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J. Biol. Chem., Vol. 281, Issue 8, 5050-5057, February 24, 2006

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Amino Acid Changes in Drosophila PS2PS Integrins That Affect Ligand Affinity^*

Thomas A. Bunch¹, Teresa L. Helsten, Timmy L. Kendall, Nikhil Shirahatti^¶, Daruka Mahadevan^¶, Sanford J. Shattil, and Danny L. Brower^||

From the Departments of Molecular and Cellular Biology, ^||Biochemistry, and ^¶Medicine, Arizona Cancer Center, Tucson, Arizona 85724 and the Department of Medicine, University of California San Diego, La Jolla, California 92093

Received for publication, August 4, 2005 , and in revised form, December 21, 2005.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
We developed a ligand-mimetic antibody Fab fragment specific for Drosophila PS2PS integrins to probe the ligand binding affinities of these invertebrate receptors. TWOW-1 was constructed by inserting a fragment of the extracellular matrix protein Tiggrin into the H-CDR3 of the v3 ligand-mimetic antibody WOW-1. The specificity of PS2PS binding to TWOW-1 was demonstrated by numerous tests used for other integrin-ligand interactions. Binding was decreased in the presence of EDTA or RGD peptides and by mutation of the TWOW-1 RGD sequence or the PS metal ion-dependent adhesion site (MIDAS) motif. TWOW-1 binding was increased by mutations in the PS2 membrane-proximal cytoplasmic GFFNR sequence or by exposure to Mn²⁺. Although Mn²⁺ is sometimes assumed to promote maximal integrin activity, TWOW-1 binding in Mn²⁺ could be increased further by the PS2 GFFNR GFANA mutation. A mutation in the PS I domain (PS-b58; V409D) greatly increased ligand binding affinity, explaining the increased cell spreading mediated by PS2PS-b58. Further mutagenesis of this residue suggested that Val-409 normally stabilizes the closed head conformation. Mutations that potentially reduce interaction of the integrin subunit plexin-semaphorin-integrin (PSI) and stalk domains have been shown to have activating properties. We found that complete deletion of the PS PSI domain enhanced TWOW-1 binding. Moreover the PSI domain is dispensable for at least some other integrin functions because PS-PSI displayed an enhanced ability to mediate cell spreading. These studies establish a means to evaluate mechanisms and consequences of integrin affinity modulation in a tractable model genetic system.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Integrins are the primary family of receptors that connects cells to the extracellular matrix (ECM)² (1). The cytoplasmic tails of integrin subunits associate with multiple intracellular components, which mediate both signaling functions and ECM-cytoskeleton connections (2). Cells can regulate the functions of integrins at least in part by inducing conformational changes in integrin structure that alter the affinity of the integrin heterodimer for ECM ligands. Similarly integrin binding to ligands can lead to outside-in signal propagation, which can regulate cellular behaviors such as growth, differentiation, and survival (3-7).

The integrin heterodimer is composed of and subunits that are nonhomologous to one another but strongly conserved structurally across the animal kingdom (8, 9). A long history of studies with conformation-sensitive antibodies indicates that integrins undergo large and concerted conformational changes as a result of cellular activation, and recent studies have begun to provide details of this structural switching (10-16). Inactive integrins are likely to be bent in the middle so that the headpiece faces in toward the membrane-proximal part of the extracellular stalks. As a result of cellular activation, the integrin adopts an extended conformation with the headpiece facing away from the cell in optimal position to engage ECM proteins. The headpiece also can adopt two states, termed the open or closed conformations, corresponding to high and low affinity states for ligand binding. Binding of substrate ligands traps the integrin in the open conformation, and this shift in the equilibrium may then trigger outside-in signaling in many cases involving integrin clustering. This view of integrin dynamics is almost certainly simplified with other intermediate states probable (for example, bent integrins can also bind ligand (17)), but overall it can provide a consistent explanation of the structure-function relationships between cellular regulation and signaling.

Invertebrates such as Drosophila melanogaster provide a system for sophisticated genetic studies of integrin function that are not practical with vertebrates (18). Genetic studies with flies can also be complimented with cell biology experiments in cell culture, and this system has become especially attractive with the simplicity of RNA interference methods in Drosophila (19). However, one persistent drawback to studies of integrin structure and function in invertebrates has been a lack of probes for measuring affinity states of integrins directly. Even in cell culture, Drosophila experiments have relied on indirect assays such as cell adhesion or spreading to quantitate integrin activity. Although these methods have generally provided a consistent set of data, a tool for measuring ligand affinity directly could permit much more reliable and rapid assays that can complement genetic analyses of integrin function in Drosophila.

Here we describe the generation of TWOW-1, a novel monovalent antibody Fab fragment that functions as a ligand-mimetic affinity sensor for PS2PS. Using this probe, we show that previously described mutants in both the PS2 and PS subunits led to changes in affinity of the fly integrins. Finally we demonstrate that complete removal of the subunit N-terminal PSI domain caused an increase in ligand affinity.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Cell Culture—Drosophila S2/M3 were cultured in Shields and Sang M3 medium supplemented with 12% heat-inactivated fetal calf serum as described previously (20). Cells were co-transfected with plasmids expressing an PS2C subunit and a PS subunit, both under the regulation of the heat shock protein 70 promoter, and with the bacterial dihydrofolate reductase selectable marker (plasmid p8HCO) as described previously (21). Transformed cells were selected in 2 x 10^-7 M methotrexate.

Prior to performing TWOW-1 assays, cells were treated with double-stranded RNA targeting the 3'-untranslated sequence of myospheroid to remove expression of endogenous PS integrin as described earlier (21); this 3'-untranslated sequence is not present in the PS transgenes. A few modifications to our previous RNA interference protocol were made. The initial 30-min treatment of cells with double-stranded RNA in serum-free medium was done in M3 medium instead of HyQ-CCM3. Also instead of treating 1 x 10⁶ cells in 0.7 ml of serum-free medium in a 6-well plate, we treated 8 x 10⁶ cells in 1.4 ml of serum-free medium (containing 42 µg of myospheroid double-stranded RNA) in a 60-mm Petri dish for 30 min followed by the addition of 2 volumes of M3 medium containing 16.7% heat-inactivated serum. Preliminary experiments demonstrated that the RNA interference effect was not diminished by the higher concentration of cells and that using Petri dishes instead of tissue culture plastic resulted in less disruption of the cells when they were removed prior to TWOW-1 binding experiments.

PS Integrin Constructs—pHSPS2C (20) or the same construct containing the GFFNR GFANA activating mutation (pHSPS2C^ANA) express the subunit (21). subunits were expressed from pHSPS4A (20) or its variants. The construct expressing PS-b58 has been described previously (22). The PSI deletion in PS was introduced using standard PCR and molecular biology protocols and removed all of the amino acids that lie between but not including Thr-39 and Pro-78.

Construction and Purification of TWOW-1—TWOW-1 Fab was constructed by replacing the DNA fragment encoding the 50-amino acid penton base 5 in the heavy chain complementarity-determining region 3 (H-CDR3) of WOW-1 fd in pMT/BiP/V5-HisB (23) with a sequence encoding 53 amino acids found in Drosophila Tiggrin. These 53 amino acids contain the Tiggrin RGD sequence and 24 amino acids prior to and 26 amino acids following the RGD. As a negative control we also constructed LGA-TWOW-1, which is identical to TWOW-1 except the RGD sequence is replaced by LGA. Standard molecular biology techniques based on PCR were used. The resulting TWOW-1 (or LGA-TWOW-1) fd, containing a His₆ tag, and PAC-1k DNA in pMT/BiP/V5-HisB (24) were co-transfected into Drosophila S2 cells with the selection vector p8HCO. Stable cell lines expressing TWOW-1 Fab were obtained and adapted to grow in CCM serum-free medium (HyClone catalog number SH30065.01) containing methotrexate.

TWOW-1 Fab expression was induced by adding 0.5 mM CuSO₄ to cells at a density of 5 x 10⁶/ml. After 2 days of induction cells were removed by centrifugation, and the supernatant was collected for purification of TWOW-1. The cells were then resuspended in fresh medium containing copper and again incubated for 2 days prior to collecting a second batch of supernatant containing TWOW-1. Typically 500 ml of medium containing TWOW-1 was centrifuged for 15 min at 30,000 x g, and the supernatant was dialyzed twice against 4 liters of PBS (0.13 M NaCl, 7 mM Na₂HPO₄, 3 mM NaH₂PO₄, pH 6.5) for 2-4 h and overnight against 4 liters of binding buffer (50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole, pH 8.0). TWOW-1 was purified by chromatography on a nickel-nitrilotriacetic acid column and dialyzed against BES-Tyrodes (137 mM NaCl, 2.9 mM KCl, 20 mM BES, 0.1% glucose, pH 7.5). Finally TWOW-1 was centrifuged to remove any potential precipitate and frozen in liquid nitrogen. Protein concentration was determined using BCA reagent (Pierce), and the purity and concentration were verified by SDS-PAGE and staining with Coomassie Blue. The typical yield was 1.5 mg of TWOW-1 from 500 ml of medium.

TWOW-1 Binding Assay—To remove pre-existing integrins and matrix and to induce high expression from the heat shock-regulated integrin transgenes, cells were incubated at 36 °C with 1 mg/ml collagenase/dispase (Roche Applied Science catalog number 1097113) in Robb's saline (minus Ca²⁺ and Mg²⁺) for 30 min as described previously (25). Cells were washed and allowed to recover in M3 + 2 mg/ml BSA for 4 h at 23°C. TWOW-1 binding was assessed by flow cytometry as follows. 1 x 10⁶ cells were washed with BES-Tyrodes and resuspended in 30 µl of BES-Tyrodes supplemented with 1 mg/ml BSA (w/v), 1.66 mM MgCl₂, and 16.6 µM CaCl₂. In some cases, 1.66 mM MnCl₂ was included in this buffer to activate the integrin. In cases where EDTA was present to assess nonspecific TWOW-1 binding, cells were washed in 10 mM EDTA (in BES-Tyrodes) and preincubated for 5-10 min in 30 µl of BES-Tyrodes containing 1 mg/ml BSA and 16.6 mM EDTA. Binding assays were initiated by adding 20 µl of TWOW-1 (in BES-Tyrodes) for 10 min at room temperature. 50 µl of 4% formaldehyde in BES-Tyrodes was added to fix the bound TWOW-1 to the cells, and after 5 min of fixation 1.5 ml of BES-Tyrodes was added and cells were centrifuged and resuspended for 25 min on ice in 100 µl of BES-Tyrodes (+1 mg/ml BSA) containing 20 µg/ml AlexaFluor488 goat anti-mouse IgG (Molecular Probes catalog number A-11029) and 10 µg/ml R-phycoerythrin-labeled streptavidin (Molecular Probes catalog number S-866). The R-phycoerythrin-streptavidin was used to detect and exclude from analysis cells that had been disrupted; the cytoplasm of these cells binds nonspecifically to secondary antibody and streptavidin, so the phycoerythrin allowed us to gate out these potentially false positive cells in the flow cytometer. After incubation with secondary antibody the cells were centrifuged and resuspended in 250 µl of PBS followed by immediate addition of 250 µl of 4% formaldehyde in PBS to fix the secondary antibody; the cells were assayed in the flow cytometer in this solution.

Levels of PS2 integrin on the surface of cells were determined by staining with the biotin-labeled anti-PS2 antibody CF.2C7 (which is not conformation-specific) as described previously (25). 5 x 10⁵ cells were incubated for 25 min at room temperature in 50 µl of biotinylated CF.2C7 in M3 + 12.5% fetal calf serum. Then R-phycoerythrin-streptavidin diluted in M3 + fetal calf serum was added to a final concentration of 10 µg/ml and incubated for an additional 30 min on ice. Cells were fixed by the addition of 0.5 ml of 2% formaldehyde in PBS. Other experiments determined that both antibody and streptavidin were present at saturating levels. Fluorescence levels for both TWOW-1 and PS2 were analyzed by flow cytometry at the Arizona Research Laboratories-Biotechnology Cell Sorting Facility at the University of Arizona.

Specific TWOW-1 binding was measured as mean fluorescence intensity minus the TWOW-1 mean fluorescence intensity of the same cells in the presence of EDTA. Typically specific binding was >90% of total binding. For our analyses, this specific binding is expressed relative to surface integrin expression (TWOW-1 mean fluorescence intensity/anti-PS2 mean fluorescence intensity), which was also measured by flow cytometry (see below).

Preliminary experiments gave the following results, which guided our design of the TWOW-1 assay. Phosphate or HEPES-buffered salines were not used for the TWOW-1 binding experiments as they result in precipitate formation when Mn²⁺ is added. Carbonate-buffered salines were avoided as their pH changes with time and exposure to CO₂ in the air. pH 7.5 gave slightly better TWOW-1 binding than pH 6.5 and pH 7.0. Simultaneous incubation of cells with TWOW-1 and secondary antibodies led to artificially high levels of binding. Varying the concentrations of the antibodies and TWOW-1 suggested that this resulted from aggregation of the primary and secondary antibodies prior to binding to the cells. Varying the TWOW-1 incubation time had relatively little effect on the results. Specifically incubations of 1, 10, or 30 min were virtually indistinguishable. If cells were centrifuged out of the TWOW-1 solution and resuspended in fixative, specific binding was reduced 20% relative to that seen when cells were diluted directly into fixative from the TWOW-1 incubation solution. Changing the BSA concentration between 0.2 and 5 mg/ml made no difference in TWOW-1 binding levels or background binding. This is not unexpected as the cells are incubated for 4 h in M3 + 2 mg/ml BSA prior to the TWOW-1 binding assay.

Transient Expression—To measure the surface expression of the activating mutants we performed transient expression experiments. To ensure uniformity of the different DNA constructs all were prepared at the same time. Cells were transformed with the indicated and subunits together with the selection plasmid pH8CO as for stable transformations. The subunits were epitope-tagged (with Myc-EQKLISEEDL or hemagglutinin (HA)-YPYDVPDYA) in the serine-rich loop between Ser-137 and Gly-138 to allow immunostaining specifically for the transfected genes. One day after transformation the medium was replaced with medium containing methotrexate (to kill non-transfected cells), and the cells were transferred from 6-well tissue culture plates to 4-ml Petri dishes. Cells do not adhere to Petri dishes so potential artifacts because of differential adherence of cells were avoided. Three days later cells were collected for staining. For HA staining, cells were incubated at room temperature in 10 µg/ml AlexaFluor488-conjugated anti-hemagglutinin (Molecular Probes catalog number A-21287). For Myc staining, cells were first incubated at room temperature with 2 µg/ml biotin-conjugated anti-c-Myc (Sigma catalog number B-7554), then centrifuged, and resuspended in 10 µg/ml R-phycoerythrin-conjugated streptavidin (Molecular Probes catalog number S-866) for 25 min on ice. Antibody and streptavidin staining was done on 1 x 10⁶ cells in 100 µl of growth medium (M3 + 12% fetal calf serum). Following antibody incubation, cells were centrifuged and resuspended in 1 ml of PBS containing 2% formaldehyde, and then 10,000 cells were analyzed by flow cytometry. PS-PSI and PS-PSI-b58 double mutants were tagged with Myc, PS-b58 was tagged with HA, and wild-type PS controls were tagged with either Myc or HA. Expression levels are given as a percentage of the mean fluorescence of the wild-type PS. The values given are the average and S.E. of three transient expression experiments for each cell line.

Cell Spreading—Cell spreading assays were done as described previously (25). Briefly cells were protease-cleared as in the TWOW binding experiments and then plated on a Tiggrin ligand (RBB-Tigg at a coating concentration of 32 ng/ml) for 4 h. Spread and round cells were visually scored. The values given are the average of two cell spreading assays. In each experiment more than 100 cells were scored from each of three separate fields.

Homology Modeling of Drosophila PS—The human V3 crystal structure coordinates (Protein Data Bank code 1JV2 [PDB] ) (10) were downloaded, and the structure was visualized in Sybyl 7.0 (Tripos). Drosophila PS sequence was aligned with the human 3 chain (ClustalW), and the alignment was adjusted by eye to obtain an optimal template for homology modeling. The model was built based on the first 434 residues of the 3 structure as the template utilizing Modeler (version 7.0). The model was energy-minimized using the Powell method (1000 cycles, -1730 kcal/mol) (Sybyl 7.0). The validity of the model was confirmed using the Ramachandran plot, which was shown to be consistent with the 3 structure. Superposition of the backbone C atoms of PS onto the 3 structure demonstrated a root mean square value of 0.75 Å; confirming the correctness of the model. Subsequently the V409D mutation was incorporated into the PS model and energy-minimized (100 cycles, -1700 kcal/mol) (Sybyl 7.0). Both PS models were utilized to evaluate potential side chain interactions in the vicinity (5-10 Å;) of the mutated residue.

View larger version (15K): [in this window] [in a new window]   FIGURE 1. TWOW-1 binding to PS2PS integrins on S2 cells. A, TWOW-1 binding to wild-type PS2PS in the absence (solid line) or presence (dashed line) of 1 mM Mn2+. In this and subsequent figures, binding is expressed as a ratio of specific TWOW-1 immunofluorescence over total integrin (as detected by an anti-PS2 antibody). "Specific" TWOW-1 binding is the total TWOW-1 immunofluorescence minus that seen in the presence of EDTA. B, similar to A but all PS2 subunits contain the activating mutation GFFNR GFANA in the cytoplasmic membrane-proximal region. Affinity for TWOW-1 is increased relative to wild type even in the presence of Mn2+. In all figures error bars represent the S.E. for three experiments unless noted.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

TWOW-1 Fab showed dose-dependent binding to cells expressing PS2PS integrins (all experiments reported here use the PS2C splice variant (31)) in the presence of Ca²⁺ and Mg²⁺ (Fig. 1A). This binding was inhibited by EDTA and RGD peptides and by mutations to the PS MIDAS ligand binding site (supplemental data). Moreover TWOW-1 in which the RGD sequence was mutated to LGA also showed reduced binding (supplemental data). These controls indicate that binding of soluble monovalent TWOW-1 Fab is a valid indicator of PS2PS affinity for ligand.

Drosophila PS2PS Is Sensitive to Affinity Modulation by Mn²⁺ and PS2 Cytoplasmic Domain Mutation—Cations form a critical part of the ligand binding region of the I domains of both and subunits, and changes in cation coordination are important mediators of the conformational changes that modulate integrin-ligand affinity (12, 14, 32, 33). Mn²⁺ is known to activate integrins generally and is believed to function primarily by binding to one or more sites on the integrin subunit (12, 34). Indeed Mn²⁺ is often used to determine a "maximal" value for integrin-ligand affinity for comparisons with various experimental conditions. We tested TWOW-1 binding in various concentrations of Mn²⁺ (see supplemental data) and found maximal stimulation at 1 mM Mn²⁺, leading to saturable binding at lower concentrations of TWOW-1. For cells expressing wild-type integrins, the Mn²⁺-induced increase in TWOW-1 binding was greater than 2-fold at 15 µg/ml TWOW-1 (Fig. 1A). Expressed another way, 8-fold more TWOW-1 was required to obtain similar levels of integrin binding in the absence of Mn²⁺. The half-maximal binding values yield apparent PS2PS-TWOW-1 K_d values of 1 and 0.12 µM in the absence or presence of Mn²⁺, respectively.

Mutations in the membrane-proximal region of the subunit cytoplasmic domain have been shown to activate integrins, and these mutations are believed to at least partially mimic the inside-out signaling important in cellular regulation of integrin function (35-38). We have shown previously that the GFFNR GFANA mutation of PS2 increases the cell spreading and cell adhesion properties of PS2PS expressed in S2 cells (25). TWOW-1 binding to these cells was also increased dramatically, demonstrating that the mutation enhances integrin affinity as for vertebrate integrins (Fig. 1, A and B, compare "no Mn²⁺" curves).

Surprisingly the GFFNR GFANA mutation also appeared to increase the affinity of PS2PS for TWOW-1 in the presence of Mn²⁺ (Fig. 1, A and B, compare Mn²⁺ curves), demonstrating that this cation does not fully activate the wild-type integrin. Affinity measurements of integrins and ligands are often measured as a ratio of binding under certain conditions relative to binding seen in the presence of optimal concentrations of Mn²⁺, which is assumed to be the maximal value. Our results indicate that this assumption is not necessarily true at least for PS2PS. Because binding of TWOW-1 to wild-type PS2PS in Mn²⁺ does not represent the maximal value, we chose throughout to express TWOW-1 binding relative to total integrin levels as measured by the PS2-specific antibody CF.2C7.

mys^b58 Increases Integrin Affinity for TWOW-1—In a screen for mutations in the Drosophila gene encoding the PS subunit (myospheroid), we found an unusual mutant that alters integrin function. The mys^b58 allele (V409D) has properties in developing animals that suggest it may enhance integrin activity, and cell spreading and adhesion experiments with mys^b58 are consistent with this proposal (22). The V409D mutation lies at the top of the 7 helix of the subunit I domain (Fig. 2A). This residue is highly conserved in integrin subunits (but not completely so; see Ref. 22 for discussion of the exception), and in the low affinity "closed headpiece" conformation Val-409 forms part of a conserved hydrophobic interface between 7 and the neighboring 1 helix (Fig. 2B). It therefore seemed likely that insertion of an aspartate residue here might disrupt this interaction, and we proposed that mys^b58 might specifically alter the equilibrium between the "open" and "closed" conformations of the I domain (22).

We found that cells expressing PS2PS-b58 showed higher affinity for TWOW-1 than cells expressing wild-type integrin (Fig. 3), indicating that the phenotypic effects of mys^b58 can be explained by increases in ligand binding activity. TWOW-1 binding to PS2PS-b58 was greater than TWOW-1 binding to GFANA-activated heterodimers and approached that obtained when wild-type cells were exposed to Mn²⁺. PS2PS-b58 binding was increased when cells were incubated with Mn²⁺, although the difference was small relative to Mn²⁺ effects on wild-type integrins. Thus, PS-b58 is a strong activator of integrin-ligand affinity.

View larger version (53K): [in this window] [in a new window]   FIGURE 2. Modeling of the PS-b58 structure. A, the I domain of PS was modeled based on the x-ray structure of 3 in the "headpiece closed" conformation (10). The 7 helix, which shifts down in the open conformation, is colored violet, and side chains are colored as follows: Val-409, red; the two nearby leucines of the 1 helix, green; and the lysine (Lys-210) that potentially may interact with Asp-409 in the b58 mutant, blue. B, same structure rotated and with the colored residues filled, showing the close apposition of the 7 helix valine with the 1 helix leucines.

View larger version (14K): [in this window] [in a new window]   FIGURE 3. The PS-b58 mutation (V409D) increases affinity for TWOW-1. Values are for binding at 15 µg/ml TWOW-1 (see Fig. 1). The PS-b58 I domain mutation increases affinity for TWOW-1, more so than the activating PS2 cytoplasmic mutation. Binding is expressed as a ratio of specific TWOW-1 immunofluorescence over total integrin. wt, wild type.

Deletion of the PS PSI Domain Increases Affinity for TWOW-1

In a screen for subunit lesions that affected integrin function, we isolated point mutations in the PSI domain (22), and preliminary experiments showed slightly increased cell spreading activity of the mutant integrins.³ This led us to question whether the PSI domain is required at all for integrin-ligand binding, and we constructed PS subunits in which this domain was deleted completely. Cells expressing PS2PS-PSI showed increased TWOW-1 binding relative to cells expressing wild-type integrins (Fig. 5A). Their affinity for TWOW-1 could be further increased by Mn²⁺. Fully functional integrins must do more than simply bind soluble ligands, so we also examined the ability of PS2PS-PSI to mediate cell spreading on a Tiggrin protein fragment. PS2PS-PSI was superior to wild type in this assay as well (Fig. 5B).

View larger version (9K): [in this window] [in a new window]   FIGURE 4. Mutations at PS 409 increase ligand binding. Binding of TWOW-1 (at 15 µg/ml) to PS2PS integrins in which Val-409 has been mutated to aspartate (as in PS-b58), lysine, alanine, or phenylalanine is shown. Any change from valine leads to increased TWOW-1 binding with Ala-409 being almost as strong as aspartate. This indicates that the increased activity of PS-b58 is not due to new specific interactions involving Asp-409. Binding is expressed as a ratio of specific TWOW-1 immunofluorescence over total integrin. wt, wild type.

View larger version (15K): [in this window] [in a new window]   FIGURE 5. PS-PSI increases integrin ligand affinity. A, complete removal of the N-terminal subunit PSI domain (PSI) greatly increases TWOW-1 binding. B, integrins without the PSI domain also show increased ability to mediate cell spreading on Tiggrin fragments even though these cells express lower levels of surface integrin compared with wild type (see Fig. 7). Data in B are means from two experiments. Binding is expressed as a ratio of specific TWOW-1 immunofluorescence over total integrin. wt, wild type.

Finally as shown below, the activating mutations often lead to reduced cell surface integrin expression. The TWOW-1 binding data shown in Fig. 6 were derived from various stably transformed cell lines that may display significantly different levels of expression. However, the ratio of TWOW-1 binding/total integrin was similar for each mutant, regardless of integrin expression levels.

View larger version (33K): [in this window] [in a new window]   FIGURE 6. PS-PSI-b58 is stronger than either individual mutation. Combining the two activating PS mutations can lead to integrins with a very high affinity for TWOW-1 compared with PS-PSI or PS-b58 alone. The upper panel shows binding at three different TWOW-1 concentrations in the absence of Mn2+; the lower panel is in the presence of 1 mM Mn2+. Binding is expressed as a ratio of specific TWOW-1 immunofluorescence over total integrin.

Integrins with activating mutations were expressed at reduced levels in transient transfectants (Fig. 7). Although the reductions were not as severe as those generally seen in stable transformed lines, the relative expression levels of the various mutants were similar in both conditions. Most strikingly, increased ligand affinity did not necessarily correlate with reduced heterodimer expression at least quantitatively. This is illustrated by the fact that PS-b58 reduced integrin expression relatively modestly despite the observation that this mutation greatly increased TWOW-1 binding. This is also consistent with observations of various stably transformed PS-b58 lines where expression often is reduced slightly but may be at levels comparable to wild type. Interestingly in mys^b58 mutant animals (or homozygous clones in heterozygous animals), we detected no difference in PS surface expression in larval tissues (22). PS-b58 did significantly exacerbate the expression reduction seen with PS2-GFANA or PS-PSI in the transient transfectants (Fig. 7). This is similar to results from mutations expected to destabilize the PS I domain (headpiece); these often show little or no reduction in cell surface expression in larval tissues on their own but lead to greatly diminished expression in combination with mutations in the PS2 GFFNR sequence (22).

View larger version (12K): [in this window] [in a new window]   FIGURE 7. Activating mutations typically lead to reduced surface integrin expression. Total surface integrin (assayed by binding to PS epitope tags) was measured for transiently transfected cell lines expressing wild type and the indicated mutants at 4 days following transfection. As typically seen in stable transformants, surface expression follows the pattern wild type > PS-b58 > PS2-GFANA > PS-PSI. Double mutants are expressed at especially low levels. wt, wild type.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Drosophila is useful as a model system because it is relatively easy to generate and study mutations in integrins or in cytoplasmic proteins that interact with integrins both in cell culture and whole developing animals. However, studies of invertebrate integrins have been hampered by a lack of probes that can directly assess integrin-ligand affinity. For example, using cell spreading or adhesion as an assay it was clear that the activity of Drosophila integrins can be affected by extracellular and cytoplasmic mutations or by culture conditions (21, 22, 25), but it was unknown whether this regulation included affinity modulation or simply changes in avidity.

Using TWOW-1, we can now measure affinity changes in the Drosophila PS2PS integrin. In this study, we describe results from a variety of previously studied and new mutations in Drosophila PS2 and PS proteins. For the most part, the data reported here are in agreement with results from previous experiments: changes that increased cell spreading (PS2 GFFNR GFANA and PS V409D) also increased integrin affinity for TWOW-1.

Mn²⁺ and Affinity Modulation—As seen for vertebrate integrins, Mn²⁺ increases the activity of PS2PS in cell spreading assays (44), and here we show that Mn²⁺ increased the affinity of PS2PS for TWOW-1. However, TWOW-1 binding in Mn²⁺ could be further increased by a GFFNR GFANA mutation in the PS2 subunit, which is expected to induce cytoplasmic tail separation (35-38), thus promoting an extended conformation of the integrin. In many published reports, integrin affinity is expressed as a fraction of the affinity seen in the presence of Mn²⁺ with the implicit assumption that Mn²⁺ ions induce maximal affinity. Our data indicate that this assumption is not always valid and suggest that in some cases measuring ligand binding relative to total integrin expression may be a more accurate reflection of overall affinity. Mn²⁺ is a general activator of integrin affinity that most likely mediates its effects by occupying one or more of the subunit cation binding sites (12, 32-34). Mn²⁺ ions have been shown to alter integrin conformations, and this appears to be a direct effect, which can be detected in isolated proteins (Ref. 14, but see also Ref. 17). Interestingly Takagi et al. (14) saw that Mn²⁺ affected the equilibrium between conformations but did not drive all of the molecules into one state. In any event, our results establish that "maximum" ligand affinity is an operationally defined entity and cannot simply be assumed to be the affinity measured in optimal Mn²⁺.

PSI and Integrin Conformation—The PSI domain is in position to make contacts with the subunit hybrid domain and stalk (32, 39). Thus, it is a candidate for transmitting structural changes in the subunit head to the stalk and then to the cytoplasmic domains of the protein. Previous data indicated that mutations in mammalian integrin PSI domains can increase integrin activity (40, 41). We found that complete deletion of the PSI domain (PS-PSI) both increased ligand affinity and reduced integrin expression.

What is the likely mechanistic explanation for these PS-PSI phenotypes? PS-PSI is similar to GFFXR alleles in that affinity and expression are altered in comparable ways. GFFXR mutations most likely affect the equilibrium between bent and extended conformations, favoring the latter, by promoting cytoplasmic tail separation. It is thus tempting to hypothesize that the PSI domain-stalk interaction helps to stabilize the bent conformation and that its absence allows the integrin to extend. Consistent with this hypothesis, combination of PS-PSI with PS-b58 (which is expected to alter the equilibrium between the open and closed conformations of the PS I domain; see below) caused further increases in affinity for ligand. Similar additive effects were also found when PS-b58 and PS2 GFFNR mutations were combined. We could not determine whether PS-PSI and PS2 GFFNR mutations were additive with respect to ligand affinity because these mutant subunits are too unstable to be expressed at detectable levels. Integrin conformations and stability appear generally to be the result of multiple weak interactions. It seems that either the associations of the transmembrane/cytoplasmic domains with one another (or other cytoplasmic proteins) or the presence of the PSI domain can provide enough heterodimer stability to result in some surface expression of integrins. However, compromising both of these stabilizing influences renders the proteins unable to be expressed on the cell surface.

PS-b58 Increases Integrin-Ligand Affinity—Mutant phenotypes of mys^b58 (PS V409D) flies and previous experiments in cell culture indicated that the mutant integrins are more biologically active than wild-type proteins (22). Here we show that PS-b58 integrins had a much higher affinity for TWOW-1 than did wild-type integrins. Indeed this mutation had a greater affect on TWOW-1 affinity than the PS2 cytoplasmic GFFNR GFANA mutation and was almost equivalent to wild-type PS activated by Mn²⁺.

Modeling of PS based on the derived structures of 3 suggested at least one possible novel molecular interaction that could result from the V409D change. However, substitution of three widely different residues into this site also increased TWOW-1 binding. The relatively large effect of the Val Ala mutation indicates that the effects of PS-b58 are mostly due to the loss of the valine at position 409 as opposed to the addition of the aspartate. It has been proposed that the closed conformation of the I domain of integrin subunits is stabilized by hydrophobic interactions between the 7 and 1 helices of the domain (45). Although not exactly in a structurally homologous position, the effects described here (and the strong conservation of these hydrophobic residues in subunits) suggest that similar forces may be at work in subunit I domains.

Barton et al. (46) also investigated a hydrophobic linkage between the 1 and 7 helices of the human 1 subunit. They found three hydrophobic residues (one in the 1 helix and two in 7) that caused activation when mutated to alanine. They did not test the residue corresponding to PS 409 but did mutate both of the neighboring conserved leucines in the 7 helix. Neither of these resulted in significant activation, although the double mutant, or changes to residues other than alanine, were not tested.

The weak effects of mys^b58 on fly viability are somewhat surprising in light of the high inherent ligand affinity of PS2PS integrins. PS2 subunits that are deleted for the entire cytoplasmic domain (PS2-cyt) or just the membrane-proximal GFFNR sequence (PS2-GFFNR) display increased integrin activity in situ as inferred by an expansion of muscle attachment sites in developing embryos and by phenotypes in developing wings (47). PS2-GFFNR can rescue most inflated (PS2) embryonic phenotypes; however, PS2-cyt is less capable of supporting development through embryogenesis. It has been suggested that the inability of the PS2 cytoplasmic mutants to support complete embryonic viability may be a consequence of a defect in inside-out signaling, resulting in an inappropriate increase in integrin-ligand affinity (47). mys^b58 also leads to a significant increase in PS2PS ligand affinity (although probably not as extreme as for PS2-cyt), but mys^b58 has very little effect on viability, not only in embryos but for complete development of fertile adults (22). Thus one must use caution in assigning apparent loss-of-function phenotypes (such as decreased viability) in PS2-GFFNR and PS2-cyt to loss of inside-out signaling; at least some phenotypes may result more directly from the deletion of required cytoplasmic residues.

The effects of PS-b58 on surface expression of integrins is not as straightforward as for other activating mutants, which reliably cause significant reductions in expression both in S2 cells and, as shown for GFFXR mutations, in developing tissues. In contrast, mys^b58 has no discernable effect on integrin expression in developing larvae (22). In S2 cell transient transfections and stable transformants, PS-b58 often shows reduced expression relative to wild-type PS, but this reduction is not as pronounced or reliable as for PS2-GFANA or PS-PSI. This difference may be because these mutations have primary effects on different aspects of integrin conformation. Both PS2-GFANA and PS-PSI are located in regions that might be expected to affect the bent extended switch, whereas the V409D mutation of PS-b58 is positioned where one would expect it primarily to alter the head open closed equilibrium, so any effects on the bent extended equilibrium are likely to be indirect.

The results with known activators and inhibitors of integrin-ligand binding as well as with integrin mutations that affect ligand binding all point to the utility of TWOW-1 as a direct reporter of PS2PS ligand affinity. In addition to studies of integrin mutants, the development of TWOW-1 will open up the possibility for future genetic and other investigations of potential cytoplasmic regulators of Drosophila integrin activity.

	FOOTNOTES

 
^* This work was supported by National Institutes of Health Grants R01GM42474 (to D. L. B.) and R01HL56595 (to S. J. S.) and American Cancer Society Grant 5P30CA023074 (to D. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental data.

¹ To whom correspondence should be addressed: Dept. of Molecular and Cellular Biology, Arizona Cancer Center, Rm. 0977, 1515 N. Campbell Ave., Tucson, AZ 85724. Tel.: 520-621-3869; Fax: 520-626-3764; E-mail: tbunch{at}u.arizona.edu.

² The abbreviations used are: ECM, extracellular matrix; BES, 2-[bis(2-hydroxyethyl)amino]ethanesulfonic acid; BSA, bovine serum albumin; HA, hemagglutinin; H-CDR3, heavy chain complementarity-determining region 3; PSI, plexin-semaphorin-integrin.

³ T. A. Bunch, unpublished results.

	ACKNOWLEDGMENTS

 
We thank Barb Carolus and Debbie Sakiestewa of the Arizona Research Laboratories Cytometry Service for much assistance and Candace George for helping to make TWOW-1. Candida Morris assisted with manuscript preparation, and Emily Smith provided useful comments on the manuscript.

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