Regulation of the avidity of integrin alpha 4 beta 7 by the beta 7

T H E JOURNAL OF Blo~ccrc~L CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 269, No. 20, Issue of May 20, pp. 14411-14418,1994 Printed in U.S.A.

Regulation of the Avidity of Integrin a$, by the p7 Cytoplasmic Domain*

(Received for publication, January 24, 1994)

David T. CroweSP, Henry Chiun, Sherman Fongn, and Irving L. WeissmanS From the Wepartments of Pathology and Developmental Biology, Stanford University School of Medicine, Stanford, California 94305 and the Wepartment of Immunology, Genentech, Inc., South Sun Francisco, California 94080

Integrins are cell-surface heterodimeric receptors with adhesive and transmembrane signaling properties. Their cytoplasmic domains can affect receptor avidity, cytoskeletal association, and post-receptor occupancy events. The a4P7 integrin mediates cell adhesion to Pey- er’s patch high walled endothelial venules (HEW, VCAM, and CS-Ufibronectin. To determine the role of the p7 cytoplasmic domain in these interactions, wild- type and truncated p7 subunits were stably expressed in the a4+/p;/p; B cell lymphoma 38C13. The cell line A727 lacks the entire p7 cytoplasmic domain, A753 lacks the 34 C-terminal residues, and IXSN is vector-transfected 38C13. Cells expressing wild-type p7 bound Peyer’s patch HEV, fibronectin, and immobilized VCAM constitutively and did not require prior activation with phor- bo1 esters. Interestingly, A753 displayed no ligand binding activity, while A727 was constitutively active for all ligands and displayed greater avidity for fibronectin and Peyer’s patch HEV than the wild-type p7. p7, A753, 6727, and IXSN were also tested for the ability to bind soluble VCAM in the presence of various divalent cations. In the presence of Ca2+, but not M e , A727 constitutively bound soluble VCAM, whereas p7, A753, and LXSN did not. p7 and A753 could bind soluble VCAM if first activated with MnZ+. The results suggest that: (i) a,& can be expressed in a constitutively active state, (ii) the p7 cytoplasmic domain regulates the avidity of a4p7, and (iii) 38C13 cell lines expressing wild-type and truncated p7 subunits define three stable activation states of a4&: inactive (A753), partially active (p,), and fully active (A727) receptors.

Integrins are a diverse family of at least 21 different cell- surface d p heterodimers that mediate adhesion to soluble, insoluble, and cellular substrates (1). Beside their adhesive properties, these receptors can also mediate both “inside-out” and “outside-in’’ transmembrane signaling. We are interested in receptors that direct lymphocyte traffic to secondary lymphoid organs and sites of inflammation. We previously identified the murine Peyer’s patch homing receptor (2) as an a4p7 integrin that mediates lymphocyte adhesion to mucosal HEV‘ (3). The

*This research was supported in part by National Institutes of Health Grant CA42551 (to I. L. W.) and Genentech, Inc. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

ence should be addressed: B259, Beckman Center for Molecular & Ge- $ Fellow of the Leukemia Society of America. To whom correspond-

netic Medicine, Stanford University Medical Center, Stanford, CA 94305. Fax: 415-723-4034.

The abbreviations used are: HEV, high endothelial venule(s); PCR,

bis-(Z-~arboxyethy1)-5 (and -6) carboxyfluorescein, acetoxymethyl ester; polymerase chain reaction; R A R , relative adhesion ratio; BCECF, 2’,7‘-

PMA, phorbol 12-myristate 13-acetate; mAb, monoclonal antibody;

murine a, (4) and p7 (5, 6) cDNAs were isolated and their expression in lymphoid cell lines conferred the ability to bind Peyer’s patch HEV in vitro (5). The ligand on HEV for this receptor is the mucosal addressin MAdCAM-1 (7, 8). Two additional ligands for a,P7, VCAM-1 and CS-1 region of fibronectin (9, lo), are also shared with the a4P1 integrin. It is possible that a,& will share many of the functional properties of a,& including the ability to mediate lymphocyte transendothelial migration (111, transmembrane signaling (12, 131, and leukocyte trafficking to sites of inflammation (14).

Many of the fundamental properties of integrins, e.g. activation by inside-out signaling, binding their cognate ligands, and association with the cytoskeleton, are in part regulated by the cytoplasmic domain of the p subunit. In general, these domains are relatively short (-40-60 amino acids), share a high degree of sequence similarity, and lack intrinsic enzymatic activity (1, 15). Initially they were shown to co-localize with focal adhesions, the membrane attachment site for the actin-based cytoskeleton (16). Such complexes also include microfilament- associated proteins such as talin, vinculin, and a-actinin (171, as well as protein-tyrosine kinases, e.g. ~ ~ 1 2 5 ~ ~ (18). p subunit cytoplasmic domains are also substrates for cellular kinases and may be phosphorylated following retroviral transfor- mation (19), phorbol ester treatment (20), and cross-linking of cell-surface proteins (21, 22).

More recently, these domains have been studied by analyzing the effects of receptor deletions and chimeras on ligand binding and integrin association in focal adhesions. Truncation of the entire p1 (23-25) or p3 (26) cytoplasmic domain prevents integrin association with focal adhesions in adherent cell lines. Replacement of the p1 transmembrane and cytoplasmic domain with the corresponding regions of p3 results in the restoration of focal contact formation (27). Reszka et a2. (28) subsequently identified three regions in the p1 cytoplasmic domain that con- tribute to integrin localization in focal adhesions. One region (cyto-1) is adjacent to the plasma membrane and is highly conserved among the PI, pz, p3, p5, &, and p7 cytoplasmic domains. Since peptides from this region in p1 and & bind a-actinin in vitro (17, 29), these p subunit residues may represent at least one site critical for the linkage of microfilaments to receptors in the plasma membrane. Interaction between integrins and the cytoskeleton are thought to be necessary for the transduction of force required for cell migration, clot retraction, and enhanced adhesion.

Truncation of integrin p subunit cytoplasmic domains has resulted in the loss of ligand binding activity or has had no im- mediately obvious effect. Expression of a truncated avian p1 cytoplasmic domain in 3T3 cells results in the formation of murine a-avian p1 hybrids that display a reduced ability to mediate cell

FITC, fluorescein isothiocyanate; HBSS, Hanks’ buffered saline solu- tion; BSA, bovine serum albumin; PAGE, polyacrylamide gel electro- phoresis; PBS, phosphate-buffered saline.

14411

14412 The p7 Cytoplasmic Domain Regulates a4P7 Avidity

adhesion to fibronectin (25). Comparably truncated hybrid p1 integrins, when solubilized in the presence of Mn2+, could, however, bind fibronectin (23), perhaps due to the activating effects of Mn2+ ions on the ligand binding activity of integrins (30). De- letion of the entire & cytoplasmic domain also results in the loss of adhesiveness of LFA-1 for ICA"1 (31). Removal of the p3 cytoplasmic domain had no apparent effect on the ability of arm& to bind soluble fibrinogen following receptor activation (26,32).

To understand the contribution of the p7 cytoplasmic domain in regulating a$, function, we made partial (11753) and complete truncations (6727) in the p, cytoplasmic domain and expressed wild-type and mutant & subunits in the murine B cell lymphoma 38C13. The ability of wild-type and truncated a$, receptors to support cell adhesion to Peyer's patch HEV, fibronectin, and both soluble and immobilized VCA"1 was tested. We show that the avidity of a& is regulated by the p7 cytoplasmic domain; unlike the PI, pz, and p3 integrins, complete removal of the p7 cytoplasmic domain results in an apparent increase in receptor function. The data support a model whereby receptor avidity is in part determined by both positive and negative regulatory elements in the p, cytoplasmic domain.

MATERIALS AND METHODS Cell Culture-The murine B cell lymphoma 38'213 (33) was main-

tained in RPMI 1640 media supplemented with 10% fetal calf serum and 5 x M 2-mercaptoethanol. The retroviral packaging cell line PA317 (34) was maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 5 x M 2-mercaptoethanol. Selection for 38'213 cells infected with recombinant retroviruses was done in medium containing 1 mg/ml G418.

Antibodies-The following ra t monoclonal antibodies (mAbs) were utilized: anti-a,, R1-2 (2) and PS/2 (35); anti-p,, M293 (36); anti-a,, M17/4.3 (from Dr. T. Springer, Harvard Medical School, Boston, MA); anti-CD44, IM781(37); anti-L-selectin, Mel-14 (38); anti-idiotype, R7D4 (39); anti-CD4, GK1.5 (40); and anti-Fc-y receptor, 2.4G2 (41). All mAbs were affinity-purified on protein A- or G-Sepharose before use (42). Polyclonal antiserum to human fibronectin (Calbiochem, San Diego, CA), murine VCAM (provided by M. Renz, Genentech, Inc.), or p, (5) was obtained from the serum of immunized rabbits. FITC-labeled goat anti-rat antibody was obtained from Caltag Laboratories (South San Francisco, CA). The FITC-labeled murine IgGl monoclonal antibody was obtained from AMAC, Inc. (Westbrook, ME). The V C M g G I chimera was labeled with a QuickTag FITC conjugation kit from Boeh- ringer Mannheim.

Mutagenesis, Subcloning, and Expression of the p7 cDNA-The sequence and characterization of the murine p7 cDNA has been previously described (5). In-frame stop codons were introduced into the 6, cytoplasmic domain at amino acid 727 or 753 by a polymerase chain reaction (PCR)-based mutagenesis strategy. The antisense primers (A753

CCCGGTCGACTCACCGGTAAGCCAGAACCAGCCAWC-3') contained sequences complementary to p,, an in-frame stop codon, and a Sal1 re- striction site. The sense primer (5"GGCGAATTCGAGAAGGGC- TGCTCCTCC-3') and appropriate antisense primer were used in PCR reactions (43) with the p7 cDNA as template. The -2.6-kilobase PCR product was subcloned into the retroviral expression vector pIXSN (44) using conventional methods (43). A753 and A727 cDNAs were sequenced to confirm the fidelity of the p7 sequence and the presence of in-frame stop codons. All DNA sequencing and primer synthesis was done by the Protein and Nucleic Acid Facility at Stanford University. Four 38C13 cell lines (referred to as p7, A753, A727, and LXSN throughout this paper) were generated by retroviral infection of 38C13 and selection with G418 as previously described (5).

Cell Sorting and Flow Cytometry-All flow cytometric analysis or sorting was carried out at the FACS Shared Users Group facility a t Stanford University using a FACStar Plus cytometer for cell sorting and a FACScan cytometer for analysis (Becton-Dickinson, Mountain View, CA). To obtain 38C13 cell lines expressing equivalent levels of either wild-type or mutant p, subunits, p7, A753, and A727 cell lines were subjected to two to three rounds of sorting after staining with M293 and a goat anti-rat FITC-labeled antibody. The fluorescein gates were ad- justed to collect cells of relatively equivalent intensities and, thus, densities of the a$, integrin. Flow cytometric analysis of various leukocyte adhesion molecules was carried out on cells stained with the appropri-

5'-CCCGGTCGACTCAGTCCTGCTTCCAGT"I'GAGT'TG-3'; A727 5'-

ate rat monoclonal antibody, followed by incubation with a goat anti-rat FITC-labeled antibody. Cells (IO4) from each sample were analyzed.

binding to soluble VCAM, -lo6 cells were first incubated with the TO determine the effect of various divalent cations on a,P,-dependent

anti-Fc-y receptor monoclonal antibody 2.4G2 to prevent capture of the FITC-labeled probes. Unbound antibody was removed by centrifugation of the cells through a bed of fetal calf serum. Cells were then resuspended in Ca2+- and Mg2"free HBSS containing 10 mM EDTA and 5 mdml BSA, for 5 min on ice. Cells were pelleted and resuspended in HBSS containing 5 mdml BSA and either 2 m~ CaCl,, 2 mM MgCl,, or 0.5 mM MnCl, as indicated in Fig. 8. Saturating amounts of either FITC-labeled VCAM/IgGl or FITC-labeled control murine IgGl were then added to the cells for 30 min on ice. Unbound ligand was removed by washing with HBSS containing 5 mg/ml BSA and the same divalent cation present in the binding reaction. Cells (lo4) were analyzed for the presence of bound ligand by flow cytometry.

Immunoprecipitation and Western Blot Analysis-For immunoprecipitation, cells were surface-labeled with lZ5I using the glucose oxidase- lactoperoxidase method (45), lysed in immunoprecipitation buffer (1% Triton X-100,50 mM Tris (pH 7.4), 150 mM NaCl, 2 mM CaCl,, and 2 mM MgCl,), precleared, and precipitated with either anti-a, (R1-2) or anti-p, (M293) monoclonal antibodies as previously described (2). Immunopre- cipitates were resolved under reducing conditions by SDS-PAGE on a 8% gel. For Western analysis, -lo6 cells were lysed in SDS-PAGE sample buffer (42) and boiled for 5 min. Lysates were resolved under reducing conditions by SDS-PAGE on a 6% gel, and transferred to nitrocellulose by electroblotting (42). After blocking with PBS containing 5% BSA, the filter was probed with a 1:5,000 dilution of anti-p, rabbit antiserum (5) followed by incubation with a 1:20,000 dilution of protein A-horseradish peroxidase (Sigma). Detection of immune complexes was performed with an ECL detection system (Amersham Corp.)

In Vitro Binding Assays-Cell adhesion assays to fibronectin and VCAM were performed as previously described (46). Human plasma fibronectin was obtained from Calbiochem (San Diego, CA). The recombinant VCAM used in these binding assays is a chimeric molecule containing the extracellular seven-domain form of murine VCAM joined to the CH2-C,3 region of murine IgG (kindly provided by M. Renz, Genentech, Inc.). The amount of ligand added to each well was previously determined by titration experiments to result in the saturable binding of 2 x lo5 cells; generally this was 1 pg of purified liganawell in a 96-well polystyrene plate. All wells were subsequently blocked with 5 mg/ml BSA in RPMI 1640.

38C13 cells were labeled with the fluorescent dye 2',7'-bis-(2-car- boxyethyl)-5 (and -6) carboxyiluorescein, acetoxpethyl ester (BCECF) (Molecular Probes, Inc., Eugene, OR) as described (46). Each binding reaction contained 2 x lo5 labeled cells, antibodies as indicated, in 0.1 ml of RPMI 1640 containing 5 mg/ml BSA. After 30 min at 37 "C, unbound cells were removed by aspiration, brief washing, and centrifugation of an inverted plate for 5 min at 200 x g. Adherent cells were lysed with 0.1% SDS in 50 mM Tris (pH 8.51, and the fluorescence was measured a t 485/535 nm in a Pandex fluorescence concentration ana- lyzer (Pandex, Mundelein, IL). Percent binding is equal to: (fluorescence measurement of adherent cells + fluorescence measurement of 2 x lo5 input cells) x 100, and is presented as the mean and standard deviation of triplicate wells.

In vitro HEV binding assays were performed as described (47) on freshly cut frozen sections of Peyer's patches isolated from C3H mice. Briefly, 38C13 cells were incubated with saturating amounts of either an anti-a, (Rl-2) or an isotype-matched, binding control monoclonal antibody (R7D4). Unbound antibody was removed, and -lo6 of both 38C13 cells and mesenteric lymph node lymphocytes were added to each cryostat section. Following incubation a t 4 "C for 30 min on a rotating platform, the cells were decanted and fmed overnight in 1.25 X

PBS containing 1% glutaraldehyde. The number of adherent 38C13 cells and mesenteric lymph node lymphocytes was determined for 30 HEV for each sample. The relative adherence ratios ( w s ) were cal- culated as described (47) and are presented as the mean of three independent assays.

RESULTS

Expression of Duncated p7 Subunits-To produce p7 subunits with truncated cytoplasmic domains, we replaced amino acids 727 and 753 with stop codons (Fig. lA) using a PCR-based mutagenesis strategy. Full-length PCR products were subcloned into the retroviral expression vector pLXSN (441, and the A753 and A727 cDNAs were sequenced to verify the loca-

The

FIG. 1. Sequence comparison and location of p, cytoplasmic domain truncations. A, the single-letter code

domain is shown beginning with the pu- amino acid sequence of the p7 cytoplasmic

tative first residue of the cytoplasmic domain. The asterisk represents the location of the in-frame stop codon. B, alignment of the putative cytoplasmic domains of

(721, human p3 (731, human ps (741, and murine p7 (51, murine p1 (i'l), murine pz

human p6 (75). A 1-amino acid gap was inserted into pz to improve its alignment with the other p subunits. The boxed regions represent identical residues present in at least four of six of the p subunits. Location of proximal and distal regions are underlined.

p7 Cytoplasmic Domain Regulates a4p7 Avidity 14413

A. 07 RLSVEIYDRREYRRFEKEQQQLNWKQDNNPLYKSAITTTVNPRFQGTNGRSPSLSLTREAD*

A753 RLSVEIYDRREYRRFEKEQQQLNWKQD*

A727 R*

tion of the introduced stop codon. Three 38C13 stable cell lines expressing wild-type and mutant p, subunits were then generated by retroviral infection and selection with G418 as previously described (5). As a control, 38C13 was also infected with retroviruses carrying the pLXSN vector alone. These four cell lines will be referred to throughout this paper as p7, A753, A727, and LXSN. To obtain cell lines with identical cell-surface densities of wild-type and mutant a,& receptors, p,-expressing cell lines were sorted by flow cytometry after staining with the anti+, mAb M293. p,, A753, A727, and LXSN are not clonal in origin, but are populations of independent transfectants. These cell lines were then analyzed by flow cytometry for the surface expression of several known leukocyte adhesion molecules (Fig. 2). All four 38C13 cell lines express nearly identical levels of a,, p7 (except LXSN), aL, L-selectin, and CD44.

These cell lines were then biochemically characterized by cell-surface labeling with lZ5I, and immunoprecipitation in the presence of divalent cations with either anti-a, or anti-p, mAbs (Fig. 3.4 1. The - 160-kDa a, subunit and the associated p7 subunit (in &, A753, and A727) were immunoprecipitated using the anti-a, mAb R1-2. Integrin subunits are thought to be expressed on the cell surface only as cr/P heterodimers (1). How- ever, these immunoprecipitation experiments suggest that a, can be expressed on the cell surface as a monomeric subunit in 38C13/LXSN. Alternately, a, may dimerize with an endogenous /3 subunit but this association may be labile under these lysis conditions. Immunoprecipitation with the anti-p, mAb M293 also recovers a,& heterodimers except for the p7- cell line LXSN. These results clearly show that the p7 cytoplasmic domain is not essential for either cell-surface expression or heterodimer formation of the a4P7 integrin.

Since subtle size differences between p,, A753, and A727 were not clearly detected when immunoprecipitates were resolved by 8% SDS-PAGE, we re-analyzed the various & subunits by Western analysis on a 6% polyacrylamide gel. The anti-p, antiserum used in these experiments was generated by immunizing rabbits with a &-glutathione S-transferase fusion protein expressed in Escherichia coli (5). This antiserum preferentially recognizes the non-glycosylated, cytoplasmic form of p7 over the glycosylated, cell-surface form of p7. Western analysis of whole cell lysates clearly resolves the size differences between the wild-type and truncated p7 subunits (Fig. 3B). The basis for the elevated intracellular levels of A727 is not under- stood. The slowest migrating band present in all four lanes is an artifact also obtained when identical blots are probed with the second-stage reagent protein A-peroxidase alone.

p7 Cytoplasmic Domain Duncations Affect Peyer's Patch HEV Binding in Vitro-We previously demonstrated that ex-

Proximal Region Distal Region

pression of p7 in the cell line 38C13 confers the ability to bind Peyer's patch HEV in vitro (5). We next determined the effect of p7 cytoplasmic domain truncations on 38C13 transfectant binding to Peyer's patch HEV. All binding assays to cryostat sections of Peyer's patches included both the 38C13 transfectants, as well as mesenteric lymph node lymphocytes, which served as an internal positive control. To ensure binding was a,P,-dependent, the 38C13 cells were preincubated with saturating amounts of either the mAb R1-2 (anti-a,) or a binding, isotype- matched control mAb R7D4. The number and type of cells bound to at least 30 HEV for each assay were scored under dark-field illumination. The RAR reflects the binding efficiency of the 38C13 transfectants compared to normal lymphocytes; normal lymphocytes have an RAR of 1.

38C13 cells expressing wild-type a4P7 bound Peyer's patch HEV in vitro with efficiencies similar to normal lymphocytes, whereas the p7- LXSN cell line displayed only background binding efficiency (Fig. 4). Partial truncation of the & cytoplasmic domain (8753) reduced binding approximately %fold, whereas cells expressing a,& receptors lacking the entire p7 cytoplasmic domain (A727) bound Peyer's patch HEV more efficiently than either normal lymphocytes or 38C13 cells expressing the wild- type p, subunit. Binding of the 38C13 transfectants to Peyer's patch HEV is clearly a,& dependent since p; cell lines fail to bind, and nearly all binding is blocked by the anti-a, mAb R1-2.

Effect of Phorbol Ester Deatment on a$,-dependent Binding to Fibronectin and VCAM-1-The a4&+ cell lines TK-1 and JY were shown to bind to the CS-1 domain of fibronectin and to human VCA"1, if first activated with phorbol esters (9, 10). However, in these experiments, the direct binding contribution by a,p7 is difficult to determine because of the lack of receptor- deficient control cell lines, or anti-& blocking antibodies. To di- rectly determine the contribution of a,& to cell adhesion to these ligands, we measured the in vitro binding of the 38C13 cell lines p7 or LXSN to fibronectin- and VCAM-coated wells. In contrast to prior reports (9, lo), we detected significant a,&-dependent binding to fibronectin and VCA"1 in the absence of PMA activation (Fig. 5). PMA treatment resulted in, at most, a 2-fold enhancement in a,&-dependent binding to fibronectin and had a negligible effect on a,P,-dependent binding to VCAM. These results suggest that the ligand binding activity of a4p7 can be inducible or constitutive, depending upon the particular cellular environment. All subsequent cell adhesion experiments described herein were performed without PMA activation.

Effect of p7 Cytoplasmic Domain Duncations on Binding to Fibronectin-We next determined the effect of the p7 cytoplasmic domain mutations on a,&dependent adhesion to fibronectin (Fig. 6). Expression of the wild-type p7 integrin increased

14414

38C1 3/p7

38C131A753

38C131A727

38C131LXSN

The p7 Cytoplasmic Domain Regulates a$, Avidity

a a4 LFA-1 L-selectin

1 10 loo 1 10 loo 1 10 l o o 1 10 IO

FLUORESCENT INTENSITY

CD44

. . 1 10 100

FIG. 2. Analysis of adhesion molecule expression on the 38C13 transfectants by flow cytometry. The p,, 8753, A727, and LXSN cell lines were incubated for 30 min on ice with the following rat monoclonal antibodies: anti-a, (R1-2), anti-p, (M293), anti-LFA-1 (M17/4.3), anti-L-selectin (Mel-14), anti-CD44 (IM781), or an IgG2b control, GK1.5 (anti-CD4). All cells were then stained with a goat anti-rat FITC-labeled antibody and analyzed by flow cytometry. Shaded histograms are the staining profiles obtained with antibodies to adhesion molecules, while the open histograms are the staining profiles of cells stained with the negative control antibody. Mean fluorescence intensity (log scale) is shown on the z axis.

fibronectin binding activity -2-fold above background levels. Partial truncation of the p7 subunit (A753) reduced a4p7-dependent adhesion to fibronectin to background levels, while A727 bound -5-fold more efficiently than 38C13 cells expressing wild-type a4p7. Preincubation of the substrate with anti- fibronectin antibodies completely blocked cell adhesion in all cases. a$,-dependent binding to fibronectin was blocked by the anti-a, mAbs PSI2 and R1-2, as has been previously described (lo), but not by the anti-CD44 mAb IM781. These results clearly demonstrate that expression of a4p7 confers the ability to bind fibronectin, and this binding activity can be modulated by the p7 cytoplasmic domain.

Effect of p7 Cytoplasmic Domain Thncations on Binding to VCAM-The third known ligand for a,& is VCAM-1. We tested the binding activity of the 38C13 cell lines to a recombinant fusion protein containing the extracellular 7-domain form of murine VCAM-1 fused to the CH2-C,3 region of mouse IgG1. We found no difference in the binding properties of this VCAM- l&Gl chimera compared to recombinant murine VCAM-1 containing only the extracellular domain (data not shown).

Expression of p7 in 38C13 confers the ability to bind to immobilized VCAM (Fig. 7). This high level of binding requires the p7 C-terminal amino acids 753-801 since their removal in A753 abolishes binding. However, complete removal of the p7 cytoplasmic domain results in an a4P7 receptor that binds VCAM with wild-type activity. Cell binding to VCAM was specifically and completely blocked by either anti-VCAM antiserum or the anti-a, mAb PSl2, demonstrating that all binding in these assays was completely VCAM- and a,p,-dependent. Although the

anti-a, mAb R1-2 blocked the a,p,-dependent binding to Peyer's patch HEV and fibronectin in vitro, i t failed to block the CY$,- dependent binding to VCAM as previously described (10). The inability of R1-2 to block a,P,-VCAM interactions is possibly due to a higher affinity of murine a,p7 for murine VCAM (used in these assays) versus human VCAM (10).

Activation of agp7 Binding to Soluble VCAM by Manganese Ions-The ligand binding properties of integrins are divalent cation-dependent (1). Different divalent cations may have either inhibitory or activating effects on integrins (30, 48, 49), perhaps via their interaction with the putative cation binding sites in the extracellular domain of the a subunit. To determine the role of specific divalent cations in a$,-dependent binding to VCAM and to test whether the conformation of A753 is permanently disrupted by the truncation, we incubated the 38C13 cell lines with various divalent cations in binding assays with soluble VCAM.

The ligand binding activity of a4P7 in the presence of various divalent cations was measured by flow cytometry, using a FITC-labeled, bivalent VCAM/IgGl chimera or a murine IgGl control mAb as probes. Flow cytometry was used to obtain a better measure of the affinity of receptor-ligand interactions, as opposed to cell binding assays that reflect multivalent interactions. In these experiments, resident divalent cations bound by a$, were first removed by treatment with EDTA, then replaced by buffers containing known concentrations of Ca2+, M e , or Mn2+. Each cell line was then incubated with saturating amounts of either FITC-labeled VCAM/IgGl or an IgGl

a4

P7

The p, Cytoplasmic Domain Regulates a4P7 Avidity

A B +M293 +R 1-2

-. .I, " . "" .- - .. -200 kD

p7 [ -97 kD

FIG. 3. Analysis of a4p, expression in 38C13 transfectants by immunoprecipitation and Western analysis. A, 38C13 transfectants were surface-labeled with ""I and lysed in immunoprecipitation buffer containing 1 mM CaCI, and 1 mM MgCI,. Cell extracts were then precleared and incubated with either anti-a, (R1-2) or anti-p, (M293) monoclonal antibodies. Immune complexes were collected by incubation with protein A-Sepharose beads saturated with goat anti-rat antibody and resolved by SDS-PAGE under reducing conditions. B, whole-cell lysates were prepared from -10' cells of each of the 38C13 transfectants by boiling in lysis buffer. Denatured proteins were then resolved by SDS-PAGE under reducing conditions, and transferred to nitrocellulose by electroblotting. The filter was then probed with rabbit anti-p, antisera, followed by incubation with protein A-horseradish peroxidase. Immune complexes on the filter were detected by chemiluminescence and autoradiography.

control mAb and analyzed by flow cytometry for the presence of bound ligand.

As expected, none of the 38C13 cell lines tested bound V C M IgGl in the absence of divalent cations (Fig. 8). Although both p7 and A727 cell lines adhered equally well t o immobilized V C M g G 1 (Fig. 71, only A727 bound soluble VCM/IgG in the presence of 2 mM CaZ+. The presence of 2 mM Mg2' alone did not support the a,P,-dependent binding of soluble V C M g G 1 , a n d the binding profiles in the presence of both Ca2+ and Mg2' (or RPMI 1640 + 5% BSA) were identical to that seen with Ca2+ alone. As has been observed with other integrins, the ligand binding activity of &, A753, and A727 was also enhanced by Mn2+. Interestingly, A727 displays an intermediate level of staining in the presence of Ca2+ that is not further enhanced by Mn2+ treatment. The addition of higher concentrations of Mn2+, although cytotoxic, led to an even greater shift in the mean fluorescence intensity for these three cell lines. The effect of Mn2+ was clearly a,P,-dependent, since LXSN failed to bind V C M IgGl even after treatment with Mn2+. These results demonstrate that the conformation of A753 is not permanently disrupted by removing the distal portion ofits cytoplasmic domain, since A753 retains ligand binding activity when activated by Mn2+. Furthermore, these results suggest that a4p7 receptors lacking the p7 cytoplasmic domain may have significantly greater affinity for VCAM (and perhaps other ligands) than wild-type receptors. The inability of p7 to bind a soluble ligand, but not one that is immobilized, will be discussed.

DISCUSSION

Through a diverse array of molecules, cells have developed the capacity to adhere to soluble, insoluble, or cellular ligands. Members of the Ig superfamily, integrin, selectin, and cadherin family of receptors mediate many of these events, although integrins are currently the only class of adhesion receptors known to modulate their ligand specificity and affinity and to transduce signals in response to ligand binding. These three characteristics of integrins are mediated in part by that portion of the receptor residing in the cytoplasmic compartment. Here we functionally characterize a4p7 receptors having partial or complete truncations of the p7 cytoplasmic domain. The results

14415

+ R7D4 + R1-2

patch KEV binding in uitro. 38C13 transfectants were incubated FIG. 4. Effect of /3, cytoplasmic domain truncations on Peyer's

with saturating amounts of either anti-a, (Rl-2) or anti-idiotypic (R7D4) monoclonal antibody on ice for 30 min. Excess antibody was removed and an equal number of mesenteric lymph node lymphocytes was added to each sample of 38C13 cells. Approximately 2 x lo5 cells were overlaid on freshly cut frozen sections of Peyer's patches and incubated a t 4 "C on a gently rotating platform for 30 min. Unbound cells were decanted and the slides fixed in 1.25 x PBS containing 1% glutaraldehyde. The type and number of cells bound to 30 HEV were scored for each sample and the RAR of three independent experiments determined. 0, LXSN; W, p,; 0, A753; El, A727.

64 FN

LXSN P7

VCAM

- LXSN I P7

fibronectin and VCAM. p7 or LXSN cell lines were labeled with FIG. 5. Effect of phorbol esters on @,-dependent binding to

BCECF and added to 96-well plates coated with BSA, fibronectin (1.0 pg/ml), or the VCAMAgG chimera (1.0 pg/well) as described under "Ma- terials and Methods." Where indicated, PMAwas present in the binding reaction a t a concentration of 50 ng/ml. Adherence was measured by fluorometry and is expressed as the mean percent of cells binding from triplicate wells. 0, - PMA, m, + PMA.

suggest that the avidity of a4P7 can be regulated by the 0, cytoplasmic domain.

The two truncations subdivide the p7 cytoplasmic domain into two segments: a proximal region and distal region, separated by the highly conserved NPLY motif (Fig. 1B). This motif is relatively equidistant from the transmembrane domains in PI, pZ, p3, ps, ps, and p7 but is absent from p4 and PR (50, 51). NPLY is similar to the NPXY motif, which is used as an inter- nalization signal for endocytosis (52). Comparison of the NPXY tetrapeptide to known three-dimensional protein structures in the Brookhaven Protein Data Bank suggests that this motif also encodes a tight turn (53). The NPIY sequence in the p, cytoplasmic domain is also a substrate for tyrosine phosphory- lation in vivo (54) and is important for integrin localization in focal adhesions (28). Reszka et al. (28) proposed that the NPJY motif permits the p1 cytoplasmic domain to adopt a folded conformation that juxtaposes the proximal and distal regions.

14416 The p7 Cytoplasmic Domain Regulates a4p7 Avidity

50

4 0

- P I

30

20

10

0

the anti-Fc-y receptor mAb 2.4G2 to prevent the nonspecific capture of cells to wells containing rabbit immunoglobulin. Cells were then added to FIG. 6. Effect of p7 cytoplasmic domain truncations on cell adhesion to fibronectin. BCECF-labeled cells were incubated for 30 min with

96-well plates coated with BSAor fibronectin (1 pg/well). Where indicated, rabbit antisera (1:200 dilution) or rat mAbs (1 or 10 pg/ml) were present during the binding reaction. Cells were allowed to adhere for 30 min at 37 "C. Nonadherent cells were removed by centrifugation of the inverted plate. Adherence was measured by fluorometry and is expressed as the mean percent of cells binding from triplicate wells. 0, LXSN; H, p,; 0, A753; a, 8727.

100

FIG. 7. Effect of & cytoplasmic domain truncations on cell adhesion to VCAM-1. BCECF-labeled cells were incubated for 30 min with the anti-Fc-y receptor mAb 2.4G2 to prevent the nonspecific capture of cells to wells containing rabbit immunoglobulin. Cells were then added to 96-well plates coated with BSA or VCAM/IgG (1 pg/well). Where indicated, rabbit antisera (1:lOO dilution) or rat mAbs (1 or 10 pg/ml) were present during the binding reaction. Cells were allowed to adhere for 30 min at 37 "C. Nonadher- ent cells were removed by centrifugation of the inverted plate. Adherence was measured by fluorometry and is expressed as the mean percent of cells bind-

0, A753; @, A727. ing from triplicate wells. 0, LXSN W, p7;

80 I F 60

C 5 i5 s 4 0

2 0

0 B S A m V C A M m V C A M m V C A M m V C A M m V C A M m V C A M m V C A M

+ + + + + a - V C A M Pre-Immune PS/2 Rl-2 R1-2 (I:IOO) (1:lOO) (tug/rnl) (10ug/rnl) (lug/ml) (luglml)

IM781

These results show that removal of the p7 cytoplasmic tail does not impair this subunit's ability to be expressed on the cell surface or to form heterodimers with a& In general, eliminating or replacing integrin cytoplasmic domains does not disrupt receptor localization or heterodimer formation (23, 25). These functions apparently reside in the extracellular and transmembrane region of the receptor. In fact, complete removal of both a, and p7 transmembrane and cytoplasmic domains results in the production of a secreted heterodimer.'

Elimination of the distal region of p7 (A753) results in the complete loss of a,&-dependent ligand binding activity. This loss of ligand binding activity is probably caused by the elimination of an essential regulatory or structural component and is not due to a nonspecific, generalized distortion of receptor conformation. This hypothesis is supported by three grounds: (i) a,& receptors on p7, A753, and A727 cells are all equiva- lently recognized by two anti-a, and four anti-& monoclonal antibodies (data not shown); (ii) the ligand binding activity of A753 can be restored by exogenous activation with Mn2+; and (iii) the cell-surface localization and ability to form heterodimers is unimpaired. It is possible that subtle alterations in the conformation of the receptor could be present in A753 and A727 that would go undetected by such criteria, yet have

dramatic effects on ligand affinity or specificity. The data presented here suggest that the distal region of p7

is required for the ligand binding activity of a4p7 and this region has a positive regulatory function. Sequences downstream of the NPLY motif may also function as positive regulatory elements in PI, pz, and p3 integrins as well. Mutations in this region impaired the ICAM-1 binding activity and phorbol ester activation of pz (20, 311, prevented p1 association with the cytoskeleton (24, 25), and blocked the activation of aIl&3s (55) . Because A753 removes 34 amino acids from the C terminus, it is difficult at this time to identify specific residues critical for the function of p7. The last 12 amino acids of p7 contain two sites, RSPS and SLTR, that are similar to the consensus phos- phorylation sites of calmodulin kinase I1 or CAMP-dependent protein kinase (R-X,-S/T), and protein kinase C (Sm-Xw, R/K_,) (56) . In addition, pl, pz, ps, and p7 share a conserved KSAXTT motif shown to be essential for the ICA"1 binding activity of LFA-1 (20).

Complete removal of the p7 cytoplasmic domain results in the expression of an a,& receptor that is constitutively active and has high avidity for all known ligands. These results are novel in that complete truncation of the p1 and pZ cytoplasmic domains generally results in a loss of ligand binding activity. The apparent acquisition of function seen with A727 us. A753 suggests that the proximal region of p7 may function in a domi- D. Crowe, unpublished results.

The p7 Cytoplasmic Domain Regulates aqp7 Avidity

+10 mM EDTA +2 mM Ca*+ +2 mM Mg2+ +2 mM CalMg2+ +0.5 mM Mn2+

14417

38C13107

38C131A753

38C1316727

38C13lLXSN

i 10 I W . .

1 IO 1w I 10 1W I IO I W i IO 1w

FLUORESCENT INTENSITY FIG. 8. Effect of divalent cations on binding of soluble FITC-labeled VCAM/IgG. p,, A753, A727, and LXSN cell lines were incubated for

30 min on ice with the anti-Fc-y receptor antibody 2.4G2 to prevent to the nonspecific capture of immunoglobulin. Cells were then incubated with Ca2+- and Mg2"free HBSS containing 10 mM EDTA and 5 mg/ml BSA for 5 min on ice. Cells were pelleted and resuspended in HBSS containing 5 mg/ml BSA and either 2 mM CaCI,, 2 mM MgCI,, or 0.5 mM MnCI, as indicated. Saturating amounts of FITC-labeled VCAMngGl or FITC-labeled control murine IgGl were then added to the cells for a 30-min incubation on ice. Unbound ligand was removed by washing with HBSS containing 5 mg/ml BSA and the same divalent cation present in the binding reaction. Cells (-104/sample) were analyzed for the presence of bound ligand by flow-cytometry.

nant, negative regulatory capacity. A negative regulatory activity has also been ascribed to the proximal region of p2 (31). Internal deletions eliminating the entire proximal region and the NPXY motif did not disrupt the constitutive ICAM-1 binding activity of LFA-1 expressed in COS cells. The net avidity a receptor displays may reflect a summation of the activity of both positive and negative regulatory regions within the p subunit cytoplasmic domain. The affinity of several leukocyte and platelet integrins (e.g. a,pl, a#*, a,pl, and a,,&) is generally low and dependent upon activation for full ligand binding activity (21, 22,49, 57). Maintenance of integrins in intrinsically low affinity states may therefore be dependent upon those sequences in the p subunit proximal region, the cytoplasmic domain of the associated a subunit (32), or both.

We propose that other integrin p subunit cytoplasmic domains are also functionally subdivided into proximal and distal regions, separated by the highly conserved NF'LY motif (Fig. 1B 1. Integrin p subunit proximal regions are relatively uniform in length (-28 amino acids), highly conserved, and are pre- dicted to have an a-helical structure when analyzed by secondary structure prediction algorithms (58). This proposal sup- ports the previous suggestions of Reszka et al. (28) that these sequences function in a generic manner, perhaps mediating integrin-cytoskeletal interactions. In contrast, the distal regions of integrin p subunits are much more heterogeneous in both length and sequence composition, which may account for the specificity or diversity of particular p chain functions. For example, the cytoplasmic domains of PI and p3 are interchange-

able with respect to ligand binding activity and cytoskeletal associations (271, but may differ in their transmembrane signaling properties (59). Additional diversity is also generated by the alternate splicing of the cytoplasmic domains of and ps (60-62). In three cases, alternate splicing occurs 2 amino acids upstream of the NPLY motif, resulting in retention of the proximal region but modification of the distal region.

Previous studies with the cell lines TK-1 (10) and JY (9) showed that a,P,-dependent binding to CS-llfibronectin and VCAM-1 required prior activation with phorbol esters. The results presented here demonstrate that a& can also be maintained in a constitutively active state, since both TK-1 (data not shown) and 38C13/P7 cells bound these ligands in the absence of phorbol ester activation. Expression of integrins in heterolo- gous cells can result in stable alterations of both the receptor's responsiveness to activation (63) and its ligand specificity (64), e.g. the ICAM-1 binding activity of LFA-1 is constitutive in transfected COS cells but PMA-responsive in lymphoid cell lines (63).

Integrin affinity can also be up-regulated in the apparent absence of transmembrane signaling by extracellular activa- tors. Divalent cations (30), activating anti-integrin mAbs (65- 67), and ligands (68) are thought to induce or stabilize confor- mational changes that mimic the inside-out signaling events mediated by integrin cytoplasmic domains. We show that inactive (A7531 and partially active (p,) forms of the a4p7 integrin display enhanced binding of soluble VCAM in the presence of Mn2+ ions. Mn2+ also strongly enhanced a,p,-dependent cell ad-

14418 The p7 Cytoplasmic Domain Regulates a4p7 Avidity

hesion to immobilized MAdCAM-1 and VCA"l(8). The inability of wild-type a4P7 to bind soluble (but not insoluble) ligands when unstimulated may be due to several potential factors, e.g. diminished receptor clustering with soluble ligands, lower ligand binding afiinity of this receptor (versus A727), or confor- mational differences between plastic-bound and soluble VCAM. That A727 was not further stimulated by treatment with Mn2+ suggests that this receptor represents a fully active a4p7 integrin. The platelet integrin a,d& can also distinguish between soluble and insoluble forms of fibrinogen; unstimulated platelets can bind to immobilized but not to soluble fibrinogen, whereas activated platelets can bind to both forms (69).

Integrins can exist in several defined states of activation with transitions between states accompanied by conforma- tional changes of the receptor (49, 70). Integrin activation can also result in the acquisition of new ligand binding properties, with the fully active receptor displaying its entire repertoire of ligand binding activity (1, 49, 64). The results presented here are consistent with A753, p7, and A727 defining at least three discrete activation states for a4& an inactive (A753), a partially active (&), and a fully active (A7271 receptor. Increases in the activation state of a4& may also result in the acquisition of additional ligands, e.g. A753 binds neither VCAM, Peyer's patch HEV, nor fibronectin; p7 is fully active for VCAM but only partially active for Peyer's patch HEV and fibronectin; A727 binds all three ligands with high avidity. These findings are analogous to those reported for a,&; this receptor exists in at least three activation states (inactive, partially active, and fully active), with the partially active receptor preferentially binding VCAM over fibronectin (49). Functional similarities between a,& and a4& are not unexpected, since these receptors share a common a4 subunit, bind two common ligands (VCAM and fibronectin), and their /3 subunits are -45% identical at the amino acid level. Future studies will be directed toward iden- tifying differences in the inward signaling properties of wild- type and mutant a4P7 receptors, as well as elucidating interactions between integrin cytoplasmic domains and intracellular components.

VCAhlL/IgGl chimera and anti-VCAM antisera. Acknowledgment-We thank Mark Renz for providing the murine

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Regulation of the avidity of integrin alpha 4 beta 7 by the beta 7