21 Kucik, D. F., Dustin, M. L., Miller, J. M. and Brown, E. J., J. Clin. Invest. 1996. 97: 2139. 22 Kassner, P. D. and Hemler, M. E., J. Exp. Med. 1993. 178: 649.
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Jonathan B. Weitzman, Cristina Pujades and Martin E. Hemler Dana-Farber Cancer Institute, Harvard Medical School, Boston, USA
Integrin a chain cytoplasmic tails regulate “antibody-redirected” cell adhesion, independently of ligand binding Here we describe a novel “antibody-redirected cell adhesion” (ARCA) assay. This assay measures heterotypic cell-cell adhesion, resulting from antibody bridging between Fcy receptors type I1 (CD32) on leukocytes, and clustered intergrins on adherent cell monolayers. This ARCA activity, facilitated by integrins a3p,or a‘p,, required an intact cytoskeleton, but did not involve typical integrin ligand binding sites or divalent cations. Furthermore, deletion of the a4 cytoplasmic tail almost completely abrogated integrin ARCA activity, suggesting an alteration of integrin recruitment into adhesive sites. If two or more tail residues were present after the conserved GFFKR motif, then ARCA activity was largely restored. Although a4tail deletion caused loss of ARCA activity, it had no effect on the binding of VCAM-1 to intact a‘-transfected K562 cells. In conclusion, the integrin a chain tail can positively regulate integrin-dependent cell adhesion by a receptor recruitment/clustering mechanism independent of conventional integrin ligand-binding considerations.
1 Introduction The ap heterodimers of the integrin family mediate both cell-matrix and cell-cell adhesion [l-31. A fundamental property of integrins is that their adhesive activities can be regulated in a cell type-specific manner and can be modulated upon cellular stimulation or differentiation [l,4-10]. Mechanisms for regulation of adhesive function may involve changes in integrin ligand binding affinity [ 11-14], or may occur independent of affinity changes [ 14-16]. Examples of affinity-independent mechanisms involve receptor clustering [ 17, 181, barrier molecules [ 191, integrin-cytoskeletal interactions [20], and altered integrin diffusion rates [21].
rin fi chain cytoplasmic tail manipulation may alter postligand binding events involving cytoskeletal organization, independently of changes in integrin affinity for ligand [20]. However, it is not yet clear whether integrin cytoplasmic domains exclusively contribute to adhesive activity by modulating ligand binding affinity, or post-ligand binding events, or whether cytoplasmic tails might modulate adhesive function through other mechanisms. In this regard, it was recently proposed that modulation of integrin recruitment into an adhesive site might also bc a key factor in regulating adhesion [21].
To investigate whether cytoplasmic domains may modulate integrin recruitment, we designed a novel FcyR11mediated antibody-redirected cell adhesion (ARCA) From mutagenesis experiments, it is clear that integrin a assay. This assay measures integrin participation in adhe[22-291 and p [20, 29-33] cytoplasmic domains can both sion that is independent of typical regulation of ligand make strong positive contributions towards cell adhesion. binding affinity or post-ligand binding events. The ARCA In several instances, cytoplasmic tail manipulation caused assay measures integrin recruitment into clusters because apparent changes in integrin ligand binding affinity [28, the FcyRII (CD32) on myeloid cells recognizes only aggre29, 34-37]. Thus, a paradigm has emerged whereby gated immunoglobulins [38]. Using this assay system, we “inside-out’’ signals act through integrin cytoplasmic found that a”, and a4P,integrins, but not the a’& integdomains to alter the extracellular integrin conformation, rin, displayed ARCA activity. Also, we showed that a4tail and thus modify affinity for ligand [4]. Alternatively, integ- deletion of more than 23 residues almost completely eliminated FcyRII-mediated cell-cell adhesion dependent on a4p1. Finally, to gain further insights into a4tail deletion [I 162841 effects, we also studied binding of purified VCAM-1 K562 cells expressing deleted and wild-type a4. The results Received September 5, 1996; accepted October 18, 1996. strongly suggest that integrin a subunit tails can regulate Present addresses: J. B. Weitzman, Unitt des Virus Oncogbnes, adhesion by a mechanism independent of direct effects on Institut Pasteur, 25 rue du Dr Roux, F-75724 Paris Cedex 15, ligand binding, and independent of typical post-receptor France; C. Pujades, Ecole Normale Suptrieure, Laboratorire de events such as cell spreading and focal adhesion formaBiologie Moltculaire, 46, rue d’Ulrn, F-75230 Paris Cedex 05, tion. France
Correspondence: Martin E. Hemler, Dana-Fdrber Cancer Institute, Room M-613, 44 Binney Street, Boston, MA 02115, USA Fax: + 1-617-632-2662;e-mail: Martin-Hemler@dfci. harvard.edu Abbreviations: ARCA: Antibody-redirected cell VCAM-1: Vascular cell adhesion molecule
adhesion
Key words: lntegrin / CD32 / Cell-cell adhesion / Fc receptor I VCAM-1 0014-2980/97/0101-78$10.00 + .25/O
2 Materials and methods 2.1 Antibodies and cell lines mAb used in this study include anti-integrin a* 5E8 [39], P1H5 [40], 12F1 [41]; anti-a3A3-IVA5, A3-IC10, A3-IIF5, and A3-X8 [42], P1F2 [43], M-KID-2 [44], 5143 [45]; anti0 VCH Verlagsgesellschaft mbH, D-6Y451 Weinheim, 1997
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a' B5G10 [46], HP1/7 and HP2/4 [47]; and anti-hamster PI 7E2 [48]. Also we used anti-CD16 mAb 3G8, anti-CD32 mAb IV.3 [49], and anti-CD64 mAb 22 [50]. Another antiCD32 mAb IgM, called 4.6.19, was newly generated, following the injection of mice with intact K562 cells (J. Weitzman, J. Bodorova, C. Pujades, and M. Hemler, unpublished). Cytochalasin D was purchased from Sigma c o.
Human A431 epidermoid carcinoma and K562 erythroleukemia cells and all other human cell lines were grown in RPMI 1640 medium supplemented with 10% fetal calf serum, 1 mM Hepes buffer and antibiotics. Chinese hamster ovary (CHO) cells transfected with human integrin a subunits have been previously described [24, 251 and were grown in MEM a medium (Gibco/BRL) with 10% dialyzed fetal calf serum containing l mg/ml G418 sulfate.
2.2 Flow cytometry and ARCA assays Flow cytometric analysis was carried out using a FACScan machine as previously described [22,42]. ARCA was modified from previous cell-substrate assays [51, 521. Briefly, cell monolayers were grown for 2-3 days in 96-well microtiter plates. Confluent cell monolayers were pre-incubated with anti-integrin mAb (ascites fluid at 1 : 100 dilution or at the antibody concentrations indicated) for 30 min at 37 "C prior to the cell-cell adhesion assay. Monolayers were washed twice to remove unbound excess antibody. Then, K562 suspension cells were labelled with the fluorescent dye 2',6'-bis(2-carboxyethyl)-5(6)-carboxy-fluorescein acetoxymethyl ester (BCECF-AM, Molecular Probes, OR) and added to cell monolayers. After 30 min at 37 "C, unbound K562 cells were removed by three washes, and cells remaining attached were detected using a CytoFluorTM2300 Fluorescent Measurement System (Millipore Co.). The number of cells bound to BSA-coated control wells (typically < 5 %) was subtracted from each value. Adhesion assays were carried out in serum-free RPMI 1640 medium unless indicated otherwise. In blocking experiments, K562 cells were incubated with anti-FcR antibodies for 20-30 min prior to the adhesion assay. Results are presented as mean number of bound cells per mm2 SD from triplicate determinations.
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3 Results 3.1 An assay to examine cell-cell adhesion mediated by an FcR To evaluate cell adhesion independent of ligand-affinity considerations, we designed the ARCA assay system (Fig. 1) in which anti-integrin IgG antibodies caused "redirected" heterotypic cell-cell adhesion by bridging between integrin and FcR. Integrin-bearing monolayers were preincubated with anti-integrin mAb at 37 "C to allow antibody-mediated receptor clustering. FcR-mediated adhesion was then measured after incubation with a suspension of K562 cells bearing FcyRII. In this system only the monolayer cell expresses the integrin under investigation and only the K562 cells express the FcyRII, thereby preventing bivalent antibody bridging. Incubation of monolayers with anti-integrin mAb (anti-a' or anti-a' mAb) caused a marked increase (5-6 fold) in cell-cell adhesion (Fig. 2). Anti-integrin antibodies induced cell adhesion to a monolayer of A431 cells, which express high levels of a3P,(Fig. 2, A-D) or to a monolayers of CHO cells transfected with either the a3or a' subunits (Fig. 2E and F). This antibody-stimulated cell-cell adhesion was dose dependent, with a maximum at 0.1-1 pg/ml of mAb (not shown). To confirm that cell adhesion resulted from the interaction of IgG Fc domains with the FcyRII (CD32) on the K562 cells, antibody-mediated cell-cell adhesion was blocked by pre-incubating K562 cells with two different anti-FcyRII antibodies (CD32, Fig. 2, C-F), but not by antibodies against two other FcyR (CD64 and CD16, Fig. 2 A and B). These other FcR are present at substantially lower levels on K562 cells, thus helping to explain why they do not contribute. In addition, antibody-induced adhesion was observed only for suspension cell lines expressing FcyRII ( i e . K562, U-937, HL-60), but not for FcyRII-negative cell lines ( i . e . JY and PEER) (Fig. 3). Together these results emphasize that this adhesion triggered by antiintegrin mAb is specifically dependent on FcyRII.
*
2.3 VCAM-1 binding assay Purified VCAM-1 mouse Cx fusion protein (VCAMl-x), and a rat mAb to mouse Cx were obtained from Sandoz Co. (East Hanover, NJ). Briefly, VCAM1-x was produced as a soluble protein from sf9 cells, that contains all seven human VCAM-1 domains, except that the transmembrane and cytoplasmic portions of domain 7 have been replaced by a 100 amino acid (aa) mouse Cx segment. For binding assays, 1.5 x 10' cells per well were washed in PBS containing 1 mM EDTA, resuspended in TBS containing 2 YO BSA, 1 mM MnClz and then incubated with increasing amounts of VCAM-lx. After 30 min at 4"C, cells were spun down, washed twice and incubated with FITCconjugated rat anti-mouse x-chain for 30 min at 4°C. Cells were again washed twice and the binding was visualized using a FACScan machine. Under these conditions, VCAMl domain 1 but not domain 4 is active [53].
Figure 1. Schematic representation of FcR-mediated ARCA assay. Cell-cell adhesion is mediated by the interaction of FcyRlI on K562 suspension cells with the Fc portions of clustered IgG bound to integrin on monolayer cells. First, integrin-bearing cell monolayers arc incubated with anti-integrin mAb (IgG, or IgG2) at 37"C, thus causing integrins to cluster. After washing away excess mAb, fluorescently labeled K562 cells arc added and FcyRII-mediated adhesion is quantitated (see Sect. 2.2).
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Figure 2. Antibody-induced cell-cell adhesion is blocked by anti-FcRyII antibodies. Adhesion of KS62 cells to adherent monolayers of A431 cells (panels A-D), or CHO cells transfected with human a' (C-A3, panel E) or a' (C-A4, panel F) was measured. Adhesion assays were performed in the absence of antibodies (black bars), following incubation of the monolayers with anti-integrin antibodies (hatched bars), or in the presence of both anti-integrin antibodies and antiFcR antibodies (gray bars). Monolayers were preincubated with anti-integrin a' mAb A3-IVAS (panels A-E) or anti-a' mAb HP117 (panel F), and K562 cells were preincubated with anti-FcR antibodies to CD64. CD32, or CD16.
3.2 IntegrinlFcyRII-mediatedadhesion differs from typical integridigand interactions Notably, FcyRII-dependent cell-cell adhesion was rnediated equally well by anti-a' antibodies that do (e.g. A3IVA5, A3-IIF5) or do not (e.g. A3-X8, [42]) block a3dependent cell adhesion to ECM (Fig. 4A). Likewise, anti-a4 antibodies that define three spatially distinct epitopes [54] and have either strong (HP2/4), intermediate (HP1/7), or minimal (B-5G10) blocking activity for typical a4integrin adhesive functions [55] also made comparable positive contributions towards FcyRII-mediated adhesion (Fig. 4B). Cell-cell adhesion could not be induced by a number of anti-a2 antibodies (Fig. 4C) or by antibodies against endogenous hamster p, (mAb 7E2) which is mostly associated with hamster a'. These results imply that a' and a" are qualitatively distinct from other integrins. Also, antibody-mediated cell adhesion was unaffected by the addition of 10 mM EDTA (Fig. SA), providing further evidence that his cell-cell adhesion is quite distinct from typical divalent cation-dependent integrin-mediated adhesion.
3.3 The a cytoplasmic tail can regulate ligandindependent cell adhesion The ARCA assay was substantially blocked by 10 nM cytochalasin D (Fig. 5B), thus suggesting a strong dependence on an intact cytoskeleton. To explore further how the cytoskeleton might influence cell-cell adhesion, we next assessed whether there was a specific requirement for the a' cytoplasmic tail, to support FcyRII-mediated adhesion through the a4P1integrin.
In marked contrast to a' wild type, a' truncated immediately after the GFFKR sequence (X4CO) failed to support FcR-mediated cell-cell adhesion when expressed in a CHO monolayer (Fig. 6). As shown previously [24], and again in Table 1, wild-type a4and X4CO were expressed at comparable levels in CHO cells. Furthermore, w e have shown
Figure 3. Cell adhesion by CD32-bearing suspension cells only. Confluent monolayers of CHO cells transfected with human a3 (panel A) or human carcinoma A431 cells (panel B) were incubated with anti-a' mAb A3-IVA5 and then tested for adhesion to suspension cell monocytic lines (KS62, U937 and HL60) or lymphocytic lines (PEER and JY). As in Fig. 2, adhesion was measured with no added antibody (black bars), in the presence of antia' mAb A3-IVAS (hatched bars), or in the presence of A3-IVA5, plus anti-FcyRII mAb 4.6.19 (gray bars). The CD32 antigen is expressed on K562, U937 and HL60 cells (mean fluorescence intensity values 17.12, 9.02 and 9.13, respectively), but not on PEER or JY cells.
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81
N
E E
Figure 5 . Effects of EDTA and cytochalasin D on FcR-mediated adhesion. FcyRII-dependent K562 cell adhesion to CHO-A3 cells, mediated by anti-a' mAb A3-IC10, was measured in the absence (hatched bars) or presence (gray bars) of 10 mM EDTA (A), or 10 nM cytochalasin D (B).
400 CHO-X4C4
300
K562 cells bound per mm2 Figure 4. Cell adhesion is induced by anti-integrin mAb recognizing different epitopes. Antibodies against human a3, a' or a*, respectively, were added to transfected CHO cells expressing the human integrin a3(panel A), a4(panel B), or a2(panel C) subunits, at similar expression levels, and then FcR-mediated adhesion of K562 cells was measured. For each cell line, controls were performed with an anti-hamster PI mAb (7E2) or no antibody treatment (none).
that truncation of the a' tail does not perturb any of the three a4epitopes defined by mAb [22,24]. Whereas '10 tail deletion essentially eliminated FcyRII-mediated cell-cell adhesion, exchange of the a4cytoplasmic domain with that of a' had no effect (Fig. 6 ) , even though the a* and a4tails are known to differ markedly in their contributions towards integrin localization into focal adhesion complexes [56]. To determine which part of the a4tail might be critical for supporting FcyRII-dependent adhesion, several truncation mutants were analyzed. As indicated (Table l), the presence of just two or more residues beyond position 974 was sufficient to recover mAb-stimulated, FcyRII-dependent cell-cell adhesion. These results parallel results from previous studies indicating that the presence of only a few residues following the GFFKR motif was sufficient to restore integrin adhesive activity and normal cellular localization [24, 251.
200 100
a S
0
. . ....-,
I-
.....
a,
Q
U
c
3
0
a
i
2001
0
10''
10'
10'
lo2
lo3
10'
H P l D ng/ml 3.4 VCAM-1 binding
Previously we assumed that a' tail deletion caused a marked reduction in cell adhesion to fibronectin fragments and VCAM-1 [22, 241, largely due to a loss of ligand binding capability. However, results shown here in Fig. 6 and
Figure 6. Effects of a' tail deletion and exchange on cell-cell adhesion. The anti-integrin a' mAb HP117 was used to trigger Fcy RII-dependent K562 cell adhesion to CHO monolayers expressing wild-type a4 (X4C4), chimeric a' (X4C2, a' tail replaced by a2 tail) or deleted a' (X4C0, a4tail deleted just after GFFKR).
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J. B. Weitzman et a1
Table 1. Determination of cytoplasmic domain residues critical
for mAb-mediated cell-cell adhesiona’ Cell line
CHO-neo CHO-974 CHO-976 CHO-977 CHO-979 CHO-980 CHO-A4
Residues after GFFKR
MFI
-
3.01 35 .54 40.49 28.79 48.53
0
2 3 5 6
25
32.17
38.73
K562 bound Fold induction pcr mm’ over CHO-neo 5 8 + 11 44+ 4 176 k 16 295 f 24 177 f 52 156 k 19 231 f 17
-
0.76 3.03 5.09 3.05 2.69
3.98
a) CHO cell monolayers expressing full length a‘ (X4C4) or a series of truncatcd a4 cytoplasmic tails (974 to 980 residues) were incubated with anti-integrin mAb (anti-a‘ mAb HP117 at 100 ng/ml) and then assayed for FcyRII-mediated adhesion of
K562 suspension cells, mcasurcd as K562 cells bound per mm’. Mutant “974” is another name for the X4CO mutant, truncated immediately following t h e GFFKR scqucncc [24]. A mock trdnsfected “neo” clone was used as a control cell line. The mean fluorescence intensity (MFI) of a“ expression on CHO cells was measured by flow cytometry using mAb HP1/7. Table 1 indicate that a‘ deletion markedly impairs cell adhesion even when it does not involve typical ligand binding. Returning to the issue of ligand binding, we investigated binding of soluble VCAMl-x ligand to K562 cells expressing wild-typc a‘ (X4C4), chimeric a‘ (X4C2), o r truncated a4(X4CO). A s seen in Fig. 7, there was essentially no difference in ligand binding between wild-type a‘ and either of the two a‘ mutants, indicating that V C A M l x binding activity is unaffected by exchange o r deletion of the a4 cytoplasmic tail. In a negative control experiment, VCAM1-x did not bind at all to untransfected K562 cells (not shown). As a positive control, the same assay condi-
Figure 7. Effccts of exchange or deletion of the a‘ tail o n VCAM1 binding. Transfected K562 cells were incubated with increasing
doses of recombinant VCAMl-x, and then bound VCAMl was detected as described in Sect. 2.3. Cells expressing wild-type a‘ (X4C4. closed circles), were compared with those expressing chimeric (x‘ (X4C2, closed squares), or deleted a‘ (X4C0, open circlcs).
tions were utilized previously to demonstrate a reduction in VCAM1-x binding, due to a4cysteine mutations [57].
4 Discussion In this report we describe a novel ARCA assay useful for examining integrin contributions to cell adhesion independent of typical ligand binding criteria. Using this assay, we then determined that the a4 cytoplasmic domain can make a strong positive contribution to integrin function by modulating receptor recruitment, independently of possible effects on conventional ligand binding activity. By a process known as “redirected lysis”, cytolytic T cells [58] o r natural killer cells [59] can utilize specific monoclonal antibodies to complex with, and then lyse target cells expressing Fc receptors. By analogy, we have developed a novel ARCA assay, that “redirects” integrins on adherent cell monolayers t o mediate adhesion to FcyRII-positive cells in suspension. This assay does not require typical integrin-ligand interactions, as confirmed by the absence of EDTA inhibition, and the comparable effectiveness of both ligand-blocking and non-blocking anti-integrin antibodies. Thus the ARCA assay is completely independent of inside out signaling mechanisms that might regulate integrin ligand-binding affinity. Furthermore, since the A R C A assay does not involve cell spreading and/or focal adhesion formation, it is also independent of typical “post-rcceptor” mechanisms thought to regulate integrin-dependent adhesion. The ARCA assay is completely inhibited by antibodies against FcyRII (CD32). CD32 is a 40-kDa integral membrane sialoglycoprotein expressed on platelets and leukocytes [60-621, and is one of three receptors for IgG [38]. CD32 binds with barely detectable affinity to monomeric IgG, but binds vcry well to immune complexes o r aggregated IgG [38, 621. Thus, the ability of integrins to form clusters is a critical aspect of our CD32-dependent ARCA assay. Inhibition of ARCA activity by cytochalasin D indicates that an intact actin cytoskeleton may be important for recruitment of integrins into clusters. Elsewhere it was shown that antibodies to allhP7could induce platelet aggregation by a mechanism involving FcyRII (CD32) [63, 641. However, interpretation of those results is more complicated since both CD32 and the integrin are present on thc same platelet. Notably, a’ integrins on C H O cells were consistently less functional than a’ o r a‘ integrins in the ARCA assay, despite being expressed at comparable levels. It is worth noting that a4p, is involved physiologically in cell-cell adhesion [51], and that a3pIis recruited to cell-cell borders [43, 651, whereas other integrins such as a’p, are involved exclusively in cell extracellular matrix adhesion. The cytoplasmic domain of a2appears not to be responsible for the rclativc lack of a’ reactivity, since an X4C2 chimera was fully functional. Also, because C H O cells d o not produce collagen or laminin, it is not likely that a’b, integrin would be engaged in matrix adhesion and thus be unavailable. Another possibility is that the epitopes recognized by antia2 mAb are in locations less available for cell-cell interactions. In this regard, we previously demonstrated that not all integrin epitopes are available for mediating cell-cell
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adhesion [66]. Future studies involving antibodies to additional integrin and non-integrin molecules will be required to determine more precisely the reasons for variable ARCA activity.
83
sion, independent of changes in soluble ligand binding activity. Just as re-directed lysis assays have delineated sets of molecules capable o r participating in the lytic mechanism [58,59], the ARCA assay should be useful for characterizing molecules as being more or less able to support cell adhesion. Also the ARCA assay, together with a VCAM-1 ligand binding assay, has provided evidence indicating that deletion of the integrin a4cytoplasmic domain causes altered integrin cell adhesion, that is likely due to altered receptor clusteringhecruitment, rather than changes in ligand binding activity. These results are of fundamental importance for understanding inside-out signaling mechanisms that regulate integrin adhesive activity.
Most importantly, our results indicate that the cytoplasmic domain of the a4subunit makes a major positive contribution to adhesion in an assay that does not involve typical integrin ligand binding. Furthermore, a4tail deletion had no effect on the binding of VCAM-1 to K562-a4 cells. Together these results are consistent with a4tail deletion causing decreased receptor clustering, and thus decreased avidity for ligand, independent of possible effects o n ligand binding activity. Thus, we can now better understand previous results in which deletion of the tails of a4 This work was supported by National Institutes of Health grant [22, 241, a’ [24, 251, a6[26], and a” [27] caused reduced GM46526 (to MEH) and by post-doctoral fellowship support from the American Cancer Society, Massachusetts Division Inc (to cell adhesion to immobilized ligands [22, 241. Prior studies showed that a4tail deletion or exchange had no effect on a4-mediated KS62 or CHO cell tethering under shear flow [56, 671. Since tethering is largely a function of ligand binding [68], these results are consistent with ligand binding results shown here. In contrast to effects o n tethering, a4tail deletion had a strong negative effect on adhesion strengthening, defined as resistance to cell detachment under shear [56, 671. Our current results, showing that a4 tail deletion may diminish recruitment of integrins into critical cell-cell adhesive sites, appear to be highly compatible with the loss of adhesion strengthening that was previously observed. Previous studies also showed that integrin a chain deletion largely eliminated responsiveness of the integrin to the adhesion-promoting activity of phorbol esters [22, 231. In this regard, it was recently demonstrated that phorbol esters may stimulate integrin-dependent adhesion by increasing integrin diffusion rates, and thus allowing increased recruitment into adhesive sites [21]. Using this model, we propose that negative constraints on diffusion cannot be easily released if a chain tails are absent. In other words, the positive role of a tails during cell adhesion may lie in their prevention of p tails from engaging in constitutive negative cytoskeletal interactions. The a chain “prevention of negative regulation” model is further supported by the results from the series of a4tail deletions. Replacement of just two or more residues after the GFFKR motif (1) restores antibody-induced adhesion (Table l), (2) largely restores cell adhesion to immobilized ligands [24], and (3) prevents unregulated integrin localization into ligand-independent focal adhesion complexes [2S]. These first two observations emphasize that regulation of ARCA activity is closely related to the regulation of cell adhcsion to immobilized ligands. The simplest model linking all three observations is that a cytoplasmic tail length is critical for masking cytoskeletal interactions of the p tail that have a negative effect on cell adhesion. The localization of a tail-deleted integrins into ligand independent focal adhesions [25, 69,701 may be a downstream consequence of unrestricted negative cytoskeletal interactions, and not surprisingly, ligand-independent focal adhesions appear to have little functional utility (561.
In summary we describe a novel ARCA assay, and utilize it to assess the contribution of integrins towards cell adhe-
JBW). We thank Dr. Clark Anderson (Ohio State University) for monoclonal antibodies to CD16, CD32 and CD64.
5 References 1 2 3 4 5 6 7 8 9
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
Hynes, R. O., Cell 1992. 69: 11. Hemler, M. E., Annu. Rev. Immunol. 1990. 8: 365. Springer, T. A., Nature 1990. 346: 425. Ginsberg, M. H., Du, X. and Plow, E. F., Curr. Opin. Cell Biol. 1992. 4: 766. Adarns, J. C. and Watt, F. M., Cell 1990. 63: 425. Van Kooyk, Y., Van DeWiel-Van Kemenade, P., Weder, P., Kuijpers, T. W. and Figdor, C. G., Nature 1989. 342: 811. Dustin, M. L. and Springer, T. A . , Nature 1989. 341: 619. Shimizu,Y., Van Seventer, G. A ., Horgan, K. J . and Shaw, S . , Nature 1990. 345: 250. Chan, B. M. C. and Hemler, M. E., J . Cell Biol. 1993. 120: 537. Masumoto, A. and Hemler, M. E., J . Biol. Chem. 1993. 268: 228. Bennett, J. S . andvilaire, G . , J. C& Invest. 1979. 64: 1393. Marguerie, G . A , , Plow, E. F. and Edgington, T. S . , J . Biol. Chem. 1979.254: 5357. Lollo, B. A . , Chan, K. W. H . , Hanson, E. M., Moy, V. T. and Brian, A. A., J . Biol. Chem. 1993.268: 21693. Faull, R. J., Kovach, N . L., Harlan, J . M. and Ginsberg. M. H., J . Exp. Med. 1994. 179: 1307. Danilov. Y. N. and Juliano, R. L., J . Cell Biol. 1989. 108: 1925. Jakubowski, A . , Rosa, M. D., Bixler, S . , Lobb, R. and Burkly, L. C., CellAdh. Comm. 1995. 3: 131. Miyamoto, S., Akiyama, S. K . and Yamada, K. M., Science 1995. 267: 883. Detrners, P. A., Wright, S. D., Olsen, E . , Kimball, B . and Cohn, Z . A . , J . Cell Biol. 1987. 105: 1137. Wesseling, J., van der Valk, S. W., Vos, H. L., Sonnenberg, A . and Hilkens, J . , J . Cell Biol. 1995. 129: 255. Peter, K. and O’Toole, T. E., J . Exp. Med. 1995. 181: 315. Kucik, D. F., Dustin, M. L., Miller, J . M. and Brown, E. J., J . Clin. Invest. 1996. 97: 2139. Kassner, P. D. and Hemler, M. E., J . Exp. Med. 1993. 178: 649. Kawaguchi, S. and Hemler, M. E., J . Biol. Chem. 1993. 268: 16279. Kassner, P. D., Kawaguchi, S. and Hemler, M. E., J . Biol. Chem. 1994. 269: 19859. Kawaguchi, S., Bergelson, J. M., Finberg, R. W. and Hemler, M. E., Mol. Biol. Cell 1994. 5: 977. Shaw, L. M. and Mercurio, A. M., J . Cell Biol. 1993. 123: 1017.
84
J. B. Weitzman et al
27 Filardo, E. J. and Cheresh, D. A., J. Biol. Chem. 1994. 269: 4641. 28 O’Toole, T. E., Mandelman, D., Forsyth, J., Shattil, S. J., Plow, E. F. and Ginsberg, M. H., Science 1991.254: 845. 29 O’Toole, T. E., Katagiri, Y., Faull, R. J., Peter, K.,Tamura, R . N., Quaranta, V., Loftus, J. C., Shattil, S. J. and Ginsberg, M. H., J. Cell Biol. 1994. 124: 1047. 30 Hibbs, M. L., Jakes, S., Stacker, S. A., Wallace, R. W. and Springer, T. A., J. Exp. Med. 1991. 174: 1227. 31 Hayashi, Y., Haimovich, B., Reszka, A , , Boettiger, D. and Horwitz, A , , J. Cell Biol. 1990. 110: 175. 32 Chen, Y. P., Djaffar, I., Pidard, D., Steiner, B., Cieutat, A . M., Caen, J. P. and Rosa, J. P., Proc. Natl. Acad. Sci. USA 1992. 89: 10169. 33 Crowe, D. T., Chiu, H., Fong, S. and Weissman, 1. L., J. B i d . Chem. 1994.269: 14411. 34 Chen, Y. P., O’Toole, T. E., Ylanne, J., Rosa, J. P. and Ginsberg, M. H., Blood 1994. 84: 1857. 35 Chen, Y.-P., O’Toole, T. E., Shipley, T., Forsyth, J., LaFlamme, S. E., Yamada, K. M., Shattil, S. J. and Ginsberg, M. H., J. Biol. Chem. 1994. 269: 18307. 36 Hughes, P. E., O’Toole, T. E., Ylanne, J., Shattil, S. J. and Ginsberg, M. H., J. Biol. Chem. 1995. 270: 12411. 37 O’Toole, T. E., Ylanne, J. and Culley, B. M., J . Biol. Chem. 1995. 270: 8553. 38 Ravetch, J. V. and Kinet, J.-P., Annu. Rev. Immunol. 1991. 9: 457. 39 Chen, F. A., Repasky, E. A. and Bankert, R. B., J. Exp. Med. 1991. 173: 1111. 40 Wayner, E. A. and Carter, W. G., J. Cell Biol. 1987.105: 1873. 41 Pischel, K. D., Hemler, M. E., Huang, C., Bluestein, H. G . and Woods, V. L., J. lmmunol. 1987. 138: 226. 42 Weitzman, J. B., Pasqualini, R., Takada, Y. and Hemler, M. E., J. Biol. Chem. 1993.268: 8651. 43 Carter, W. G., Wayner, E. A., Bouchard,T. S. and Kaur, P., J. Cell Biol. 1990. 110: 1387. 44 Bartolazzi, A., Fraioli, R., Tarone, G . and Natali, P. G., Hybridoma 1991. 10: 707. 45 Fradet, Y., Cordon-Cardo, C., Thomson, T., Daly, M. E., Whitmore, W. F. J., Lloyd, K. O., Melamed, M. R. and Old, L. J., Proc. Natl. Acad. Sci. USA 1984. 81: 224. 46 Hemler, M. E., Huang, C., Takada, Y., Schwarz, L., Strominger, J. L. and Clabby, M. L., J. Biol. Chem. 1987.262: 11478. 47 SBnchez-Madrid, F., De Landazuri, M. O., Morago, G . , Cebrian, M., Acevedo, A. and Bernabeu, C., Eur. J. Immunol. 1986. 16: 1343. 48 Brown, P. J. and Juliano, R. L., Exp. CellRes. 1988. 177: 303. 49 Looney, R. J., Ryan, D. H., Takahashi, K., Fleit, H. B., Cohen, H . J., Abraham, G. N. and Anderson, C. L., J. Exp. Med. 1986. 163: 826.
Eur. J. Immunol. 1997.27: 78-84 50 Guyre, P. M., Graziano, R. F., Vance, B. A., Morganelli, P M. and Fanger, M. W., J . Immunol. 1989. I43: 1650. 51 Elices, M. J., Osborn, L.,Takada, Y., Crouse, C., Luhowskyj, S., Hemler, M. E. and Lobb, R. R., Cell 1990. 60: 577. 52 Chan, B. M. C., Elices, M. J., Murphy, E. and Hemler, M. E., J . Biol. Chem. 1992. 267: 8366. 53 Needham, L. A., Van Dijk, S., Pigott, R., Edwards, R. M., Shephard, M., Hemingway, I., Jack, L. and Clements. J. M., Cell Adh. Comm. 1994. 2: 87. 54 Schiffer, S. G., Hemler, M. E., Lobb, R. R., Tizard, R. and Osborn, L., J. Biol. Chem. 1995. 270: 14270. 55 Pulido, R., Elices, M. J., Campanero, M. R., Osborn, L., Schiffer, S., Garcia-Pardo, A , , Lobb, R., Hemler, M. E. and Sbnchez-Madrid, F., J. Biol. Chem. 1991.266: 10241. 56 Kassner, P. D., Alon, R., Springer, T. A. and Hemler, M. E., Mol. Biol. Cell 1995. 6: 661. 57 Pujades, C., Teixidb, J., Bazzoni, G. and Hemler, M. E., Biochem. J. 1996. 313: 899. 58 Kovacsovics, T. J., Bachelot, C., Toker, A , , Vlahos, C. J., Duckworth, B., Cantley, L. C. and Hartwig, J. H., J. Biol. Chem. 1995. 270: 11358. 59 Abrams, C., Deng, J., Steiner, B., O’Toole, T. and Shattil, S. J., J . Biol. Chem. 1994. 269: 18781. 60 Karas, S. P., Rosse, W. F. and Kurlander, R. J., Blood 1982. 60: 1277. 61 Cohen, L., Sharp, S. and Kulczycki, A. Jr., J. Immunol. 1983. 131: 378. 62 Rosenfeld, S. I., Looney, R. J., Leddy, J. P., Phipps, D. C., Abraham, G. N. and Anderson, C. L., J. Clin. Invest. 1985. 76: 2317. 63 Anderson, G . P., van de Winkle, J. G. J. and Anderson, C. L., Br. J . Haernatol. 1991. 79: 75. 64 Rubinstein, E., Kouns. W. C., Jennings, L. K., Boucheix, C. and Carroll, R. C., Br. J. Haematol. 1991. 77: 80. 65 Kaufmann, R., Frosch, D., Westphal, C., Weber, L. and Klein, C. E., J. Cell Biol. 1989. 109: 1807. 66 Weitzman, J. B. and Hemler, M. E., in Schlossman, S. F., Boumscll, L., Gilks, W., Harlan, J. M., Kishimoto, T., Morimoto, C., Ritz, J., Shaw, S., Silverstein, R. L., Springer, T. A., Tedder, T. F. and Todd, R. F. (Eds.), Leukocyte Typing V White cell differentiation antigens, Oxford University Press, Oxford 1995, p. 1634. 67 Alon, R., Kassner, P. D., Carr, M. W., Finger, E. B., Hemler, M. E. and Springer, T. A., J. Cell Biol. 1995. 128: 1243. 68 Alon, R., Hammer, D. A. and Springer, T. A., Nature 1995. 374: 539. 69 Ylanne, J . , Chen, Y., O’Toole, T. E., Loftus, J. C., Takada, Y. and Ginsberg, M. H., J . Cell Biol. 1993. 122: 223. 70 Briesewitz, R., Kern, A. and Marcantoni, E. E., Mol. Biol. Cell 1993. 4: 593.
