations of splice variants and ligands; all cell spreading by PS2 integrins reported here is similarly inhibited by GRGDSP. Abbreviations are as for Fig. 2. Ligands illustrated here are as follows: CA, 3 µg/ml TIG-RGD; CB, 10 µg/ml TENM-RGD; m8A, 5 µg/ml DLAM-RGD.

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 beta 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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  		Fig. 4.   Spreading of integrin-expressing cells on recombinant TENM-RGD fragment. Abbreviations are as for Fig. 2. Spreading levels recorded are the average of at least three experiments; error bars represent S.E.

A Laminin Fragment Promotes PS2 m8-Mediated Cell Spreading-- Analysis of the predicted coding sequence of D-laminin alpha 2 indicates that this protein is a member of the laminin alpha  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 alpha 2 is similar to murine laminin alpha 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 alpha 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.

We generated a recombinant protein fragment that includes the D-laminin alpha 2 RGD sequence (DLAM-RGD), and this 342-amino acid fragment was purified and plated on microtiter plates to be used as a ligand in integrin-mediated cell spreading assays. All PS2 integrin-expressing cells spread on D-laminin alpha 2, in contrast to the parental S2 cell line (Fig. 5). However, alpha PS2m8beta PS4A cells spread 2-3 times better on D-laminin alpha 2 than all other PS2 integrin-expressing cells, including alpha PS2m8beta PS4B cells. This was in contrast to PS2-mediated cell spreading on TIG-RGD and on TENM-RGD, where PS2C cells always spread better than PS2 m8 cells, and the beta  subunit splice variant made little difference. Again, spreading on DLAM-RGD was inhibited by RGD peptides (Fig. 3).


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  		Fig. 5.   Spreading of integrin-expressing cells on recombinant DLAM-RGD fragment. Abbreviations are as for Fig. 2. Spreading levels recorded are the average of at least three experiments; error bars represent S.E.

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 alpha PS2Cbeta PS4A-expressing and alpha PS2Cbeta 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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  		Fig. 6.   FACS analysis of integrin expression on the various cell lines. Cells were protease treated and heat shocked as for cell spreading experiments and stained with anti-PS2 monoclonal antibody after a 3-h recovery period. Expression was heterogeneous for the different cell lines and did not correlate with cell spreading behavior in general, indicating that simple differences in protein expression are not responsible for the ligand specificity observed. Abbreviations are as for Fig. 2.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Integrin Structure and alpha PS2 Isoforms-- Using structural homology arguments, Springer (30) has generated a model to describe the organization of the integrin alpha 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 beta -propeller, and each "blade" of the beta -propeller is postulated to be composed of four strands of anti-parallel beta  sheet. For alpha PS2, sequence alignments place the residues encoded by the alternatively spliced exon 8 in the loop connecting beta  sheet strands two and three of the third propeller blade (Fig. 7). Recently, mutagenesis studies have demonstrated that residues in the corresponding loops of alpha IIb, alpha 4, and alpha 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 beta -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 alpha PS2m8, but it may provide an additional surface that adds to the stability of binding (17).


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  		Fig. 7.   Structural predictions for the splice variants of PS2 integrin subunits. A, alignment of the four strands of beta  sheet composing the third blade of the alpha  subunit beta -propeller structure proposed by Springer (30). Underlined residues for human alpha 4, alpha IIb, and alpha M are predicted to have the potential to form beta  sheet (30). For alpha PS2m8, the underlined residues scored above an arbitrarily chosen number for beta  sheet prediction, using the PHD algorithm (31, 32) in all tested sequence alignments. B, residues encoded by the fourth exon of beta PS, aligned with corresponding regions of beta 1 and beta 3. Below the sequence are shown regions predicted to form alpha -helix (h) or beta  sheet (e) according to models of Collins Tozer et al. (42) (upper line) or Tuckwell and Humphries (38) and Takagi et al. (39) (lower line).

Alternatively, exon 8 could encode a new strand of beta  sheet that directs the polypeptide chain down to the lower side of the beta -propeller, leaving the ligand binding surface relatively intact but adding new residues to potential regulatory sites. For example, alternative splicing of the alpha 7 subunit alters residues in the same part of the protein as does the alpha PS2 alternative splicing, and the alpha 7 isoforms have been shown recently to affect the activity state of the alpha 7beta 1 integrin (36). In this vein, it is noteworthy that PS2m8 and PS2C can display different cation requirements in cell spreading experiments (19).

In an attempt at gaining further insights into these possibilities, we have used a number of different algorithms to predict potential secondary structures in this region of alpha PS2. Unfortunately, no consistent pattern was seen in the predictions for the residues encoded by exon 8; of particular relevance to the above discussion, residues at the beginning of exon 8 show beta  sheet potential in some predictive paradigms but not in others. Overall, there is no consistent pattern that would allow one to prefer one structural model over the other. In any case, the residues encoded by exon 8 are likely to be involved in specific protein interactions, since they are highly conserved in the distantly related dipteran Ceratitis capitata (17).

Integrin Structure and beta PS Isoforms-- An overall similarity in hydropathy profiles suggested that the ligand binding domain of beta  subunits would fold into a structure similar to the I domains of some alpha  subunits, with a cation-containing pocket that is expected to be directly involved in ligand association (37). Recently, models for beta  subunit I domain-like structures have been proposed, and these models differ significantly in the predicted tertiary structure for the region encoded by beta PS exon 4. (A comparison of the sequences encoded by the alternatively spliced forms of beta 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 beta  I domain and then run via a beta  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 alpha  subunit I domains (42). According to this model, the exon 4-encoded domain begins low in the structure, and then, via an alpha  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 beta 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.2

D-Laminin alpha 2 as a PS2 Integrin Ligand-- The Drosophila wing blister locus encodes a new laminin alpha  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 alpha 2 can function as an RGD-dependent ligand for PS2 integrins.

Overall sequence comparisons indicate that D-laminin alpha 2 resembles the mouse laminin alpha 2 chain, as exemplified by the length of the protein and the signature laminin domain structure of the molecule.4 For our purposes, however, a more notable comparison is with the recently cloned mouse laminin alpha 5 chain (49). Although the overall domain structure of alpha 5 more closely resembles the previously described Drosophila alpha  chain (28), potential integrin binding sites of D-laminin alpha 2 and mouse alpha 5 are very similar. In D-laminin alpha 2, the RGD tripeptide sequence is in region IVb between the third and fourth laminin EGF-like repeats in the N-terminal (short arm) portion of the molecule. In murine laminin alpha 5, there are two RGD sequences, located in domains IVa and IIIa. A 50-amino acid region of D-laminin alpha 2 domain IVb and murine laminin alpha 5 domain IVa show 39% identity and 54% similarity to each other when rooted in the RGD sequence (Fig. 1B), suggesting a functionally conserved portion of the proteins. To date, integrin association with this portion of vertebrate laminin alpha 5 has not been reported.

Previous experiments using portions of the original Drosophila laminin chains as substrates for PS1-mediated cell spreading indicated the necessity for the native heterotrimeric molecule.2 Our results with DLAM-RGD demonstrate that a portion of this laminin chain alone has an inherent PS2 integrin binding domain. Presumably, D-laminin alpha 2 forms a trimer with laminin beta  and gamma  chains in situ. The domain containing the RGD motif is in the exposed short arm of D-laminin alpha 2 (based on homology to other laminins) and should not be obscured by association with other subunits.

As the name implies, mutations in the wing blister locus can lead to blistering of the wing, where the dorsal and ventral wing surfaces separate (25). This phenotype is frequently associated with mutations in integrins (50-52), and D-laminin alpha 2 and integrins co-localize in many fly tissues.4 These observations suggest that the interaction between the RGD-containing domain of D-laminin alpha 2 and PS2 integrins that we find in cell culture is important for morphogenesis in vivo. This proposal is supported strongly by genetic interactions between myospheroid (beta PS) and wing blister mutations in situ.2 For example, wing blister mutations enhance the partial lethality of weak myospheroid mutations, and even wing blister heterozygosity can lead to blisters in myospheroid mutants that normally show insignificant frequencies of wing defects. Conversely, weak myospheroid alleles can greatly increase the blistering of wing blister flies. Thus, D-laminin alpha 2 and PS2 integrins have the biochemical potential to recognize one another, are in many of the same places, and function in at least some of the same morphogenetic events. From this we infer that D-laminin alpha 2 serves as a PS2 integrin ligand in vivo (see also below).

alpha PS2 Splice Variant Isoforms Affect Ligand Preference-- Results presented here indicate that alpha 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 alpha PS2m8beta 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.

Levels of the alpha PS2 alternatively spliced transcripts vary during development (17), which may imply different roles for the different alpha PS2 isoforms. Genetic data also support the notion that ligand preferences will have functional significance in situ. Although transgenic expression of either form of the alpha PS2 subunits in flies is sufficient for viability in alpha PS2 (inflated) null backgrounds, the two isoforms are not equivalent (53). alpha PS2C rescues overall viability better than alpha PS2m8, whereas expression of alpha PS2m8 is more efficient at rescuing some specific mutant phenotypes, such as wing blisters. This latter result is particularly noteworthy, in light of the preference we find for a PS2m8 integrin in mediating cell spreading on polypeptides from the product of the wing blister (D-laminin alpha 2) gene.

beta PS Splice Variant Isoforms Affect Ligand Preference-- S2 cells expressing alpha PS2m8beta PS4A integrins spread more efficiently on recombinant DLAM-RGD protein fragments than did cells expressing alpha PS2m8beta PS4B. One potential trivial explanation for the preference for beta PS4A is that the cells might be making more beta PS4A than beta PS4B. However, we did not see large differences in expression between alpha PS2m8beta PS4A and alpha PS2m8beta 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 beta 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 beta PS isoforms.

Although we can state unequivocally that the isoform of beta PS can affect function, it is difficult to apply any precise quantitative interpretations to these data. The S2 cell line makes a relatively small amount of endogenous beta PS, which appears to be mostly beta PS4A. Following the proteolysis and induction protocol, the beta PS produced from the multiple copies of the heat shock-induced transgenes would be expected to be present in large excess relative to that generated from endogenous genes, and this expectation is borne out by Western blot data (18). Our functional results further indicate that there is relatively little alpha PS2m8beta PS4A present on the surface of alpha PS2m8beta PS4B-transformed cells; otherwise, this line would be expected to spread much better on DLAM-RGD. It should also be noted that the data overall indicate that associations of the various alpha /beta subunits are not grossly disturbed by isoform composition, since in vivo and in vitro, all combinations tested either rescue mutant phenotypes or demonstrate ability to spread on at least some ligands.

In flies carrying mutations in the beta PS subunit, rescue experiments with beta PS transgenes indicate that beta PS4A and beta PS4B are both capable of rescuing the postembryonic mutant phenotypes in the eye and wing (16). Rescue of embryonic lethality, on the other hand, is efficient only if both isoforms are expressed (16). This would lead one to expect that another ligand, as yet uncharacterized, may show preference for beta PS4B, in combination with one or more alpha  subunits.

    ACKNOWLEDGEMENTS

We thank Frances Fogerty for the gift of TIG-RGD fusion protein and Norma Seaver for help with the FACS analyses.

    FOOTNOTES

* 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.

Dagger Dagger 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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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
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