Cell, Vol. 67, 275-282, October 18, 1991,Copyright© 1991 by Cell Press

Ligand-Sensitive Binding of Actin-Binding Protein to Immunoglobulin G Fc Receptor I (FcyRI) Yasutaka Ohta,* Thomas P. Stossel,* and John H. Hartwig*t Division of Experimental Medicine Brigham and Womens' Hospital *Department of Medicine tDepartment of Anatomy and Cellular Biology Harvard Medical School Boston, Massachusetts 02115

Summary The high affinity receptor that binds the Fc domain of immunoglobulin G (IgG) subclasses 1 and 3 (FcyRI) mediates important immune defense functions by inducing cell surface changes on human leukocytes. In this article, we document direct high affinity binding of FcyRI to the actin filament cross-linking protein, actin-binding protein (ABP). In the absence of IgG, all FcyRI molecules in undifferentiated cells of myeloid line U937 bound to ABP over a 9-fold range of Fc~,RI expression induced by human IFN-y. Binding of IgG to U937 cells constitutively expressing FcyRI or to COS cells genetically transfected to express FcyRI rapidly decreased the avidity of FcyRI for ABP. This finding suggests the existence of a pathway communicating a signal between a functional IgG receptor and intracellular components involved in the effector responses to FcyRl-ligand interaction. Introduction Immunoglobulin G (IgG) antibodies account for much of the specificity of the human immune response by identifying microbial and neoplastic targets slated for destruction and instructing phagocytes and cytotoxic lymphocytes to clear, digest, and kill them by phagocytosis, pinocytosis, or antibody-dependent cellular cytotoxicity. The effector aspect of this immune surveillance involves the interaction of the Fc domain of the IgG molecules with several species of homologous glycoprotein receptors, designated Fc'yRI, Fc.yRII, and FcyRII. These receptors are expressed to various extents on the plasma membrane of neutrophils, monocytes, and natural killer cells (Kinet, 1989; Odin et al., 1990). The way that binding of IgG to Fc receptors elicits the cellular mechanics that bring about phagocytosis and cytotoxicity is an interesting and important probJem in immunobiology. No structural or functional linkages between Fc receptors and the intracellular machinery involved in IgG-mediated effector responses, however, have hitherto been clearly identified. This paper reports that the actin cross-linking protein actin-binding protein (ABP) specifically binds Fc,fRI and that this interaction is susceptible to inhibition by IgG molecules that ligate FcyRI, thereby introducing a signal transduction pathway of possible relevance for antibody-mediated functions.

Results ABP Binds to the Human High Affinity IgG Fc Receptor (FcyRI) of U937 Cells Figure 1 shows that the monoclonal anti-ABP antibody designated MAb5 specifically precipitated a 1251-labeled band from a Triton X-100 extract of 12Sl-labeledU937 cells containing radioactive polypeptides of many mobilities. The single ~2Sl-labeledband migrated with an apparent M, of 72 kd. This polypeptide, which was designated p72, was also detected in a MAb5 anti-ABP IgG immunoprecipitate of [3H]glucosamine-labeled U937 cells, suggesting that it is a glycoprotein (data not shown). The ABP-p72 complex was partially purified by sequential affinity column chromatographies. ABP and p72 in detergent lysates of iodinated U937 cells bound to a MAb5 anti-ABP IgG-Sepharose column and eluted together in 3 M MgCI2,suggesting an association between p72 and ABP (data not shown). As shown in Figure 2, the p72 polypeptide was the major labeled species in this mixture eluted by N-acetyI-D-glucosamine (GIcNAc) from a wheat germ agglutinin (WGA)-Sepharose affinity column after extensive washing, demonstrating that p72 is a glycoprotein. Most of the ABP applied to this column was recovered in the flowthrough and wash fractions, but it was also detectable in GIcNAc-eluted fractions containing p72 (Figure 2, inset). Since only a small fraction of total ABP applied to the WGA column was bound, p72 binds to a minor fraction of the total ABP in the cell extract. As indicated below, however, all p72 binds to ABP under these conditions. No actin was detectable in silver-stained polyacrylamide gels resolving polypeptides of these fractions, indicating direct association between ABP and p72 rather than association through the intermediary of actin filaments (Figure 2, inset). Fc'yRI is a lectin-binding glycoprotein with a mobility similar to p72 in Na dodecyl sulfate-polyacrylamide gel electrophoresis analyses (SDS-PAGE) (Anderson, 1982). We performed the following experiments to show that p72 is FcyRI. Separate detergent extracts of surface-radioiodinated U937 cells were incubated with MAb5 anti-ABP IgG and subsequently with sedimentable protein A-Sepharose or human IgG-Sepharose. The polypeptides adsorbed by the Sepharose bead conjugate were analyzed by SDS-PAGE and autoradiography. Human IgG-Sepharose absorbs both FcyRI and FcyRII (Anderson et al., 1986) (Figure 3, lanes g and h), and the migration of the p72 coprecipitating with ABP (Figure 3, lane a) is indistinguishable from that of FcyRI affinity-absorbed on human IgG-Sepharose beads. Since Fc-yRI has very low affinity for the murine IgG~ subclass of the MAb5 monoclonal anti-ABP IgG (Lubeck et al., 1985), it was unlikely that FcyRI precipitated with ABP through the Fc portion of the anti-ABP antibody. The following experiments support this conclusion. First, FcyRI was recovered only in MAb5 immunoprecipitates (Fig-

Cell 276

Starting Anti-ABP material Immunoprecipitate 1251

CBB

ure 3, lane a) and was absent when nonspecific mouse IgG1 was used as an immunoprecipitant (Figure 3, lane b). Second, Sepharose beads conjugated to the Fab portion of MAb5 or to purified human ABP bound FcTRI (Figure 3, lanes c and d). Third, removal of FcTRI from the detergent extracts of radioiodinated U937 cells with human IgGSepharose resulted in FcTRI being no longer recoverable with anti-ABP MAb5 Fab or purified ABP (compare lanes c and d with lanes e and f in Figure 3). In addition, following immunoprecipitation of U937 cell extracts with anti-ABP antibody, the FcTRI polypeptide was no longer recoverable with IgG-Sepharose from the extracts, indicating that all Fch,RI in the extract bound ABP (data not shown). To confirm that the Fc~,RI-binding glycoprotein was Fc~,RI, COS cells were transfected with human FcyRI cDNA-containing plasmids, and the detergent extracts of radioiodinated transfectants were immunoprecipitated with MAb5 anti-ABP or MAb197 anti-FcTRI antibodies. No 12Sl-labeled p72 was detected in the MAb5 immunoprecipitates from control COS cells (Figure 4, lanes a and b). On the other hand, the 72 kd labeled protein was recovered in MAb5 anti-ABP IgG immunoprecipitates (Figure 4, lanes c and d) from Fc-yRI-transfected cells. The migration of p72 by S D S - P A G E was indistinguishable from that of F ~ R I absorbed in anti-FcyRI antibody from these cells (Figure 4, lanes e and f). These results demonstrate that ABP binds to FcyRI and that this binding is not restricted to ABP of myeloid cells.

1251 ~--

ABP

200K-

94K-

68K-

< _ p72 ~-" IgG(h)

45K-

30K-

~-- IgG(l)

Figure 1. Precipitation of p72 with ABP from U937 Cell Extract U937 cells were surface labeled with Na~l and lysed in a buffer (see Experimental Procedures) containing 1.0%oTriton X-100 and 2 mg/ml of DNAase I. ABP was immunoprecipitated from the cell extract with mouse monoclonal MAb5 anti-ABP antibody and protein A-Sepharose beads. The immunoprecipitate was washed with the Triton X-100containing lysis buffer, bound protein was eluted in 1% SDS, and it was displayed by SDS-PAGE (5%-15% gradient gel). Bound proteins were visualized by Coomassie brilliant blue staining (CBB), and nSl-labeled proteins were detected by autorediography (1251).On the left, lZSl-labeledtotal cell surface protein is shown, demonstrating that p72 is one of many ~251-1abeledproteins on the surface of U937 cells and that coprecipitation of p72 with ABP reflects a specific interaction (middle and right). Molecular weight markers include myosin (200 kd), phosphorylase b (94 kd), BSA (68 kd), ovalbumin (45 kd), and carbonic anhydrase (30 kd). The migration of the ABP subunit (calculated molecular weight, 280 kd), p72, and the heavy (h) and light chains (I) of IgG are indicated.

Characteristics of Reconstituted and Endogenous FcyRI-ABP Complexes 12Sl-labeled Fch,RI purified from U937 cells by affinity ligation to human IgG saturably bound to human splenic ABP immobilized on Sepharose with an apparent Kd of 3.1 x

Figure 2. The ABP-p72 Complex Binds to a WGA-Sepharose 6MB Column The 3 M MgCI2eluates containing p72 and ABP from MAb5 anti-ABP IgG-Sepharose column were dialyzed against buffer B (20 mM TrisHCI [pH 7.4], 0.15 M NaCI, 0.1 mM EGTA, and 0.1% Triton X-100) in the presence of 0.1 mM phenylmethylsulfonyl fluoride. The dialysate was applied to a 0.5 ml WGA-Sepharose 6MB column and washed with 20 vol of buffer B (fractions 2-11, 1.0 ml/tube), and lectin-bound glycoprotein was eluted using 0.2 M GIcNAc in buffer B (fractions 12-21,0.5 ml per tube). The elution profile of l~l-labeled proteins was monitored by direct liquid scintillation counting of a portion of each fraction. The inset shows the polypaptide composition of the peak fraction (fraction 14) eluted with GIcNAc monitored by SDS-PAGE, followed by silver staining and autoradiography for l~l-labeled proteins. The migration of p72 and ABP subunit are indicated.

I

.~

=

10

t

c¢3 200 K

X

-

94 K -

6

68K-

V

45 K -

4 30 K

-

0.2 M GlcNAc

2

1 5

10

15

Fraction Number

20

Ligand-Sensitive Binding of ABP to FcTRI 277

e~ o=

o~

120 K94 K 68K-

I

g

8

~u

t,.

m

m

,,~

•.~

.,,7_,

"(--- FcyRI

~

a 45 K -

b

c

d

e

f

120 K 94 K -

.(-- FcyRII 30 K 68 K -

abcde

fgh

Figure 3. Demonstration that p72 is FcyRI of U937 Cells and Characterization of Its Association with ABP Samples of the '2Sl-surface-labeled cell extract were immunoprecipirated with MAb5 anti-ABP antibody (lane a), nonspecific mouse IgG1 (lane b), MAb5 Fab-Sepharose bead conjugates (lanes c and f), or ABP-Sepharose bead conjugates (lanes d and e). FcyRI and FcyRII were removed from a portion of the radioiodinated U937 cell lysate with human IgG-Sepharose bead conjugates (lanes e-h). The IgG beads were pelleted from the extracts by centrifugation, removing the FcyRI and FcyRII receptors (lanes g and h). The IgG-precleared extracts were further incubated with MAb5 Fab-Sepharose beads (lane f) or human ABP-Sepharose beads (lane e). Immunoprecipitates were analyzed by SDS-PAGE (10%) followed by autoradiography.

10-8 M and with a stoichiometry of ,~1 FcyRl:17 ABP dimers (Figure 5). The basis of this relatively low molar ratio of binding is unclear, but possibilities include inaccessibility of immobilized ABP to Fc~RI binding sites and selectivity imposed by regulatory modifications of a subset of ABP molecules. U937 cells express about 2 x 104 FcTRI (Kurlander and Batker, 1982) and 1.5 x 105 ABP dimers per cell (see Experimental Procedures), representing a ratio of about 1 FcTRI to 8 ABP molecules. These estimates are consistent with the findings that all U937 cells extract FcyRI-bound ABP and that only a small fraction of total U937 cell ABP molecules eluted together with FcTRI from WGA-Sepharose after exposure to GIcNAc. The estimates also indicate that free ABP is in excess of FcTRI in U937 cells." Whether the substoichiometric binding of FcyRI to ABP in cell extracts is related to that seen in our reconstitution studies remains to be determined.

Effect of IFN-y on the ABP-FcyRI Complex in U937 Cells IFN-y increases the expression of FcyRI by 2- to 10-fold on the cell surface of myeloid cells (Guyre et al., 1983). We examined how much the linkage of the Fc-yRI to ABP is modified when the expression of FcyRI is increased by

45 K-

Figure 4. Fc-fRIExpressedbyTransfectionofCOSCellsBindstoABP COS cells were transfected using the DEAE-dextran method with a • human Fc'fRI cDNA-containing CDM8 plasmid (10 mg per dish) (Allen and Seed, 1989). They were cultured for 48 hr in the growth medium with 5% FCS. Control, untransfected COS cells (lanes a and b) and FcyRI-transfected cells (lanes c, d, e, and t) were surface labeled with Na'ZSl, solubilized, and incubated with MAb5 anti-ABP antibody (lanes a, b, c, and d) or MAb 197 anti-FcTRI antibody (lanes e and f). Antibodies were immobilized with protein A-Sepharose beads, immunoprecipitates were washed to remove nonspecific radioactivity, and bound protein was eluted in SDS-sample buffer and analyzed by SDS-PAGE and autoradiography.

IFN-y. U937 cells were cultured in the presence or absence of human recombinant JFN-y.As previously demonstrated, IFN-y stimulated the expression of Fc-yRI 3- to 9-fold, but did not stimulate the expression of FcyRJl (Figures 6A and 6B). IFN-7 also increased the amount of Fc'yRI recovered from immunoprecipitates with anti-ABP MAb5. The range of increments was equivalent (3- to 9-fold) to the quantity of FcyRI recovered using human IgG-Sepharose beads, indicating that all surface FcyRI links to ABP in U937 cells over a wide FcyRI concentration range.

Effect of IgG on the ABP-FcTRI Complex Addition of monomeric human IgG to either cellular extracts or to intact cells diminished FcyRI association with immunoprecipitated ABP in a concentration-dependent manner (Figure 7). This loss of association of FcyRI with ABP occurred rapidly (within 1 min) after exposure of intact cells to human IgG. Mouse IgG1, which has little affinity for human FcyRI (Lubeck et al., 1985), had no effect on the recovery of ABP-FcyRI complexes (Figure 7). Human IgG but not mouse IgG1 also decreased the association of the receptor expressed in COS cells with immunoprecipitated

Cell 278

I

~

0.02

Figure5. Binding of Purified n%Labeled FcyRI to ABP-Sepharose Bead Conjugates Specific binding of nSl-labeled FcvRI to ABPcoupled Sepharose beads after 80 min at 22°C (circles). Nonspecific binding of l~l-labeled FcTRI to Sepharose beads was determined using ethanolamine-blockedcyanogen bromideactivated beads (squares). Nonspecific binding was subtracted from total binding to obtain the specific binding data. The inset shows Scatchard plot analysis of the speci~c binding of FcyRI to the ABP-Sepharose beads. On the ordinate, "R" is the bound FcvRI per 0.3 mM ABP (total per assay). Specific binding data are representative of three independent analyses.

I

u4

ool

, !

M

M

L2

1,6

gab =

0.01

= o

!

r

0.00

l

,

I

0.1

0.0

0.2

0.3

Fc~RI (I~M) ABP. IgG (50 mg/ml) added to COS cells at 5 0 % confluency diminished by 9 5 % (mean of two separate experiments) the a m o u n t of 12~l-labeled FcvRI recovered from cell extracts by MAb5. These results suggest that engagement of the Fc,fRI by the Fc domain of IgG dissociates it

A

anti-ABP I g G (mAbS)

TIFN

-

"4-

huIgG -

+

120 K 94 K FcTRI

68 K -

45 K

-

O

FcTRII

30 K -

B

3O0

O

2oo

o

0

•tI~

-

"l"

anti-ABP IgG (mAbS)

-

+ hulgG

from ABP both in intact cells and cell extracts. The results further confirm that FcTRI binds ABP directly and does not bind to murine MAb5 (an IgG1), since FcyRI could not be dissociated from the immunoprecipitates with excess nonspecific m o u s e IgGl. To address the possibility that the dissociating effect of IgG ligand on the ABP-Fc~RI c o m p l e x m e a n t that ABP was actually bound to an extracellular d o m a i n of FcTRI, we incubated intact U937 cells with an anti-FcyRI antibody (MAb197), which recognizes Fch, RI through its Fab as well as Fc portion, in the presence or a b s e n c e of purified ABP. We then d e t e r m i n e d whether purified ABP c o m p e t e s with the anti-FcTRI monoclonal antibody for the binding to the external d o m a i n of FcTRI. A 100-fold molar excess of ABP did not alter the binding of the anti-FcyRI monoclonal anti-

Figure 6. IFN-7 Increases the Amount of FcyRI Complexed to ABP in U937 Cells U937 cells (2 x 107cells per dish) were incubated for 18 hr with (lanes b and d) or without (lanes a and c) IFN-7 (100 U/mI) in Iscove's modified Eagle's medium containing 10% FCS. Cells were washed twice with PBS and surface labeled with NanSl as described, n%labeled cells were solubilized as described, and aliquots of soluble proteins were immunoprecipitated with either MAb5 anti-ABP antibody (lanes a and b) or human IgG (lanes c and d). Bound protein was eluted in a SDSsample buffer and subjected to SDS-PAGE (5O/o-15O/ogradient gel), silver staining, and autoradiography. (A) Autoradiographs of immunoprecipitated proteins. In this experiment, the expression of FcyRI in IFN-7-treated cells (lane d) is 9-fold compared with untreated cells (lane c), as determined by densitometric scanning of the corresponding bands in the autoradiograph. (B) Quantitation of the amounts of FcyRI immunoprecipitated. The amounts of immunoprecipitated Fc'f RI was quantitated by densitometric scanning of the corresponding bands in the autoradiograph. The data are expressed as mean _+ S. E. from three separate experiments. Values are expressed as percent of increase in the amount of FcTRI relative to the IFN-y-minus cells.

Ligand-SensitiveBindingof ABP to Fc.fRI 279

. . . . . . . .

i

. . . . . . . .

i

. . . . . . . .

i

. . . . . . . .

=

. . . .

=

. . . . . . . .

lOO-"

g

60

,< 20

0

. . . . . . . .

.1

1

10

H . I

100

i

i

=l...

1000

lgG (p.g/ml) Figure 7. Dissociation of Fc'fRI from ABP-FcRI Complex by Human IgG Effect of loG on ABP-FcTRI complex. Samples (6.7 x 106 cells per tube) of the l~l-labeled U937 cell extract were incubated with 50 ml of MAb5 anti-ABP IgG-Sepharose beads in the presence or absence of increasing concentrations of monomeric human 10G (circles) or moose IgG1 (squares) for 90 rain at 4°C. Samples (8 x 10Bcells per tube) of the 12Sl-labeled intact U937 cells were incubated in the presence or absence of increasing concentrations of monomeric human loG (triangles) for 10 rain at 37°C with gentle stirring. Cells were washed quickly

by PBSand lysedwithTritonX-100lysisbuffer.The cellextractswere incubatedwith 50 ml of MAb5anti-ABPIgG-Sepharosebeadsfor 90 min at 4°C. Bound proteinswere eluted in SDS-samplebuffer and analyzedby SDS-PAGEand autoradiography.The amountof bound Fc'7RIwasquantitatedbydensitometricscanningof thecorresponding bands in the autoradiograph.Valuesare meansof two experiments and expressedas percentageof controls.

body to Fc'yRI, as quantitated by fluorescence-activated cell sorting (data not shown).

Discussion

Human Fc"yRI (CD64) is a transmembrane glycoprotein member of the immunoglobulin superfamily that exhibits high affinity binding (Ka = "~108 x M-1) to monomeric human IgG1 and murine IgG2aand IgG3. IFN-7 increases the basal content of Fch,RI receptors in monocytes and macrophages and increases the level from virtually undetectable to measureable amounts in neutrophils in vitro (Akiyama et al., 1984; Guyre et al., 1983; Perussia et al., 1983); bacterial infection has the same inducing effect on FcTRI expression on leukocytes in vivo (Guyre et al., 1990). FcTRI can mediate the capacity of mononuclear phagocytes and interferon 7 (IFN-7)-treated polymorphonuclear leukocytes to bind and engulf various antibodycoated particles, to perform antibody-dependent cellular cytotoxicity against normal and neoplastic cells, and to secrete superoxide anion and tumor necrosis factor (Graziano and Fanger, 1987; Gomez et al., 1989; Pfefferkorn and Fanger, 1989a; Akerley et al., 1991; Debets et al., 1990). Monocytes and U937 cells express about 20,000 FcTRI receptors per cell (Kurlander and Batker, 1982) and are equivalent in interferon responsiveness, making the

latter cells an appropriate and convenient system for the study of Fc'yRI receptor function (Odin et al., 1990). We report in this article that Fc'TRI expressed constitutively in myeloid U937 cells and by genetic transfection on COS fibroblastic cells binds ABP, a cytoplasmic homodimer that promotes the gelation of actin filaments into orthogonal networks in the periphery of eukaryotic cells (Stossel and Hartwig, 1976; Hartwig and Shevlin, 1986; Gorlin et al., 1990). Of the several FcTR classes, only Fc-fRI demonstrably binds to ABP, a finding consistent with the low degree of primary sequence relationship among the different types of FcTR (Kinet, 1989). ABP binds the transmembrane glycoprotein complex Gplb/9 in human blood platelets (Fox, 1985; Okita et al., 1985; Ezzell et al., 1988; Hartwig and DeSisto, 1991), which establishes a precedent for a membrane-binding role for this protein. Binding of the GP1 b/9 complex occurs at the carboxyl third of the ABP molecule (Ezzell et al., 1988). Like the ABPFcyRI interaction, the binding of GPlb/9 complexes to ABP has also been reconstituted in vitro using a solid phase assay (Andrews and Fox, 1991). The affinity of the GPlb/9 complex for ABP (K~ = 10-7 M) is about 10-fold lower than that of the FcTRI for ABP. It is not known where along the ABP molecule these glycoproteins interact, and sequence comparison reveals no regions of sequence conservation between the cytoplasmic tails of the GPlb~ chain and the Fc~,RI (Allen and Seed, 1989). The detection of the FcyRI-ABP association was dependent upon approaching it from the inner side of the membrane, because ligands that bind to the receptor's extracellular domain lowered its affinity for ABP. This observation may explain why previous research has not identified an association between FcyRI and ABP. ABP dimers are predicted to have two glycoprotein-binding sites in close proximity (Ezzell et al., 1988; Gorlin et al., 1990), suggesting that FcyRI molecules bound to these sites could be sufficiently close together for ligand binding to change the orientation of their cytoplasmic domains, thereby causing a change in their affinity for ABP. The finding of IgG-induced dissociation of the ABP-Fc'yRI complex is consistent with the observation of others that Fc~RI bound to monomeric IgG is solubilized by detergents from U937 cells; and it is not contradictory to documentation of transient association with a detergent-insoluble residue of U937 cells of Fc~RI bound to cross-linking IgG (Pfefferkorn and Fanger, 1989a). Detergent insolubility is not equivalent to binding of FcTRI to the cytoskeleton in general or to actin via ABP in particular, and the observation that cells internalize Fc~,RI bound to cross-linking IgG but not monomeric IgG (Jones et al., 1985; Pfefferkorn and Fanger, 1989b)suggests that some of the detergent resistance may result from enclosure of Fc.yRI within vesicles. The discovery of at least one molecular interaction in the FcyRI pathway provides a firmer basis for exploring the function of this important immune receptor. Control of the avidity of Fch,RI for ABP by IgG suggests some molecular mechanisms by which these antibodies might influence cellular function. One possibility concerns the stability of the ventral surface of spread FcyRI-bearing cells such as macrophages. Regions of macrophage membranes tightly

Cell 280

adherent to sinusoidal endothelial cells in the liver, spleen, or lymph nodes or to extracellular matrix fibers might not be exposed to ambient IgG molecules. In addition, macrophages attempting to ingest surfaces opsonized with C3 fragments or neutrophils migrating on fibrinogen films exclude molecules the size of IgG from the adherent surface (Wright and Silverstein, 1984; Weitz et al., 1987). Associations between FcTRI, ABP, and actin filaments on such surfaces could establish a continuous lattice that stabilizes the membrane, with the submembrane actin skeleton composed of actin filaments cross-linked by A B P (Hartwig and Shevlin, 1986). A B P molecules would function in this case both to cross-link actin filaments on the cytoplasmic face of the membrane and to link the cross-linked actin gel tightly to the membrane bilayer by association with Fc3,RI. Exposure of the cell edge to agonists that disrupt the actin filament network--for example, by an elevation of calcium, which would activate gelsolin to sever actin filaments (Stossel, 1 9 8 9 ) - c o u l d cause the cell edge to lift, permitting IgG to percolate underneath part of the cell and bind to FcTRI receptors. The s u b m e m b r a n e lattice would then be separated from the membrane bilayer, increasing its flexibility at that point. In speculating about the possible role of F c y R I - A B P interactions in phagocytosis, an IgG-mediated function requiring that the plasma membrane be sufficiently flexible to deform itself around a particle undergoing ingestion, it is important to note that the nonequilibrium conditions imposed by our sedimentation assay do not rule out lower affinity ABP binding to FcyRI bound to monomeric IgG in vivo. This is especially so, because A B P is present in the cell at micromolar concentrations. Such low affinity binding might be sensitive to the aggregation state of the receptor, and Fc~RI clustered by ligation to IgG-opsonized particles or to immune c o m p l e x e s might, in comparison with dispersed FcTRI, more persistently dissociate the plane of the membrane bilayer from the submembrane actin lattice and thereby facilitate the membrane invagination mediating endocytosis. A finding consistent with this formulation was the observation that A B P was more easily extractable by sucrose solutions from membranes derived from macrophages undergoing phagocytosis than from m e m b r a n e s of resting cells (Stossel and Hartwig, 1976). Experimental Procedures Reagents Na~l, 140 mCi/mMol, [3H]glucosamine, 27 Ci/mMol, and EnSHance were purchased from DuPont-New England Nuclear Corp., Boston, Massachusetts; Iscove's modified Eagle's medium from Gibco, Gaithersburg, Maryland; fetal calf serum (FCS) from Irvine Scientific, Santa Ana, California; protein A-Sepharose 4B, cyanogen bromidHctivated Sepharose 4B, and WGA-Sepharose 6MB from Pharrnacia-LKB, Piscataway, New Jersey; murina IgG1 and human IgG, Aspergillus niger glucose oxidase, benzamidine, aprotinin, phenylmethylsulfonyl fluoride, leupeptin, GIcNAc, and V8 protease from Sigma Chemical Corp., St. Louis, Missouri; monoclonal antibodies MAb 32.2 and 197 against FcyRI (Anderson et al., 1986; Guyre et al., 1989) from Madarex Co., West Lebanon, New Hampshire; bovine lactoperoxidase from Calbiochem Corp., La Jolla, California; FITC-conjugated F(ab')=fragments of goat anti-mouse IgG from Jackson Immuno Research, West Grove, Pennsylvania; and DNAase I from Boehringer Mannheim Corp., Indianapolis, Indiana. Human recombinant IFN-7, 3.4 x 107 U/mg, was a gift of Dr. David

Thomas, Biogen Corporation, Cambridge, Massachusetts. Full-length human FcyRI cDNA in the CDM8 vector (Seed, 1987) was generously provided by Dr. Brian Seed, Department of Genetics, Massachusetts General Hospital. An IgG, mouse monoclonal antibody against human ABP, MAb5, has been previously characterized in our laboratory (Ezzell et al., 1988). Fab fragments of MAb5 were prepared as described by Unkeless (1979). Monomeric antibody solutions were centrifuged at 100,000 x g for 30 rain to remove aggregates. ABP was purified from human uteri and spleen (Gorlin et al., 1990; Hartwig and Stossel, 1981). ABP, MAb5, MAb5 Fab, and human IgG were coupled to cyanogen bromide-activated Sepharose 4B according to themanufacturer's specifications at concentrations of 2.4 mg/ml, 5 mg/ml, 0.3 mg/ml, and 5.0 mg/ml, respectively. Cell Culture, Surface Labeling, and Isolation of ABP-Binding Surface Components Suspensions of myeloid cell line U937 cells were grown at 37°C in plastic tissue culture dishes (13.5 cm diameter) in 40 ml of Iscove's modified Eagle's medium containing 10% FCS (Kwiatkowski, 1988). Cells were diluted to 0.3 x 108/ml and grown to a density of 2 x 106/ ml. In some experiments, human IFN-7 was added at a concentration of 100 U/ml for 18 hr. The following procedures were performed at room temperature. U937 cells (8 x 107)were centrifuged for 5 min at 500 x g, washed gently twice with 0.154 M NaCI and 10 mM sodium phosphate (pH 7.4) (PBS), and incubated with 2 ml of PBS containing 5 mM glucose, 2 mCi of Nal~Sl,40 mg of lactoperoxidase, and 0.7 U of glucose oxidase for 10 rain with gentle stirring. The reaction was stopped by adding 30 ml of 0.15 M Nal with 10 mM sodium phosphate (pH 7.4). The cell suspension was centrifuged for 5 rain at 500 x g and washed twice with 50 ml of the Nal solution, l~l-labeled cells were suspended in 2 ml of a lysis buffer composed of 100 mM Tris-HCI, 20 mM EGTA, 1.0°/0 Triton X-100, 2.0 mg/ml of DNAase I, 42 mM leupeptin, 1.0 mM benzamidine HCI, 12 mM aprotinin, and 2.0 mM phenylmethylsulfonyl fluoride (pH 7.4). They were incubated for 10 min with gentle stirring at room temperature and then centrifuged at 100,000 x g for 30 rain at 22°C. In some experiments, 0.5 ml samples of the supernatant fluids were incubated at 0°C for 2 hr with various soluble antibodies, followed by incubation at 4°C for 15 min with 100 ml of a solution containing 50o/0 (v/v) protein A-Sepharose beads in 0.15 M NaCI buffered with 10 mM Tris-HCI (pH 7.4). In other experiments, 0.5 ml supernatant samples were incubated with rotation for 2 hr at 4°C, with the indicated concentrations of Sepharose beads coupled to various reagents. All precipitates were washed six times with 20 vol of 100 mM Tris-HCI, 20 mM EGTA, and 1.0O/oTriton X-100 (pH 7.4). Bound proteins were eluted from the Sepharose beads by adding 60 ml of gel sample buffer (Laemmli, 1970) containing 10/0SDS and boiling them for 2 min. Beads were removed by centrifugation at 12,300 x g for I min, supernatants werec011ected, and eluted proteins were displayed by SDS-PAGE (Laernmli, 1970). Acrylamide gels were stained with Coomassie brilliant blue or silver (Galcode, Pierce), dried, and exposed to Kodak X-Omat film for autoradiography of ~l-labeled proteins. The relative amount of ~1 incorporation into each band was determined by dsnsitometry of the autoradiographs using an LKB gel densitometer. In some experiments, ~l-labeled cells were suspended at a density of 8 x 10ecells per ml of Iscove's modified Eagle's medium containing 1% bovine serum albumin (BSA). Cells were incubated for various time with or without monomeric human IgG at 37°C. After the cell suspensions were centrifuged at 1000 x g for 20 s and the incubation medium was aspirated, cells were washed quickly with PBS and solubilized in 0.5 ml of the lysis buffer. The cell extracts were clarified by centrifugation and the supematants incubated with MAb5-Sepharose beads (50 ml) for 90 min at 4°C. The immunoprecipitates were washed with the lysis buffer six times, and bound proteins were analyzed by SDS-PAGE followed by autoradiography. For analysis of total cell proteins, 12Sl-labeledcells were suspended in PBS containing 2°/0 SDS and 2 mM phenylmethylsulfonyl fluoride, boiled for 5 min, and centrifuged at 100,000 x g for 30 min at 22°C. The suparnatants were then collected and analyzed by SDS-PAGE followed by autoradiography. For labeling of cells with [3H]glucosamine, cells (0.5 x 107)were incubated with D-[6-3H(N)]-glucosamine(100 mCi/ml) in minimum essential medium (1000 rag/liter glucose) containing 10% FCS for 20 hr

Ligand-Sensitive Binding of ABP to FcyRI 281

in 35 mm plastic tissue culture dishes. Labeled cells were washed twice with PBS, solubilized with Triton X-100 lysis buffer, and immunoprecipitated with anti-ABP IgG MAb5. Bound proteins were analyzed by SDS-PAGE. Acrylamide gels were stained with Coomassie brilliant blue, dried, and exposed to Kodak X-Omat film for fluorography (En3Hance) of 3H-labeled proteins.

Affinity Chromatography of Labeled Surface Components All procedures were performed at 4°C. 12el-surface-labeled extracts from 8 x 107 U937 cells in one culture dish were combined with unlabeled extracts of cells from four other dishes and incubated with rotation for 2 hr with 0.5 ml of anti-ABP IgG MAb5-Sepharose 4B conjugates (5 mg/ml) equilibrated with 20 mM Tris-HCI, 0.15 M NaCI, 1 m M EGTA, and 0.1%0Triton X-100 (pH 7.4) (buffer A) in the presence of 42 mM leupeptin, 1.0 mM benzamidine HCI, 12 mM aprotinin, and 2.0 mM phenylmethylsulfonyl fluoride. The Sepharose bead-containing suspension was poured into a column (0.9 x 6.0 cm), the column was washed free of unbound radioactivity with 10 vol of buffer A, and bound protein was sequentially eluted with 10 vol of 1.0 M NaCI in buffer A and 5 ml of 3 M MgCI2 in buffer A (Chaponnier et al., 1986). Analysis of the eluates by SDS-PAGE followed by silver staining revealed that most of the ABP eluted by the 3.0 M MgCI2 solution. In addition, most of the radioactivity in the 3.0 M MgCI2 eluate moved as a single broad band with a molecular weight of 72,000 (p72). The MgCI2 eluate was dialyzed against 20 mM Tris-HCI, 0.15 M NaCI, 0.1 mM EGTA, 0.1 mM phenylmethylsulfonyl fluoride, and 0.1O/o Triton X-100 (pH 7.4) (buffer B) and passed over a 0.5 ml column of WGA-Sepharose 6MB previously equilibrated with buffer B. The column was washed with 10 vol of buffer B, and bound glycoproteins were eluted with a 0.2 M GIcNAc in buffer B. Eluted protein was monitored by SDS-PAGE followed by silver staining and by autoradiography of the dried gel and direct counting of 1251radioactivity in each fraction.

Binding Assays 12el-labeledFcyRI was purified from U937 cells according to Anderson (1982) with some modification. The detergent lysates of 1251-labeled U937 cells were prepared as described above and incubated with 2.0 ml of human IgG-Sepharose beads (5 mg/ml) with rotation for 2 hr at 4°C. The Sepharose bead-containing suspension was poured into a 9 x 6 cm column, which was washed free of unbound radioactivity with 40 ml of 20 mM Tris-HCI, 0.15 M NaCI (pH 7.4) containing 1 mM phenylmethylsulfonyl fluoride and 0.1% Triton X-100, then washed with 10 ml of the same buffer containing 1.0 M NaCI to elute FcTRI. FcyRI was eluted with 0.5 M acetic acid containing 0.1%o Triton X-100 (0.5 ml per tube), and the eluates were immediately neutralized by adding 0.1 ml of 2 M Tris-HCI containing 0.1% Triton X-100. The specific activity of radiolabeled FcyRI purified by this procedure was 1 x 103 cpm/mg protein. Binding of ABP to purified FcyRI was measured by incubating lzSi-labeled FcyRI protein with 10 ml of human ABP-Sepharose bead conjugates (1.7 mg/ml) in a final volume of 100 ml of buffer C containing 100 mM Tris-HCI, 20 mM EGTA, 1.00/o Triton X-100, and 0.1% (w/v) BSA (pH 7.4). In control experiments, ethanolamine-blocked cyanogen bromide-Sepharose beads were substituted for the ABP-Sepharose beads. ABP beads-FcyRI mixtures were incubated for 80 min at 22°C with gentle stirring, and then washed twice with buffer C. Bound ~1 was counted in a gamma counter.

COS Cell Expression of Human FcyRI and Analysis of ABP-FcyRI Association COS cells (Allen and Seed, 1989) at 50% confluency were transfected by the DEAE-dext~'an method (Kwiatkowski et al., 1989) using 10 mg of CDM8-FcyRI plasmid per 10 cm petri dish. The cells were grown for 48 hr in medium containing Dulbecco's modified Eagle's medium (4.5 g/I glucose) with 5% FCS. Transfected COS cells were surface labeled with Nal~l, solubilized, and immunoprecipitated by anti-ABP (MAb5) or anti-FcyRI (MAb197) antibodies as described above. Imm unoprecipitates were analyzed by SDS-PAGE and autoradiography.

taining 5% BSA (0.7 x 105 cells per ml) at 4°C for 30 min. Cells were washed twice with PBS and incubated with 10 mg/ml FITC-conjugated F(ab~2 fragments of goat anti-mouse IgG in PBS containing 5°/o BSA at 4°C for 30 min. Cells were washed twice with PBS, fixed with 1% paraformaldehyde in PBS, washed twice again to remove unbound IgG in PBS, and resuspended in 1.5 ml of PBS. The fluorescence signal from 2,000 cells per sample was measured in a fluorescence activated cell sorter (FACscan, Becton Dickinson). Other Methods SDS-PAGE was performed by the method of Laemmli on 5O/o-150/o gradient or 10o/0 slab gels (Laemmli, 1970). Protein was determined by the method of Bradford (1976) with BSA as standard.

Acknowledgments We wish to acknowledge the excellent technical assistance of Ms. Andrea Francisco and Ms. Micheile DeSisto; Dr. Brian Seed for human FcyRI cDNA; Dr. David Thomas for human recombinant IFN-7; Dr. David Kwiatkowski and Dr. Jed Gorlin for help in COS cell transfection; and Ms. Darlene Jackson for her editorial and secretarial skills. Supported by U. S. Public Health Service grants HL-19429, HL-45154, AI-28465 and DK-38452. 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 USC Section 1734 solely to indicate this fact. Received April 22, 1991; revised July 29, 1991.

References Akerley, W. L., III, Guyre, P. M., and Davis, B. H. (1991). Neutrophil activation through high-affinity Fcy-receptor using a monomeric antibody with unique properties. Blood 77, 607-615. Akiyama, Y., Lubeck, M. D., Steplewski, Z., and Koprowski, H. (1984). Induction of mouse igG2a- and IgG3-dependent cellular cytotoxicity in human monocytic cells (U937) by immune interferon. Cancer Res. 44, 5127-5131. Allen, J. M., and Seed, B. (1989). Isolation and expression of functional high-affinity Fc receptor complementary DNAs. Science 243, 378-381. Anderson, C. L. (1982). Isolation of the receptor for igG from a human monocyte cell line (U937) and from human peripheral blood monocytes. J. Exp. Med. 156, 1794-1805. Anderson, C. L., Guyra, P. M., Whitin, J. C., Ryan, D. H., Looney, R. J., and Fanger, M W. (1986). Monoclonal antibodies to Fc receptors for IgG on human mononuclear phagocytes: antibody characterization and induction of superoxide production in a monocyte cell line. J. Biol. Chem. 261, 12856-12864. Andrews, R. K., and Fox, J. E. B. (1991). Interaction of purified actinbinding protein with the platelet membrane glycoprotein Ib-IX. J. Biol. Chem. 266, 7144-7147. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Anal. Biochem. 72, 248-254. Chaponnier, C., Janmey, P. A., and Yin, H. L. (1986). The actin filament-severing domain of plasma gelsolin. J. Cell Biol. 103, 14731481. Debets, J. M. H., Van de Winkel, J. G. J., Cueppens, J. L., Dietteren, I. E. M., and Buurman, W. A. (1990). Crosslinking of both FcyRq and Fc'yRI induces secretion of tumor necrosis factor by hu man monocytes, requiring high affinity Fc-FcyR interactions. Functional activation of FcyRII bytreatment with proteases or neuraminidase. J. Immunol. 144, 1304-1310. Ezzell, R., Kenney, D., Egan, S., Stossel, T., and Hartwig, J. (1988). Localization of the domain of actin-binding protein that binds to membrane glycoprotein Ib and actin in human platelets. J. Biol. Chem. 263,

Flow Cytometrlc Analysis for ABP Binding to Intact Cells

13303-13309.

IFN-y-treated U937 cells (3.3 x 105 cells) were washed twice in PBS and incubated with 0.1-10 mg/ml of anti-FcyRI (MAb197) IgG in the presence or absence of 20 mg/ml of purified human ABP in PBS con-

Fox, J. E. B. (1985). Identification of actin-binding protein as the protein linking the membrane skeleton to glycoproteins on platelet plasma membranes. J. Biol. Chem. 260, 11970-11977.

Cell 282

Gomez, F., Chien, P., King, M., McDermott, P., Levinson, A. I., Rossman, M. D., and Schreiber, A. D. (1989). Monocyte Fc7 receptor recognition of cell-bound and aggregated IgG. Blood 74, 1058-1065. Gorlin, J., Yamin, R., Egan, S., Stewart, M., Stossel, T., Kwiatkowski, D., and Hartwig, J. (1990). Human endothelial actin-binding protein (ABP-280, non-muscle filamin): a molecular leaf spring. J. Cell Biol. 111, 1089-1105. Graziano, R. F., and Fanger, M. W. (1987). Human monocyte-mediated cytotoxicity: the use of Ig-bearing hybridomas as target cells to detect trigger molecules on the monocyte cell surface. J Immunol. 138, 945954. Guyre, P. M., Morganelli, P. M., and Miller, R. (1983). Recombinant immune interferon increases immunoglobulin G Fc receptors on cultured human mononuclear phagocytes. J. Clin. Invest. 72, 393-397. Guyre, P. M., Graziano, R. F., Vance, B. A., Morganelli, P. M., and Fanger, M. W. (1989). Monoclonal antibodies that bind to distinct epitopes on FcyRI are able to trigger receptor function. J. Immunol. 143, 1650-1655. Guyre, P. M., Campbell, A. S., Kniffin, W. D., and Fanger, M. W. (1990). Monocytes and polymorphonuclear neutrophils of patients with streptococcal pharyngitis express increased numbers of type 1 IgG Fc receptors. J. Clin. Invest. 86, 1892-1896. Hartwig, J. H., and DeSisto, M. (1991). The cytoskeleton of the resting human blood platelet: structure of the membrane skeleton and its attachment to actin filaments. J. Cell Biol. 112, 407-425. Hartwig, J. H., and Shevlin, P. (1986). The architecture of actin filaments and the ultrastructural location of actin-binding protein in the periphery of lung macrophages. J. Cell Biol. 103, 1007-1020. Hartwig, J. H., and Stossel, T. (1981). The structure of actin-binding protein molecules in solution and interacting with actin filaments. J. Mol. Biol. 145, 563-581. Jones, D. H., Nusbacher, J., and Anderson, C. L. (1985). Fc receptormediated binding and endocytosis by human mononuclear phagocytes: monomeric IgG is not endocytosed by U937 cells and monocytes. J. Cell Biol. 100, 558-564. Kinet, J.-P. (1989). Antibody-cell interactions: Fc receptors. Cell 57, 351-354. Kurlander, R. J., and Batker, J. (1982). The binding of human immunoglobulin G1 monomer and small, covalently cross-linked polymers o1 immunoglobulin G1 to human peripheral blood monocytes and polymorphonuclear leukocytes. J. Clin. Invest. 69, 1-8. Kwiatkowski, D. J. (1988). Predominant induction of gelsolin and actinbinding protein during myeloid differentiation. J. Biol. Chem. 263, 13857-13862. Kwiatkowski, D. J., Janmey, P., and Yin, H. (1989). Identification of critical functional domains in gelsolin. J. Cell Biol. 108, 1717-1726. Laemmli, U. (1970). Cleavage of structural proteins during the assembly o1 the head of bacteriophage T4. Nature 227, 680-685. Lubeck, M. D., Steplewski, Z., Baglia, F., Klein, M. H., Dorrington, K. J., and Koprowski, H. (1985). The interaction of murine IgG subclass proteins with human monocyte Fc receptors. J. Immunol. 135, 12991304. Odin, J. A., Painter, C. J., and Unkeless, J. C. (1990). Fcy receptors: a diverse and multifunctional gene family. In Cellular and Molecular Mechanisms of Inflammation; Receptors of Inflammatory Cells: Structure-Function Relationships (New York: Academic Press, Inc.). Okita, L., Pidard, D., Newman, P., Montogomery, R., and Kunicki, T. (1985). On the association of glycoprotein Ib and actin-binding protein in human platelets. J. Cell Biol. 100, 317-321. Perussia, B., Dayton, E. T., Lazarus, R., Fanning, V., and Trinchieri, G. (1983). Immune interferon induces the receptor for monomeric IgG1 on human monocytic and myeloid cells. J. Exp. Med. 158, 1092-1113. Pfefferkorn, L. C., and Fanger, M. W. (1989a). Cross-linking of the high affinity Fc receptor from human immunoglobulin G1 triggers transient activation of NADPH oxidase activity. J. Biol. Chem. 264, 1411214120. Pfefferkorn, L. C., and Fanger, M. W. (1989b). Transient activation of the NADPH oxidase through Fc'fRl: oxidase deactivation precedes internalization of cross-linked receptors. J. Immunol. 143, 2640-2649.

Seed, B. (1987). An LFA-3 cDNA encodes a phospholipid-linked membrane protein homologous to its receptor CD2. Nature 329, 840-842. Stossel, T. P. (1989). From signal to pseudopod: how cells control cytoplasmic actin assembly. J. Biol. Chem. 264, 18261-18264. Stossel, T. P., and Hartwig, J. H. (1976). Interactions of actin, myosin and an actin-binding protein of rabbit lung macrophages. II. Role in cytoplasmic movement and phagocytosis. J. Cell Biol. 68, 602-614. Unkeless, J. C. (1979). Characterization of a monoclonal antibody directed against mouse macrophage and lymphocyte Fc receptors. J. Exp. Med. 150, 580-596. ,, Weitz, J. I., Huang, A. J., Landman, S. L., Nicholson, S. C., and Silverstein, S. C. (1987). Elastase-mediated fibrinogenolysis by chemoattractant-stimulated neutrophils occurs in the presence of physiologic concentrations of antiproteinases. J. Exp. Med. 166, 1836-1850. Wright, S. D., and Silverstein, S. C. (1984). Phagocytosing macrophages exclude proteins from zones of contact with opsonized targets. Nature 309, 359-361.