L-plastin Peptide Activation of alpha vbeta 3-mediated Adhesion Requires Integrin Conformational Change and Actin Filament Disassembly*

Jun WangDagger , Hua Chen§, and Eric J. Brown§

From the Dagger  Program in Molecular Cell Biology, Division of Biology and Biomedical Sciences, Washington University School of Medicine, St. Louis, Missouri 63110 and the § Program in Host-Pathogen Interactions, University of California, San Francisco, San Francisco, California 94143

Received for publication, August 11, 2000, and in revised form, January 23, 2001




    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

L-plastin (LPL) is a leukocyte actin binding protein previously implicated in the activation of the integrin alpha Mbeta 2 on polymorphonuclear neutrophils. To determine the role for LPL in integrin activation, K562 cell adhesion to vitronectin via alpha vbeta 3, a well-studied model for activable integrins, was examined. Cell permeant versions of peptides based on the N-terminal sequence of LPL and the LPL headpiece domain both activated alpha vbeta 3-mediated adhesion. In contrast to adhesion induced by treatment with phorbol 12-myristate 13-acetate (PMA), LPL peptide-activated adhesion was independent of integrin beta 3 cytoplasmic domain tyrosines and was not inhibited by cytochalasin D. Also in contrast to PMA, LPL peptides synergized with RGD ligand or Mn2+ for generation of a conformational change in alpha vbeta 3 associated with the high affinity state of the integrin, as determined by binding of a ligand-induced binding site antibody. Although LPL and ligand showed synergy for ligand-induced binding site expression when actin depolymerization was inhibited by jasplakinolide, LPL peptide-induced adhesion was inhibited. Thus, both actin depolymerization and ligand-induced integrin conformational change are required for LPL peptide-induced adhesion. We hypothesize that the critical steps of increased integrin diffusion and affinity enhancement may be linked via modulation of the function of the actin binding protein L-plastin.




    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

A fundamental property of leukocyte integrins is the ability to modulate their adhesive functions. As cells circulate through blood and lymph, adhesion is minimal. In response to inflammatory signals, integrin-mediated adhesion is markedly augmented. This property of the integrins is physiologically critical, because it is required for appropriate migration out of the vasculature into sites of inflammation and at the same time limits the potentially host-damaging inflammatory response to those sites alone. Because this activity of integrins is so important to their role on leukocytes, much attention has been devoted to the molecular mechanisms by which integrin adhesion is regulated. Two distinct alterations in integrins are likely to be involved in enhancement of adhesion. A rapid response to cell activation is an increase in integrin diffusion, due to loss of cytoskeletal constraint of integrin mobility. Increased diffusion may lead in turn to integrin clustering at sites of cell interaction with ligand. A second response to activation is a conformational change in the integrins, which often reflects increased affinity for ligand. This conformational change may require initial interaction with ligand and can be reflected in the generation of new epitopes recognized by monoclonal antibodies, the so-called ligand-induced binding sites (LIBS1). Although a number of signaling molecules, such as PKC and phosphatidylinositol 3-kinase, have been implicated in this process (1-3), these enzymes may be many steps upstream from the actual change in integrin behavior. GTPases of the Ras and Rho families also have been implicated in regulation of integrin function (4-6), but their downstream targets for this function have not been identified. Therefore, much remains to be learned about the mechanisms involved in regulation of integrin avidity.

Our own studies have implicated the actin-binding protein L-plastin (LPL) in regulation of integrin function (7, 35). Recently, we have shown that cell-permeant peptides from the N terminus of LPL can rapidly activate alpha Mbeta 2 (Mac-1)-mediated adhesion in polymorphonuclear neutrophils (PMN). When the peptides introduced into PMN cytosol contained phosphoserine at position 5, which is the major if not exclusive site of phosphorylation in LPL (7, 35), integrin activation was not inhibited by blockade of PKC or phosphatidylinositol 3-kinase, suggesting that LPL phosphorylation might be a mechanism by which these enzymes signal changes in integrin function. Thus, LPL is likely a downstream effector of multiple signaling pathways leading to integrin activation, and LPL peptide induction of adhesion is a useful experimental system in which to begin to understand regulation of integrin avidity.

To pursue these mechanistic questions, we turned to a well-characterized model for studying integrin activation. alpha vbeta 3 is not normally expressed in undifferentiated K562 cells, but when expressed through transfection of alpha v and beta 3 cDNAs, alpha vbeta 3-mediated adhesion in K562 cells is dependent on cell activation (8). Moreover, adhesion in response to PMA or thrombin is dependent on phosphorylation of Tyr-747 in the beta 3 cytoplasmic tail, an event that is itself dependent on the presence of the alpha v cytoplasmic tail (8, 9). Using these cells, we have examined the requirements for LPL peptide-induced adhesion. We have found that a cell permeant version of the entire headpiece domain of LPL, as well as synthetic LPL peptides, can induce adhesion. In contrast to PMA, LPL peptide-induced adhesion does not require tyrosine phosphorylation of the beta 3 cytoplasmic tail, suggesting that LPL is downstream of the tyrosine signal in the activation cascade. Nonetheless, LPL peptide-induced adhesion does require actin depolymerization, presumably to induce integrin release from cytoskeletal constraint (10-12), and LPL peptide does cooperate with Arg-Gly-Asp (RGD) ligand to induce LIBS epitope expression. These data suggest that the steps of increased integrin diffusion and conformational change may be linked via modulation of the function of the actin binding protein L-plastin.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Cells and Reagents-- The human erythroleukemic cell line K562 transfected with cDNA encoding alpha vbeta 3 (Kalpha vbeta 3) and alpha vbeta 3 in which the tyrosine residues at position 747, 759, or both were mutated to phenylalanine (Y747F, Y759F, and Y747F/Y759F, respectively) were derived as described (8, 9). Cells were maintained in Iscove's modified Dulbecco's medium (Life Technologies, Inc., Gaithersburg, MD), containing 10% heat-inactivated fetal calf serum (HyClone, Logan, UT), 2 mM L-glutamine, 100 µg/ml penicillin, and streptomycin under a 5% CO2 atmosphere. JY B lymphoblastoid cells were from ATCC (Rockville, MD) and maintained in RPMI 1640 medium (Life Technologies, Inc.) with the same supplements as Iscove's modified Dulbecco's medium. Jasplakinolide was from Molecular Probes (Eugene, OR).

Monoclonal antibodies L230 (anti-human alpha v), 1A2.1 (anti-human beta 3), AP3 (anti-human beta 3), 7G2 (anti-human beta 3), W6/32 (anti-human HLA), P1F6 (anti-alpha vbeta 5), mAb16 (anti-alpha 5), TS2/16 (anti-beta 1), and LPL4A.1 (anti-human LPL) have been previously described (8, 13-15). Monoclonal mouse antibody against the tat-(49-58) epitope (19) was a gift from Hybridolab, Institut Pasteur, Paris, France. Calcein was purchased from Molecular Probes. 96-well Immulon 2 plates were from Dynatech (Chantilly, VA). Vitronectin (Vn), RGD (GRGDSP) and RAD (GRADSP) peptides, and phosphate-buffered saline (PBS) were purchased from Life Technologies, Inc. (Gaithersburg, MD). Human serum albumin (HSA) was from Therapeutic Corp. (Los Angeles, CA). Purified human recombinant osteopontin was a gift from Simon M. Blake, SmithKline Beecham, King of Prussia, PA.

LPL peptides were described previously (7). The sequences of the LPL-related peptides used in this study were LPL (ARGSVSDEEMMELREAFA), LPLtat (ARGSVSDEEMMELREAFAYGRKKRRQRRRG), tatLPL (YGRKKRRQRRRGARGSVSDEEMMELREAFA), SCRtat (AGDESEMEFVMASALRREYGRKKRRQRRRG), and TPLtat (ATTQISKDELDELKEAFAYGRKKRRQRRRG).

Tat Fusion Protein Purification-- The cDNA containing the LPL N-terminal Ca2+-binding domain (headpiece fragment, amino acids 1-105) was cloned into pBluescript KS+ as an XhoI/BamHI fragment. An myc tag was fused in-frame with the C terminus of the headpiece sequence (Headmyc). To obtain the headpiece fragment with the tat sequence at the C terminus (Headtat), the myc sequence was replaced by an oligonucleotide encoding the 12-amino acid tat peptide sequence. The Headmyc fragment also was subcloned into the pTAT vector (a gift of Dr. Steven Dowdy, Washington University, St. Louis, MO) (16) to obtain a headpiece with the tat sequence at the N terminus (tatHead). The constructs were transformed into BL-21 LysS cells (Novagen). Bacteria from 1-liter cultures were resuspended in 50 ml of PBS + 50 µg/ml lysozyme. The bacteria were lysed with three freeze-thaw cycles and sonication, and lysates were clarified by centrifugation. The supernatant was run through an immunoaffinity column of LPL4A.1 mAb coupled to Sepharose 4B and eluted with PBS containing 10 mM EDTA as LPL4A.1 binds to LPL in a Ca2+-dependent manner. EDTA was removed by dialysis.

Adhesion Assay-- Kalpha vbeta 3 cells or JY cells (1 × 107/ml) were washed once with HBSS (1× Hanks' buffered salt solution with 20 mM Hepes and 8.9 mM sodium bicarbonate) and incubated with 2 µg/ml calcein in HBSS for 30 min at RT. The cells were washed three times and resuspended in HBSS2+ (HBSS with 1.0 mM Mg2+ and 1.0 mM Ca2+) at 2 × 106/ml. Cells were subjected to various treatments as detailed in respective figure legends. 1 × 105 cells/well were added to HSA-, Vn (10 µg/ml or as indicated in the figures)-, or osteopontin (10 µg/ml)-coated 96-well Immulon 2 plates. Cells were allowed to settle for 10 min before L-plastin peptides (50 µM), PMA (50 ng/ml), or MnCl2 (1 mM) were added to the wells. Cells were incubated at 37 °C for 1 h. After the incubation period, the fluorescence (485-nm excitation and 530-nm emission wavelengths) was measured using an fMax fluorescence plate reader (Molecular Devices, Sunnyvale, CA) before and after washing four times with 180 µl of PBS. Percent adhesion was calculated by dividing the fluorescence after washing by the fluorescence before washing. The assays were performed in triplicate, and data are presented as mean ± S.D. All experiments were performed at least three times with similar results. In preliminary experiments, fluorescence was shown to be linearly related to cell number.

FACS Analysis of Ligand-induced Binding Site (LIBS) Expression-- Binding of mAb 7G2 (13) to Kalpha vbeta 3 cells was assessed by fluorescence flow cytometry (FACS). Kalpha vbeta 3 cells were washed once with HBSS and resuspended in HBSS2+. Cells were treated with control buffer, PMA (50 ng/ml), MnCl2 (1 mM), or 50 µM LPL peptides (LPLtat, SCRtat, TPLtat, tatLPL, or LPL) at 37 °C for 20 min. Cells were then incubated with RGD or RAD peptides (100 µM or as specified in the figures) together with the 7G2 antibody (5 µg/ml) for 60 min at RT. After washing three times with FACS buffer (PBS + 200 µg/ml bovine serum albumin + 130 µg/ml NaN3), cells were stained with fluorescein isothiocyanate-conjugated Fab-specific anti-mouse IgG (Sigma Chemical Co., St. Louis, MO) for 45 min on ice. Cells were washed with FACS buffer and analyzed by flow cytometry. For MnCl2-treated samples, 250 µM MnCl2 was always present in the subsequent incubation and washing buffers.

F-actin Content-- F-actin content of Kalpha vbeta 3 cells was determined as previously described (41) with minor modifications. Briefly, cells were incubated with buffer, 50 µM LPLtat, 1 µM jasplakinolide, or both for 15 min at 37 °C and then solubilized by incubation with an equal volume of Tris-buffered saline, pH 7.4, containing 4% Triton X-100, 4 mM EDTA, and 5 µg/ml leupeptin. After centrifugation at 38,000 rpm for 30 min in a TL100.2 rotor (Beckman Optima TL Ultracentrifuge, Beckman Instruments, Fullerton, CA), the supernatant was carefully removed, and the gelatinous pellet was dissolved in SDS-polyacrylamide gel electrophoresis sample buffer. After separation by SDS-polyacrylamide gel electrophoresis, actin content of the Triton-insoluble pellet was analyzed by Western blot with rabbit anti-actin (Sigma) and quantitated by densitometry.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

LPL N-terminal Peptide Induces alpha vbeta 3-mediated Adhesion-- We previously demonstrated that a cell permeant peptide containing LPL amino acids 2-19 fused at the C terminus with the HIV tat sequence (LPLtat) induces alpha Mbeta 2-mediated adhesion in PMN (7). LPLtat also causes alpha Mbeta 2-dependent adhesion to C3bi, an alpha Mbeta 2-specific ligand, in Jurkat cells transfected with alpha Mbeta 2 integrin.2 These data suggest that LPL has a role in alpha Mbeta 2 activation in leukocytes. We next asked whether LPL was specifically involved in alpha Mbeta 2 activation or if it was involved in the regulation of other integrins. The K562 cell, transfected with exogenous integrins, is a well-established model system for studying integrin functions in a myeloid background (8, 9, 14, 17), and our laboratory has shown previously that cell activation is required for adhesion mediated by alpha vbeta 3 expressed in these cells (8). Therefore, we tested the effects of LPLtat on alpha vbeta 3-mediated adhesion to Vn in stable K562 transfectants (Kalpha vbeta 3). LPLtat induced Kalpha vbeta 3 adhesion to Vn but not to HSA (Fig. 1A). Although LPLtat did not induce cell adhesion in the absence of ligand, its effect was apparent even at a Vn coating concentration of 0.1 µg/ml (Fig. 1B). This effect was specific to LPLtat, because control peptides, including tatLPL, in which the tat sequence mediating membrane permeability of the peptide was N-terminal to the LPL sequence, SCRtat, in which the LPL sequence was scrambled, and TPLtat, which contained amino acids 2-19 of the LPL homologue T-plastin, did not increase adhesion (Fig. 1C). LPLtat did not induce adhesion of the parental K562 cells, which do not express alpha vbeta 3, to Vn (K562+LPLtat in Fig. 1C). LPLtat-induced adhesion of Kalpha vbeta 3 was inhibited by the anti-alpha v antibody L230 and the anti-beta 3 antibody 1A2.1 (Fig. 1D), further demonstrating the requirement for the alpha vbeta 3 integrin. LPLtat, but not tatLPL, SCRtat, or TPLtat, also induced Kalpha vbeta 3 adhesion to osteopontin (data not shown). Like adhesion to Vn, Kalpha vbeta 3 adhesion to osteopontin was inhibited by an anti-beta 3 mAb but not by antibodies recognizing alpha 5, beta 1, or HLA. Thus, LPLtat activates alpha vbeta 3- as well as alpha Mbeta 2-mediated adhesion. In contrast, alpha 5beta 1-mediated adhesion to fibronectin does not require activation in K562 cells (14), and LPLtat had no significant effect on adhesion of untransfected K562 to fibronectin (data not shown). Thus, LPLtat affects integrin activation for adhesion, rather than adhesion by already active integrins.



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Fig. 1.   LPLtat induces alpha vbeta 3-mediated adhesion to Vn in Kalpha vbeta 3 cells. A, Kalpha vbeta 3 cells were allowed to adhere to HSA (open bar)- or Vn (filled bar)-coated surfaces, treated with buffer or 50 µM LPLtat. B, Kalpha vbeta 3 cells were allowed to adhere to surfaces coated with various concentrations of Vn as indicated and treated with buffer (triangle) or 50 µM LPLtat (circle). C, Kalpha vbeta 3 cells were allowed to adhere to Vn-coated surfaces and treated with buffer or 50 µM peptides as specified. The bar labeled K562+LPLtat depicts the binding of untransfected K562 cells treated with LPLtat. D, Kalpha vbeta 3 cells were incubated with 10 µg/ml monoclonal antibodies W6/32 (HLA), L230 (alpha V), 1A2.1 (beta 3), P1F6 (complex-specific for alpha vbeta 5), or mAb16 (alpha 5) for 30 min at RT before addition to Vn-coated surfaces and activation with 50 µM LPLtat. Adhesion assays were performed exactly as described under "Experimental Procedures." Data are representative of at least four independent experiments.

To determine whether LPLtat could activate alpha vbeta 3 in a cell constitutively expressing this integrin, we examined adhesion of the JY B lymphoma line, which is known to have activable alpha vbeta 3 (23, 37). These cells also adhered to Vn when treated with LPLtat (Fig. 2A), in a manner dependent on Vn-coating concentration (Fig. 2B) and beta 3 integrin (Fig. 2C).



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Fig. 2.   LPLtat activates alpha vbeta 3-mediated adhesion in JY cells. A, JY cells were incubated with Vn-coated surfaces and treated with buffer or 50 µM peptides as specified. B, JY cells were allowed to adhere to surfaces coated with the indicated concentrations of Vn and treated with buffer (triangle) or 50 µM LPLtat (circle). C, JY cells were incubated with 10 µg/ml monoclonal antibodies W6/32 (HLA), P1F6 (alpha Vbeta 5), 1A2.1 (beta 3), or mAb16 (alpha 5) for 30 min at RT before addition to Vn-coated surfaces and activation with 50 µM LPLtat. Adhesion assays were performed as described under "Experimental Procedures." Data are representative of three independent experiments.

The LPL Headpiece Induces alpha vbeta 3-mediated Adhesion in Kalpha vbeta 3 Cells-- In addition to the site for phosphorylation, the N-terminal domain, or headpiece, of LPL contains two EF-hand-type Ca2+ binding motifs (18), and Ca2+ markedly affects the conformation of this domain. To determine whether LPLtat induction of adhesion could be recapitulated with the entire LPL N-terminal domain, we expressed an LPL truncation mutant that contained the entire headpiece domain fused to the tat peptide sequence either at the C terminus (Headtat) or the N terminus (tatHead) (Fig. 3A). The purified proteins were recognized by specific monoclonal antibodies directed toward the tat-(49-58) epitope (19) or toward the LPL N terminus (Fig. 3B). As in the case of LPL peptides, Headtat, but not tatHead induced Kalpha vbeta 3 adhesion to Vn. Headtat-induced adhesion was completely inhibited by both anti-alpha v and anti-beta 3 antibodies (Fig. 3C). These data demonstrate that, in addition to short peptides, the intact headpiece domain of LPL can activate alpha vbeta 3-dependent adhesion in Kalpha vbeta 3 cells.



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Fig. 3.   LPL headpiece domain induces alpha vbeta 3-mediated adhesion in Kalpha vbeta 3 cells. A, schematic representation of the recombinant tat-expressing domains. TatHead had an myc epitope at its C terminus, whereas Headtat had the tat peptide in that position. B, 15 µl of purified Headtat and tatHead proteins were Western-blotted with anti-tat mAb (upper panel) or the anti-LPL mAb LPL 4A.1 (lower panel). C, Kalpha vbeta 3 cells were treated with W6/32 (control antibody), L230 (anti-alphaV), or 1A2.1 (anti-beta3) mAb as in Fig. 1 prior to measuring adhesion to Vn in the presence of buffer (open bar), 2 µM tatHead (hatched bar), or 2 µM purified Headtat (filled bar). The bar labeled K562+Headtat depicts adhesion to the Vn-coated surface of parental untransfected K562 cells activated by Headtat. Adhesion assays were performed as described under "Experimental Procedures." Shown is a representative of three independent experiments with similar results.

LPLtat-induced Adhesion Does Not Require the beta 3 Cytoplasmic Tail Tyr-747 or Tyr-759-- PMA- and thrombin-activated Kalpha vbeta 3 adhesion to Vn requires Tyr-747 of the beta 3 cytoplasmic tail, because Y747F mutants fail to adhere in response to these stimuli (8). To determine whether LPLtat-induced adhesion was also dependent on Tyr-747, we examined the effect of LPLtat on adhesion to Vn in K562 cells expressing alpha vbeta 3 with single amino acid mutations Y747F, Y759F, and the double mutant Y747F/Y759F. As previously observed (8), the Y747F but not the Y759F mutation abolished PMA-stimulated adhesion to Vn (Fig. 4). However, Kalpha vbeta 3Y747F, Kalpha vbeta 3Y759F, and Kalpha vbeta 3Y747F/Y759F adhered equally well as cells expressing wild-type alpha vbeta 3 when treated with LPLtat (Fig. 4). Thus, unlike PMA, LPLtat-activated adhesion does not require beta 3 Tyr-747 or its phosphorylation. This demonstrates that the LPLtat effect on alpha vbeta 3 is either downstream or independent of the Tyr-747-dependent signaling cascade (20, 21) involved in PMA-induced adhesion.



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Fig. 4.   LPLtat-induced adhesion does not require beta 3 cytoplasmic tail Tyr-747. K562 cells transfected with alpha v and normal beta 3 (WT), beta 3 encoding a Y to F mutation at Tyr-747 (Y747F), beta 3 encoding a Y to F mutation at Tyr-759 (Y759F), or beta 3 expressing the Y747F/Y759F double mutation (Y747/759F) have been described (8). Cells of each type with equal surface expression of alpha vbeta 3 were treated with buffer control (open bar), 50 ng/ml PMA (hatched bar), or 50 µM LPLtat (filled bar). Adhesion assays were performed as described under "Experimental Procedures." Data shown are representative of at least three independent experiments.

LPLtat and RGD Act Synergistically to Increase Ligand-induced Binding Site (LIBS) Expression-- Leukocyte integrin activation can be achieved by several mechanisms. For many integrins, binding of ligand or exposure to the divalent cation Mn2+ induces a conformational change that results in increased adhesion because of enhanced affinity for ligand (22). In the case of alpha vbeta 3, RGD peptide or Mn2+ binding can induce this conformational change, which can be identified by the binding of monoclonal antibodies (mAb) that recognize LIBS epitope (8, 23, 24). This conformational change is associated with increased affinity for Vn, apparently due to a decreased off-rate of the receptor-ligand interaction (25). As shown in Fig. 5A, the anti-beta 3 mAb 7G2 (13) recognizes an LIBS epitope as its binding to alpha vbeta 3 increased ~10-fold in response to RGD peptide. Maximal 7G2 reactivity was achieved with 25 µM RGD peptide (Fig. 5A), and increasing the RGD peptide concentration to as high as 2 mM did not induce further 7G2 reactivity (data not shown). Control RAD peptide did not enhance 7G2 binding (Fig. 5B). Treatment of Kalpha vbeta 3 with LPLtat alone only minimally increased 7G2 binding (Fig. 5, A and B). However, even at concentrations of RGD peptide that caused maximum 7G2 binding, 7G2 reactivity was further increased when cells were treated with RGD in combination with LPLtat, demonstrating marked synergy between the cytoplasmic peptide and the ligand (Fig. 5A). Treatment of Kalpha vbeta 3 with RGD together with control peptides such as LPL (LPL aa 2-19 without associated tat), or other peptides bearing the tat sequence, TPLtat, tatLPL, and SCRtat, did not cause 7G2 reactivity to increase over the level induced by RGD alone (Fig. 5B). Total alpha vbeta 3 expression on the surface of the transfected K562 cells was not altered by LPLtat treatment as assessed by the (non-LIBS) anti-beta 3 antibody AP3 (data not shown). LPLtat synergized with RGD to induce LIBS expression in alpha vbeta 3 with the Y747F and Y759F mutations (Fig. 5C), consistent with the finding that LPLtat-induced adhesion does not require these beta 3 cytoplasmic tail tyrosines. LPLtat also synergized with Mn2+ to increase LIBS expression in wild type and mutant receptors (Fig. 5D and data not shown). In contrast, PMA treatment did not induce 7G2 binding either with or without LPLtat (Fig. 5D). These data indicate that LPLtat induces alpha vbeta 3-mediated adhesion by cooperating with ligand to induce expression of the high affinity conformation of the integrin, independent of Tyr-747 or Tyr-759.



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Fig. 5.   LIBS recognition by mAb 7G2. A, Kalpha vbeta 3 cells were treated with buffer control (triangle) or 50 µM LPLtat (circle) for 20 min at 37 °C. The expression of an LIBS epitope in the presence of indicated amounts of RGD peptide was assessed by 7G2 staining and FACS analysis as described under "Experimental Procedures." The mean fluorescence of each histogram was taken for numerical comparison. B, Kalpha vbeta 3 were treated with buffer (None), 50 µM RGD or control RAD peptide, and/or 50 µM LPL peptides, singly or in combinations as in the figure. LIBS epitope expression was assessed by 7G2 binding as in A. C, Kalpha vbeta 3Y747F (open bar) and Kalpha vbeta 3Y759F (filled bar) were treated with 50 µM RGD or control RAD peptide, and/or 50 µM LPLtat singly or in combination as in the figure. LIBS epitope expression was assessed as in the previous panels. D, Kalpha vbeta 3 were treated RGD (50 µM), MnCl2 (1 mM), PMA (50 ng/ml), and/or LPLtat (50 µM), alone and in combinations as indicated in the figure, and LIBS epitope expression was assessed as above. The data in each panel are representative of three independent experiments with similar results.

Inhibition of LPLtat-induced Adhesion Requires a Higher Concentration of RGD Peptide than PMA- or Mn2+-induced Adhesion-- As has been shown repeatedly, RGD peptide competes with Vn for the ligand binding site in alpha vbeta 3 and inhibits adhesion to this protein. Because LPLtat cooperated with RGD to induce an LIBS epitope and presumably the high affinity state of the integrin, we asked whether adhesion induced by PMA, Mn2+, and LPLtat had similar sensitivity to RGD peptide inhibition. If LPLtat increased the number of high affinity receptors, it should shift the ID50 for RGD to a higher concentration. As this hypothesis predicted, LPLtat-activated adhesion was much more resistant to RGD peptide inhibition than either PMA or Mn2+ stimulation (Fig. 6A). Mn2+- or PMA-induced adhesion was completely inhibited by 50 µM RGD peptides, with an ID50 at about 20 µM (Fig. 6, B and C). LPLtat-induced adhesion, however, required 100-fold more RGD peptide for equivalent inhibition, with an ID50 of ~2.5 mM RGD (Fig. 6A). This result is consistent with the higher LIBS expression induced by RGD and LPLtat than by RGD and Mn2+ or RGD and PMA (Fig. 5D).



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Fig. 6.   RGD peptide inhibition of alpha vbeta 3-mediated adhesion. A, Kalpha vbeta 3 cells were incubated with buffer (the y intercepts for each curve) or the indicated concentration of RGD peptide for 20 min at RT prior to assessment of cell adhesion to Vn-coated surfaces upon treatment with buffer (open circle), 50 ng/ml PMA (filled circle), 1 mM MnCl2 (filled triangle), or 50 µM LPLtat (filled square). For B and C, cells were treated with indicated amount of RGD (square) or RAD (triangle) as control and then activated with 50 ng/ml PMA (B) or 1 mM MnCl2 (C). Adhesion assays were performed as in Fig. 1. Data are representative of three separate experiments.

LPLtat-induced Adhesion Is Inhibited by Jasplakinolide, but Not Cytochalasin D-- It is generally thought that integrins require attachment to actin filaments for firm adhesion. However, adhesion mediated by high affinity integrin receptors can be unaffected by cytochalasin D (26), implying that new actin microfilament formation is not required for this mechanism of attachment. Therefore, the sensitivity of LPLtat-induced adhesion to cytochalasin D was tested. As expected, PMA-induced adhesion, which requires the actin cytoskeleton in post-receptor events, was completely inhibited by cytochalasin D at 1 µg/ml. In contrast, cytochalasin D had no inhibitory effect on either LPLtat or Mn2+-induced alpha vbeta 3-mediated adhesion even at 10 µg/ml (Fig. 7A). These results demonstrate that LPLtat induces adhesion mediated by high affinity receptors, independent of actin microfilaments.



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Fig. 7.   Effects of cytochalasin D on alpha vbeta 3-mediated adhesion. A, LPLtat-induced adhesion is not inhibited by cytochalasin D. Kalpha vbeta 3 were treated with Me2SO (the y intercepts for each curve) or indicated concentrations of cytochalasin D for 30 min at 37 °C before measurement of adhesion to Vn upon incubation with buffer (open circle), 50 ng/ml PMA (filled circle), 50 µM LPLtat (filled square), or 1 mM MnCl2 (filled triangle). Adhesion was assessed as described under "Experimental Procedures." B, low concentration cytochalasin D stimulates alpha vbeta 3-mediated adhesion. Kalpha vbeta 3 were treated with varying concentrations cytochalasin D as indicated prior to measurement of adhesion to Vn-coated surfaces upon exposure to buffer (open circle) or 50 ng/ml PMA (filled circle). Representative of three independent experiments.

It is recognized that actin microfilaments can also negatively regulate integrin activation by restricting integrin lateral mobility in resting cells (10). As a result, low doses of cytochalasin D actually induces LFA-1- and Mac-1-mediated adhesion by releasing integrins from cytoskeletal constrains leading to integrin activation (10, 27-29). In this study, low concentration of cytochalasin D (0.1 µg/ml) activated alpha vbeta 3-mediated adhesion as well (Fig. 7B). Apparently, as in the case of LFA-1 and Mac-1, release of unactivated integrins from cytoskeletal constraint can lead to alpha vbeta 3-mediated adhesion.

Because actin depolymerization can induce alpha vbeta 3-mediated adhesion, the requirement for actin depolymerization in alpha vbeta 3-mediated adhesion was examined. Jasplakinolide stabilizes pre-existing F-actin, prevents actin depolymerization, and induces a net increase in actin polymerization (30). It was previously shown to inhibit LFA-1-dependent adhesion (12). Similarly, jasplakinolide inhibited alpha vbeta 3-mediated adhesion induced by LPLtat, Mn2+, or PMA in Kalpha vbeta 3 (Fig. 8A) and in JY cells (data not shown). Taken together, the cytochalasin D and jasplakinolide data suggest that, although actin polymerization is not required for LPLtat-initiated alpha vbeta 3-mediated adhesion, actin depolymerization is. F-actin content of cells treated with LPLtat and jasplakinolide was determined (Fig. 8B). Although LPLtat caused about a 50% decrease in total F-actin, jasplakinolide treatment more than doubled cellular F actin. Jasplakinolide also prevented the LPLtat-induced decrease in F-actin.



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Fig. 8.   Effect of jasplakinolide on alpha vbeta 3-mediated adhesion, F-actin, and LIBS expression. A, Kalpha vbeta 3 were treated with 1 µM jasplakinolide (filled bar) or an equivalent amount of Me2SO vehicle as the control (open bar) for 45 min at 37 °C and incubated with buffer, 50 µM LPLtat, 1 mM MnCl2, or 50 ng/ml PMA as indicated. Adhesion assays were performed exactly as described under "Experimental Procedures." B, F-actin content of Kalpha vbeta 3 cells treated with buffer (control), 50 µM LPLtat, 1 µM jasplakinolide, or the combination of LPLtat and jasplakinolide was determined as described under "Experimental Procedures." The ordinate is in arbitrary densitometric units. Shown is the average of two independent experiments. C, Kalpha vbeta 3 cells were treated with Me2SO (open bar) or 1 µM jasplakinolide (filled bar) for 45 min at 37 °C. Cells were then incubated with 50 µM RGD peptide, 50 µM LPLtat, or 1 mM MnCl2 singly or in combinations as indicated in the figure, and 7G2 LIBS expression assessed by FACS analysis as described in Fig. 4. Shown are data representative of three independent experiments.

Because both actin depolymerization and increase in receptor affinity are involved in LPLtat-induced adhesion, we asked whether actin depolymerization was required for the generation of high affinity alpha vbeta 3. Surprisingly, jasplakinolide had little effect on synergistic expression of the 7G2 epitope by RGD and LPLtat (Fig. 8C). In addition, cytochalasin D had no effect on the ability of RGD, LPLtat, or their combination to induce the LIBS epitope recognized by 7G2 (data not shown). Thus, although actin depolymerization is required for alpha vbeta 3-mediated adhesion, it is neither necessary nor sufficient for achieving a high affinity conformation of the receptor.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The term "integrin activation" is sometimes used to mean the modulation of affinity that can be induced in many integrins by Mn2+, activating antibodies, ligand, and sometimes other physiologic or pharmacologic stimuli. If used in this way, integrin activation is thought be related to a conformational change in the extracytoplasmic domain of the integrins and can occur in a wide variety of cell types. However, beta 2 and beta 3 integrins on bone marrow-derived cells, including leukocytes and platelets, are different from integrins on other cell types, because without physiologic or pharmacologic activation, they do not mediate adhesion. Thus, for these integrins, cell activation is an absolute requirement for function. Activation of adhesion likely results from integrin clustering within the membrane as well as changes in affinity of individual integrins (10-12). Moreover, leukocyte integrins can be distinguished from platelet integrin alpha IIbbeta 3 because, although alpha IIbbeta 3 activation for ligand binding is irreversible, activation of leukocyte integrins is a reversible process. Indeed, reversible activation of integrin-mediated adhesion is thought to be important in a variety of critical leukocyte functions, including migration, phagocytosis, and cell-mediated cytotoxicity.

Despite the importance of this reversible activation of leukocyte integrin-mediated adhesion, the mechanisms involved in regulation of leukocyte integrin function remain obscure. Although a number of signaling molecules have been identified that can be involved in the signal transduction pathways of inside-out signaling, the effector mechanisms leading to changes in integrin function have not been elucidated. In general, two not mutually exclusive models have been invoked, one involving receptor rearrangement on the plasma membrane and another involving conformational change in the integrin itself (31, 32). Our previous work implicates LPL phosphorylation in a final common pathway influencing integrin function after cell activation with G-protein coupled, tyrosine kinase-mediated, and PKC-dependent stimuli (7, 33-35). The fact that peptides that mimic the phosphorylation site in the N terminus of LPL rapidly stimulate integrin activation when introduced into PMN or monocyte cytoplasm (7) presents an excellent opportunity to gain insight into the effector mechanisms of inside-out signaling. Therefore, in this work we have investigated the mechanism by which the LPL N terminus activates integrin-mediated adhesion, using the well-characterized model of alpha vbeta 3-mediated adhesion in K562 cells.

A cell-permeant version of the LPL N-terminal domain (headpiece) activated alpha vbeta 3-mediated adhesion as well as the 18-amino acid LPL peptides previously shown to activate alpha Mbeta 2-mediated adhesion. As previously discussed (7), integrin activation by the LPL N-terminal peptides requires their entry into the cytoplasm, because peptides of identical sequence without the tat addition have no effect on integrin function. Placing the tat sequence at the N-terminal end of either the peptide or the headpiece domain abrogates function even though this placement does not decrease entry into the cytoplasm.3 This suggests the possibility that a free LPL N terminus is essential for its integrin regulatory function and that addition of the 12-amino acid tat peptide blocks some essential function of this region of the protein. However, this domain binds neither actin nor vimentin (36), and there are no known cytoplasmic proteins that interact with the LPL headpiece. Although the headpiece does bind Ca2+ through its tandem EF-hand domains, the active peptides do not, and modulation of intracytoplasmic Ca2+ by itself is not known to affect integrin function. Thus, the molecular interactions by which the LPL N terminus activates integrin avidity are unknown. Nonetheless, both the headpiece domain and the N-terminal peptides induce alpha vbeta 3-mediated adhesion, and the peptides clearly synergize with RGD ligand to activate a conformational change in alpha vbeta 3 associated with increased ligand affinity. We hypothesize that this occurs as the result of an interaction between the N-terminal domain of LPL and an as yet unknown target, which could be the integrin itself.

The requirements in the beta 3 cytoplasmic domain for LPLtat induction of adhesion are distinct from PMA or thrombin, because the beta 3 Y747F mutant, which fails to support adhesion stimulated by PMA, supports normal adhesion induced by the LPL peptides. Because the Y747F mutant also supports adhesion in cells with constitutively active integrins (8), it is clear that phosphorylation of this tyrosine is not required for alpha vbeta 3-mediated adhesion. Rather, Tyr-747 phosphorylation is likely required in a signal transduction pathway involving the integrin, perhaps by recruiting an SH2- or PTB-containing protein, that leads to a final common pathway of integrin adhesion that does not itself require Tyr-747. If this is the case, then LPL functions downstream of Tyr-747 phosphorylation.

LPLtat acts synergistically with RGD ligand to induce the expression of an LIBS epitope on beta 3 recognized by mAb 7G2. The RGD peptide can induce 7G2 binding in a dose-dependent manner, as is expected for a LIBS mAb; in contrast, at optimal concentration, LPLtat induces little 7G2 binding. Thus, LPLtat does not act like an activating antibody or certain alpha IIbbeta 3 chimeras, for which LIBS expression becomes independent of ligand. Instead, LPLtat appears to augment the conformational change induced by RGD. In K562, as in other cells, only a minority of integrins expresses the LIBS epitope in the presence of ligand. Virtually nothing is known about what distinguishes receptors undergoing conformational change from those that do not. LPLtat seems to increase the fraction of receptors capable of achieving this conformational change when exposed to ligand. In studying purified alpha vbeta 3, Orlando and Cheresh (25) noted that prior exposure to RGD markedly decreased the off-rate of its binding to Vn and suggested that this was the mechanism by which the ligand-induced conformational change in the integrin induced stable cell adhesion. It is likely that, in the context of an intact cell, LPLtat exaggerates this normal response, leading to marked strengthening of adhesion. It is noteworthy that LPLtat-mediated adhesion is insensitive to cytoskeleton disruption with cytochalasin D, suggesting that the LPL effect on integrin interaction with ligand dominates any potential indirect effect of adhesion strengthening through modulation of actin-integrin interactions.

Although the change in integrin conformation is essential for LPLtat-induced adhesion, it is not sufficient, because jasplakinolide blocks adhesion without significant inhibition of the conformational change. It is now clear that integrin release from cytoskeletal constraint is an early and essential aspect of activation of adhesion (10-12). We hypothesize that, in addition to recruiting new alpha vbeta 3 to respond to a ligand-induced conformational change, LPLtat induces integrin release from cytoskeletal constraint. Consistent with this, treatment of cells with LPLtat diminishes total F-actin content and simultaneous treatment with jasplakinolide inhibits this effect of the peptide. Effects on both cytoskeleton and integrin conformation are required for stable adhesion induced by LPLtat, even though cytoskeletal release is not required for the peptide effects on integrin conformation. Moreover, cytoskeletal release occurs in the absence of ligand (10), so conformational change is not a prerequisite for release. In platelets, alterations in integrin-cytoskeleton interaction are important for inside-out signaling (38), and an increase in intracytoplasmic Ca2+ can activate alpha IIbbeta 3 by releasing it from cytoskeletal constraints (39). Release of integrins from cytoskeletal constraint may require phosphorylation of the myristoylated alanine-rich protein kinase C substrate (40) as well as LPL. Thus, LPL peptide appears to induce two necessary but potentially independent events in control of integrin function. It is intriguing to speculate that the peptide may interact directly with integrin cytoplasmic tails to achieve these effects. Because phosphorylation of LPL may activate its release from actin filaments,2 it is possible that signal transduction leading to LPL phosphorylation frees the N terminus to interact with integrins, leading in turn to their release from cytoskeleton and diffusion to sites of ligand concentration, where a conformational change leading to high affinity interaction occurs upon ligand binding.


    ACKNOWLEDGEMENTS

We thank Dr. Terry Woodford-Thomas and the late Dr. Matthew Thomas for many useful discussions during the course of these experiments, and Dr. Woodford-Thomas for her careful critique of the paper. We also thank Dr. F. Patrick Ross for providing the osteopontin.


    FOOTNOTES

* This work was supported by Grant AI35811 from the National Institutes of Health (to E. J. B.).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.

To whom correspondence should be addressed: Program in Host Pathogen Interactions, Campus Box 0654, University of California at San Francisco, 513 Parnassus Ave., San Francisco, CA 94143-0654. Tel.: 415-514-0167; Fax: 415-514-0169; E-mail: ebrown@medicine.ucsf.edu.

Published, JBC Papers in Press, January 30, 2001, DOI 10.1074/jbc.M007324200

2 J. Wang and E. J. Brown, unpublished data.

3 S. L. Jones and E. J. Brown, unpublished data.


    ABBREVIATIONS

The abbreviations used are: LIBS, ligand-induced binding site; LPL, L-plastin; Headtat, a recombinant protein containing amino acids 1-105 of L-plastin fused at the C terminus to 12 amino acids of the HIV tat sequence; HSA, human serum albumin; Kalpha vbeta 3, K562 cells transfected with alpha vbeta 3 integrin; LPLtat, a synthetic peptide based on amino acids 2-19 of L-plastin followed by 12 amino acids of the HIV tat sequence; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; PMN, polymorphonuclear neutrophils; RAD, Arg-Ala-Asp peptide; RGD, Arg-Gly-Asp peptide; SCRtat, a synthetic peptide in which amino acids 2-19 of L-plastin have been scrambled, followed by 12 amino acids of the HIV tat sequence; tatHead, a recombinant protein containing amino acids 1-105 of L-plastin fused at the N terminus to 12 amino acids of the HIV tat sequence; tatLPL, a synthetic peptide based on amino acids 2-19 of L-plastin fused at the N terminus to 12 amino acids of the HIV tat sequence; TPLtat, a synthetic peptide based on amino acids 2-19 of T-plastin followed by 12 amino acids of the HIV tat sequence; Vn, vitronectin; FACS, fluorescence flow cytometry; PBS, phosphate-buffered saline; mAb, monoclonal antibody; HBSS, Hanks' balanced salt solution; RT, room temperature; HIV, human immunodeficiency virus.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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