Presentation of Integrins on Leukocyte Microvilli: A Role for the Extracellular Domain in Determining Membrane Localization

M. Abi Abitorabi, Russell K. Pachynski, Ronald E. Ferrando, Mark Tidswell, and David J. Erle

The Lung Biology Center, Department of Medicine, University of California, San Francisco, California 94143

Abstract
Materials and Methods
Results
Discussion
Footnotes
Acknowledgements
Abbreviations used in this paper
References


Abstract

Adhesion of blood leukocytes to the endothelium involves multiple steps including initial attachment (tethering), rolling, and firm arrest. Presentation of adhesion molecules on leukocyte microvilli can substantially enhance tethering. Localization of L-selectin to microvilli and of CD44 to the planar cell body have been shown to depend upon their transmembrane and cytoplasmic domains. We investigated the role of leukocyte integrin transmembrane and cytoplasmic domains in initiating adhesion under flow and in microvillous localization. Integrins alpha 4beta 7, alpha Lbeta 2, and alpha Mbeta 2 were heterologously expressed in K562 cells. alpha 4beta 7 initiated adhesion under flow and localized to microvilli, whereas beta 2 integrins did not initiate adhesion and localized to the cell body. Chimeric integrins were produced by replacing the alpha 4beta 7 cytoplasmic and/or transmembrane domains with the homologous domains of alpha Lbeta 2 or alpha Mbeta 2. Unexpectedly, these chimeras efficiently mediated adhesion to the alpha 4beta 7 ligand mucosal addressin cell adhesion molecule-1 under flow and localized to microvilli. Therefore, differences between the transmembrane and cytoplasmic domains of alpha 4 and beta 2 integrins do not account for differences in ability to support attachment under flow or in membrane localization. Integrins alpha 4beta 1, alpha 5beta 1, alpha 6Abeta 1, alpha vbeta 3, and alpha Ebeta 7 also localized to microvilli. Transmembrane proteins known or suspected to associate with extracellular domains of microvillous integrins, including tetraspans and CD47, were concentrated on microvilli as well. These findings suggest that interactions between the extracellular domains of integrins and associated proteins could direct the assembly of multimolecular complexes on leukocyte microvilli.


LEUKOCYTE recruitment to tissues from blood involves a series of adhesive interactions between leukocytes and the vascular endothelium (Springer, 1994; Butcher and Picker, 1996). In some cases, initial binding of leukocyte adhesion molecules to their endothelial ligands can lead to the transient arrest, or tethering, of the leukocyte followed by leukocyte rolling. Rolling cells can then be "activated" via incompletely understood mechanisms, which lead to an increase in the activity of certain adhesion molecules and the arrest of the leukocyte on the lumenal surface of the endothelium. In other cases, leukocytes may arrest immediately, without rolling. After arrest, leukocytes can extravasate into the underlying tissue. Different leukocyte adhesion molecules are used for different steps in this process (von Andrian et al., 1991). L-selectin and the E- and P-selectin ligands are expressed on some leukocytes and mediate initial adhesion (tethering and rolling), but do not support firm arrest. In contrast, the leukocyte beta 2 integrins alpha Lbeta 2 (LFA-1, CD11a/CD18) and alpha Mbeta 2 (Mac-1, CD11b/CD18) mediate firm arrest but not initial adhesion. Another integrin subfamily, the alpha 4 integrins alpha 4beta 1 (VLA-4, CD49d/CD29) and alpha 4beta 7 (LPAM-1), can support both initial adhesion and firm arrest (Sriramarao et al., 1994; Alon et al., 1995; Berlin et al., 1995).

Presentation of certain leukocyte adhesion molecules on microvilli substantially enhances the ability of these molecules to support tethering and rolling on endothelial ligands. The importance of receptor distribution was highlighted by studies of the adhesion molecules L-selectin and CD44 (von Andrian et al., 1995). L-selectin is located primarily on microvilli, whereas CD44 is concentrated on the planar cell body. L-selectin-CD44 chimeras were used to examine the role of cytoplasmic and transmembrane domains in receptor localization and the ability to roll on ligands. A chimera comprising the L-selectin extracellular domain fused to the CD44 transmembrane and cytoplasmic domains (L/CD44) localized to the cell body, and a CD44 extracellular, L-selectin transmembrane and cytoplasmic domain chimera (CD44/L) localized to microvilli. Although replacement of the transmembrane and cytoplasmic domains of L-selectin or CD44 did not alter their ability to adhere under static (no flow) conditions, it affected adhesion under flow. L-selectin (on microvilli) supported initial attachment better than the L/CD44 chimera (cell body), whereas CD44/L (microvilli) supported initial attachment better than CD44 (cell body). These results indicate that the transmembrane and/or cytoplasmic domains account for the differences in localization of L-selectin and CD44, and strongly suggest that microvillous localization is important for optimal initial adhesion under flow.

Available evidence about the distribution of integrins on the leukocyte surface is also consistent with a role for microvillous presentation in initial adhesion under flow. Integrins alpha Lbeta 2 and alpha Mbeta 2 are concentrated on the planar cell body and do not support initial adhesion, whereas alpha 4beta 1 and alpha 4beta 7 localize primarily to microvilli and do support tethering and rolling (Erlandsen et al., 1993; Berlin et al., 1995). The mechanism underlying the differential topography of these integrins on nonadherent leukocytes is not known. However, studies of other integrins have established a central role for the cytoplasmic domains of integrin beta  subunits in localization of integrins on membranes of adherent cells (LaFlamme et al., 1992, 1994; Briesewitz et al., 1993; Sastry and Horwitz, 1993; Ylanne et al., 1993; Pasqualini and Hemler, 1994). Interactions with several cytoskeletal proteins such as talin and alpha -actinin (demonstrated in vitro) are suggestive of links to microfilament fibers that may regulate protein localization. In addition, several transmembrane proteins, such as tetraspan proteins (including CD9, CD53, CD63, CD81, and CD82) (Slupsky et al., 1989; Rubinstein et al., 1994; Berditchevski et al., 1995, 1996, 1997), CD32 (Fcgamma RIIA) (Worth et al., 1996), and CD47 (integrin-associated protein) (Lindberg et al., 1993), have been shown to associate with integrins. These interactions are known or suspected to involve the extracellular domains of these proteins, and their role (if any) in integrin localization is unknown.

We sought to examine the role of leukocyte integrin transmembrane and cytoplasmic domains in microvillous localization and in initial adhesion under flow. Here we report that replacement of alpha 4beta 7 transmembrane and cytoplasmic domains with the homologous domains of beta 2 integrins does not alter membrane localization or initiation of adhesion under flow. This unexpected result suggests that differences in localization of leukocyte integrins to microvilli are determined by the extracellular domain.


Materials and Methods

Cell Lines

K562 human erythroleukemia cells (CCL 243; American Type Culture Collection, Rockville, MD) were maintained in growth medium: RPMI 1640 supplemented with 10% FBS, penicillin (50 IU/ml), streptomycin (50 µg/ml), and glutamine (2 mM). Stably transfected K562 lines expressing human integrin alpha 4 (K562-alpha 4beta 1) and alpha 4beta 7 (K562-alpha 4beta 7) were described previously (Tidswell et al., 1997). Additional K562 integrin transfectants were provided by other investigators: K562-alpha Lbeta 2 and K562-alpha Mbeta 2 (I. Graham, Washington University, St. Louis, MO) (Graham et al., 1994); K562-alpha 6Abeta 1 (A. Sonnenberg, The Netherlands Cancer Institute, Amsterdam, The Netherlands) (Hogervorst et al., 1993); K562-alpha vbeta 3 (S. Blystone, Washington University) (Blystone et al., 1994).

cDNAs

The pCDM8-integrin alpha 4 cDNA plasmid (Kamata et al., 1995) was a gift from Y. Takada (Scripps Research Institute, La Jolla, CA). The cloning of the beta 7 cDNA has been previously described (Erle et al., 1991). Integrin beta 2 cDNA (Hickstein et al., 1988) was provided by D. Hickstein (University of Washington, Seattle, WA). Integrin alpha L (Larson et al., 1989) and alpha M (Corbi et al., 1988) cDNAs were provided by T. Springer (Center for Blood Research, Boston, MA). alpha E cDNA (Shaw et al., 1994) was a gift from G. Russell and M. Brenner (Harvard Medical School, Boston, MA). Chimeric integrin subunits were constructed using splice overlap extension PCR (Horton et al., 1989). The amino acid splice sites for each construct are shown in Fig. 1. Chimeric alpha  subunits were subcloned into pCDM8 (Invitrogen, San Diego, CA) and chimeric beta  subunits were subcloned into pCEP4 (Invitrogen). The integrity of the constructs was confirmed by DNA sequencing.


Fig. 1. Schematic representation of wild-type and chimeric alpha 4beta 7 integrins. The amino acid sequence at the splice site is shown with conserved regions in bold, and the alpha Lbeta 2 or alpha Mbeta 2 sequences underlined.
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Transfections and Expression

K562 cells in log phase growth were washed twice in electroporation buffer (HBSS, 20 mM Hepes, 6 mM dextrose). 8 × 106 cells were resuspended in 0.2 ml buffer and transferred to 2 mm electroporation cuvettes (BTX, San Diego, CA). Stable transfections were performed using the pCDM8-alpha 4 and pCEP-beta 7 constructs (25 µg each) plus 5 µg pBK-neo (Stratagene, La Jolla, CA) at 900 µFarad, 200 V, 13 Omega  (Electro Cell Manipulator 600; BTX). Samples were left at room temperature for 10 min before and after electroporation. Cells were then placed in 20 ml growth medium and maintained at 37°C in a 5% CO2 incubator. After 48 h, transfectants were selected using growth medium containing 500 µg/ml each of Hygromycin B (Calbiochem-Novabiochem Corp., La Jolla, CA) and G418 (GIBCO BRL, Gaithersburg, MD). Stably transfected clones were obtained by limiting dilution and analyzed by flow cytometry as previously described (Tidswell et al., 1997). Transfectants were maintained in selection medium.

Antibodies

Fib 504 (anti-integrin beta 7) was a gift of E.C. Butcher (Stanford University, Palo Alto, CA) (Andrew et al., 1994). HP1/2 was used to detect the integrin alpha 4 subunit (Pulido et al., 1991). 7E4 (anti-beta 2) and GoH3 (anti-alpha 6) were purchased from Immunotech (Westbrook, ME). L230 (anti-alpha v) and B11G2 (anti-alpha 5) were provided by D. Sheppard and C. Damsky (University of California, San Francisco, CA). Antibodies against the integrin-associated proteins CD53 (clone HI29; PharMingen, San Diego, CA), CD63 (MAB1787; Chemicon International, Inc., Temecula, CA), and CD32 (clone IV.3; Medarex, East Annandale, NJ) were obtained from commercial sources. The anti-CD47 antibody B6H12 was a gift from E. Brown (Washington University). Hybridoma supernatants or purified IgG preparations were used for flow cytometry analysis and immunoelectron microscopy. Gold-conjugated secondary antibodies (6- or 12-nm particles) were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA).

Immunoelectron Microscopy

Transfectants were immunolabeled in suspension. Cells were prefixed for 20 min in 0.2% paraformaldehyde/PBS at 4°C. After washing twice with PBS containing 10% goat serum, cells were incubated with primary antibody diluted in PBS containing 10% goat serum. After a 30-min incubation at 4°C and a wash with PBS, gold-conjugated secondary antibody in PBS was added. Cells were incubated for 30 min at 4°C, washed twice with PBS, and fixed in 0.1 M sodium cacodylate buffer, pH 7.4, with 3% glutaraldehyde. Before embedding, cells were rinsed in 0.1 M cacodylate buffer and postfixed in 1% osmium tetroxide/0.1 M cacodylate buffer. After rinsing, cells were dehydrated through a graded series of acetone washes, and infiltrated and embedded in Spurr's epoxy resin (Ted Pella, Inc., Redding, CA). Sections (70-nm thick) were stained with uranyl acetate and lead citrate, and examined with a CM120 Phillips electron microscope (Philips Electron Optics, Inc., Mahwah, NJ). 50-100 cells were examined and representative cells were photographed. Each experiment was repeated at least twice. Colloidal gold distribution on immunolabeled cells was determined by analysis of electron micrographs (×19,500-×40,000). Gold particles associated with cell body or microvilli were counted from 3-11 micrographs that represented different individual cells.

Static Adhesion Assay

Static adhesion assays were performed as previously described (Tidswell et al., 1997). Briefly, 21-well glass slides (Structure Probe, West Chester, PA) were coated with mucosal addressin cell adhesion molecule-1 (MAdCAM-1)1-IgG fusion protein (4 ng/well) or intercellular adhesion molecule-1 (ICAM-1)-Ckappa fusion protein (0.23 µg/well) overnight at 4°C. MAdCAM-1-IgG (Tidswell et al., 1997) was a gift of M. Briskin (Leukosite Inc., Boston, MA). ICAM-1-Ckappa (Piali et al., 1995) was a gift of B. Imhof (Centre Medicale Universitaire, Geneva, Switzerland). After blocking with 4% BSA for 2 h, 4 × 104 cells were resuspended in 15 µl of 10 mM Hepes, 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, with or without 1 mM MnCl2, and added to the wells. Cells were allowed to adhere for 90 min at room temperature. After washing, cells were fixed with 3% glutaraldehyde and stained with 0.5% crystal violet. Adherent cells were counted using a microscope. Assays were performed in triplicate.

Adhesion under Flow

Capillary tubes (100-µl capacity; Drummond, Broomall, PA) were coated at 4°C overnight with 20 µl of solution containing MAdCAM-IgG (0.2 µg/ ml) or ICAM-1-Ckappa (0.7 mg/ml) and blocked with 4% BSA for 2 h at 37°C. K562 transfectants were washed with HBSS containing 1 mM EDTA, and resuspended at 5 × 105 cells/ml in HBSS/10 mM Hepes containing 1 mM each of Ca2+ and Mg2+. To measure adhesion under flow, cells were perfused through the coated capillary tube using a syringe pump (74900 series; Cole-Parmer Instrument Co., Vernon Hills, IL) at a flow rate of 0.67 ml/ min. Calculated shear stress was 1.0 dyne/cm2 according to Pousille's law of dynamic shear (Berlin et al., 1995). Results were captured using a TMS microscope (Nikon, Garden City, NJ), CCD video camera (Sony, Park Ridge, NJ), and time lapse SVMS videocassette recorder (Panasonic, Secaucus, NJ). After 2 min of flow, five randomly chosen fields from the coated region were analyzed for 5 s each. All adherent cells (rolling or arrested) were counted. Cells did not adhere to areas coated with 4% BSA alone (control).


Results

Generation of Transfectants Expressing Chimeric and Mutant alpha 4beta 7 Constructs

To examine the role of cytoplasmic and transmembrane domains of alpha 4beta 7 in adhesion and membrane localization, we expressed chimeric integrin heterodimers (Fig. 1). Each construct included the extracellular domains of alpha 4 and beta 7. In one construct, designated alpha 4beta 7(alpha Lbeta 2c), most of the cytoplasmic domains of alpha 4 and beta 7 were replaced with homologous regions of alpha L and beta 2. In a second construct, alpha 4beta 7(alpha Mbeta 2tc), all of the transmembrane and cytoplasmic domains of alpha 4 and beta 7 were replaced with homologous domains of alpha M and beta 2. The integrin alpha  and beta  subunit cDNAs were cotransfected into K562 human erythroleukemia cells, which do not normally express alpha 4, alpha L, alpha M, beta 2, or beta 7. The levels of protein expression on the transfectants K562-alpha 4beta 7, K562-alpha 4beta 7(alpha Lbeta 2c), and K562-alpha 4beta 7(alpha Mbeta 2tc) were determined to be similar by flow cytometry (Fig. 2, A-C). Two truncated cDNAs, alpha 4Delta (truncated after amino acids GFFKR) and beta 7Delta (truncated after amino acids VLAYR), were also produced. The alpha 4Delta was expressed in combination with beta 7 on transfected K562 cells (K562-alpha 4Delta beta 7), although at levels somewhat below those seen with other constructs (data not shown). We were unable to detect expression of beta 7Delta on cells cotransfected with alpha 4, despite a previous report that the homologous truncation mutant of mouse beta 7 was expressed on transfected cells (Crowe et al., 1994). We were able to document heterologous expression of other wild-type integrins, including alpha Lbeta 2, alpha Mbeta 2, alpha 6Abeta 1, alpha vbeta 3, and alpha Ebeta 7, on appropriate K562 transfectants by flow cytometry (Fig. 2, D-H).


Fig. 2. Cell surface expression of wild-type and chimeric integrins. K562-alpha 4beta 7 (A), K562-alpha 4beta 7 (alpha Lbeta 2c) (B), and K562-alpha 4beta 7 (alpha Mbeta 2tc) (C) cells were stained with the anti-beta 7 antibody, Fib 504, as shown. Each of these three transfectants was also recognized by other antibodies specific for the alpha 4 subunit or the alpha 4beta 7 heterodimer, but were not recognized by anti-alpha E antibodies (not shown). K562-alpha Lbeta 2 were recognized by antibodies to beta 2 (D) and alpha L (not shown). K562-alpha Mbeta 2 cells were recognized by antibodies to beta 2 (E) and alpha M (not shown). K562-alpha Ebeta 7 cells stained with antibodies to beta 7 (F) and alpha E, but not with anti-alpha 4 antibodies (not shown). K562-alpha 6Abeta 1 cells were stained with the anti-alpha 6 antibody GoH3 (G). There was low level expression of alpha v on nontransfected K562 cells, and higher expression on K562-alpha vbeta 3 cells as determined using the anti-alpha v antibody L230 (H). Fluorescence intensity is shown on a log scale (one log per division). Dotted and solid histograms represent staining with nontransfected and transfected K562 cells, respectively.
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Adhesion of Transfectants to MAdCAM-1 under Static Conditions

We analyzed the ability of integrin-transfected K562 cells to adhere to MAdCAM-1, an alpha 4beta 7 ligand, and ICAM-1, an alpha Lbeta 2 ligand, under static (no flow) conditions (Fig. 3). After transfection with alpha 4 cDNA alone, K562 cells express alpha 4beta 1 but not alpha 4beta 7 (Tidswell et al., 1997). These cells failed to adhere to MAdCAM-1. In contrast, cells transfected with both alpha 4 and beta 7 (K562-alpha 4beta 7) adhered efficiently to MAdCAM-1. As previously reported, adhesion was increased in the presence of Mn2+. The chimeric transfectants, K562-alpha 4beta 7(alpha Lbeta 2c) and K562-alpha 4beta 7(alpha Mbeta 2tc), also adhered to MAdCAM-1, and the extent of adhesion was very similar for chimeric and wild-type alpha 4beta 7 transfectants. As expected, K562-alpha 4beta 7 cells did not adhere to ICAM-1, whereas K562-alpha Lbeta 2 cells did.


Fig. 3. Static adhesion of transfectants to immobilized ligands. Adhesion of various integrin transfectants to MAdCAM-1 (top) and ICAM-1 (bottom) was measured in the presence and absence of Mn2+. Bars indicate SEM. nd, not determined.
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Adhesion of Transfectants to MAdCAM-1 under Flow

We next examined the ability of the wild-type and chimeric integrins to initiate adhesion under flow (Fig. 4). K562-alpha 4beta 7 cells adhered to MAdCAM-1 under flow. This adhesion was dependent upon both alpha 4beta 7 and MAdCAM-1 because K562 cells transfected with alpha 4 alone (K562-alpha 4beta 1) did not adhere to MAdCAM-1, and K562-alpha 4beta 7 cells did not adhere to capillary tubes coated with other ligands, such as ICAM-1. The chimeric integrins, alpha 4beta 7(alpha Lbeta 2c) and alpha 4beta 7 (alpha Mbeta 2tc), both supported adhesion to MAdCAM-1 under flow. Wild-type alpha 4beta 7 and the chimeric integrins were very similar in their ability to initiate adhesion under flow. We also examined the resistance to detachment from MAdCAM-1 at increasing shear stress conditions (up to 10 dynes/cm2). We did not find differences between wild-type and chimeric transfectants in this assay (data not shown). At a shear stress of 1 dyne/cm2, most cells remained adherent during the time interval of cell counts. Transfectants expressing a truncated alpha 4 subunit (K562-alpha 4Delta beta 7) also adhered to MAdCAM-1 under flow, although at a somewhat lower rate (perhaps related to lower levels of expression of this construct, data not shown). As expected from previous reports (von Andrian et al., 1991), K562-alpha Lbeta 2 cells were unable to initiate adhesion to their ligand, ICAM-1, under flow (Fig. 4).


Fig. 4. Adhesion to ligands under flow. Adhesion of various integrin transfectants to MAdCAM-1 (top) and ICAM-1 (bottom) measured at a wall shear stress of 1 dyne/cm2 in the absence of Mn2+, as described in Materials and Methods. Bars indicate SEM.
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Membrane Localization of Wild-type and Chimeric alpha 4beta 7, alpha Lbeta 2, and alpha Mbeta 2 Integrins on Transfected K562 Cells

Previous reports demonstrated that integrin alpha 4beta 7 localizes primarily to microvilli of mouse TK-1 lymphoma cells, whereas integrins alpha Lbeta 2 and alpha Mbeta 2 localize primarily to the cell body of TK-1 cells and human neutrophils respectively (Erlandsen et al., 1993; Berlin et al., 1995). We began by using immunoelectron microscopy to determine whether these integrins would localize similarly in transfected K562 cells. K562 cells had numerous microvillous projections. alpha 4beta 7 was found primarily on microvilli (Fig. 5 A). In contrast, alpha Lbeta 2 and alpha Mbeta 2 integrins were located primarily on the cell body (Fig. 5, B-D). Both alpha 4beta 7(alpha Lbeta 2c) and alpha 4beta 7(alpha Mbeta 2tc) localized to microvillous projections (Fig. 5, E and F). A quantitative analysis of the distributions of these integrins is shown in Table I. The alpha 4Delta beta 7 construct was also concentrated on microvilli (not shown). These results indicate that the transmembrane and cytoplasmic domains are not responsible for the differential localization of alpha 4beta 7 versus alpha Lbeta 2 and alpha Mbeta 2.


Fig. 5. Localization of alpha 4beta 7, alpha Lbeta 2, alpha Mbeta 2, and chimeric integrins by immunoelectron microscopy. K562 transfectants were stained for protein expression using 12-nm gold particles (arrows) as described in Materials and Methods. Wild-type alpha 4beta 7 (identified using the anti-beta 7 antibody, Fib 504) was localized predominantly to microvilli of K562-alpha 4beta 7 tranfectants (A). K562-alpha Lbeta 2 (B and C) and K562-alpha Mbeta 2 (D) were stained with antibodies to alpha L (B) or beta 2 (C and D), demonstrating that alpha Lbeta 2 and alpha Mbeta 2 were expressed mostly on the cell body. K562 cells transfected with the chimeric integrins alpha 4beta 7(alpha Lbeta 2c) (E) and alpha 4beta 7(alpha Mbeta 2tc) (F) were stained with Fib 504. These chimeric integrins were found predominantly on microvilli. Photomicrographs are representative of integrin distribution on the 50-100 cells examined in each sample. Bar, 0.5 µm.
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Table I. Integrin Distribution on Transfected K562 Cells

[View Table]

Presentation of Other Leukocyte Integrins on Microvilli

In addition to alpha 4beta 7, alpha Lbeta 2, and alpha Mbeta 2, leukocytes express other integrins that play roles in adhesion to endothelial cells, to other cells, and to extracellular matrix proteins. One of these, alpha 4beta 1, can initiate adhesion to vascular cell adhesion molecule-1 (VCAM-1) under flow and has been reported to be expressed on lymphocyte microvilli (Alon et al., 1995; Berlin et al., 1995). We confirmed that alpha 4beta 1 was also expressed preferentially on microvilli of K562-alpha 4beta 1 transfectants (Table I). We found that the T cell integrin alpha Ebeta 7 (Cepek et al., 1994), which can mediate adhesion to epithelium but has no established role in endothelial adhesion, was also localized to microvilli of K562-alpha Ebeta 7 cells (Fig. 6 A, and Table I). Integrin alpha 6Abeta 1, a laminin receptor which is expressed on monocytes and some lymphocytes, was concentrated on microvilli of K562 transfectants (Fig. 6 B, and Table I). The vitronectin receptor, integrin alpha vbeta 3, is also a receptor for the endothelial cell ligand platelet/endothelial cell adhesion molecule-1 (Piali et al., 1995) and is expressed on monocytes and other cells. We found that alpha vbeta 3 localized predominantly to microvilli (Fig. 6 C, and Table I). The fibronectin receptor, integrin alpha 5beta 1, is constitutively expressed on K562 cells and was also expressed predominantly on microvilli (not shown).


Fig. 6. Localization of alpha Ebeta 7, alpha 6Abeta 1, and alpha vbeta 3 integrins by immunoelectron microscopy. K562 transfectants were stained for protein expression using 12-nm gold particles (arrows) as described in Materials and Methods. Staining of K562-alpha Ebeta 7 with Fib 504 (anti-beta 7, A), K562-alpha 6Abeta 1 with anti-alpha 6 (GoH3, B), and K562-alpha vbeta 3 with L230 (anti-alpha v, C) revealed that each of these integrins was found primarily on microvilli. Bar, 0.5 µm.
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Localization of Transmembrane Proteins Known to Associate with Integrins

Integrins have been shown to associate with a variety of intracellular and transmembrane proteins. Some of these interactions occur within the cell and are mediated by integrin cytoplasmic domains, whereas others are known or suspected to be extracellular. Since our results suggested that extracellular (and not transmembrane or cytoplasmic) domains determine integrin localization, we performed immunoelectron microscopy to localize several cell surface proteins known or suspected to interact with integrin extracellular domains. Several members of the tetraspan family of transmembrane proteins have been shown to associate with alpha 4beta 1, alpha 4beta 7, alpha 6beta 1, and some other integrins (Berditchevski et al., 1996; Mannion et al., 1996). These associations are likely to involve the extracellular domains of tetraspans and integrins (see Discussion). Several tetraspans are constitutively expressed on K562 cells. CD53 (82 ± 5% on microvilli, Fig. 7 A), CD63 (92 ± 4% on microvilli, Fig. 7 B), and CD81 and CD82 (data not shown) are all localized predominantly to microvilli. Another transmembrane protein, CD47 (integrin-associated protein), has been shown to associate with alpha vbeta 3 via its extracellular domain. CD47, like alpha vbeta 3, was distributed mostly on microvilli (Fig. 7 C). CD32 is a transmembrane protein that has been reported to associate with alpha Mbeta 2 (which is on the cell body; Fig. 5 C) and possibly with the tetraspan CD82 (found on microvilli, see above) (Lebel-Binay et al., 1995). CD32 was expressed on both microvilli (62 ± 15%) and the cell body (38 ± 15%) (Fig. 7 D).


Fig. 7. Localization of integrin-associated proteins by immunoelectron microscopy. The tetraspan proteins CD53 (A) and CD63 (B), the alpha vbeta 3-associated protein CD47 (C), and CD32 (D) were localized by immunoelectron microscopy using 12-nm (A, C, and D) or 6-nm (B) gold particles (arrows). The tetraspan proteins and CD47 localized primarily to microvilli, whereas CD32 was found in substantial amounts on both the cell body and microvilli. Nontransfected K562 cells (A) and K562-alpha 4beta 7 (B), K562-alpha Vbeta 3 (C), and K562-alpha Mbeta 2 (D) transfectants were used for staining. Bar, 0.5 µm.
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Discussion

In this study, we examined the role of leukocyte integrin cytoplasmic and transmembrane domains in adhesion under flow and microvillous localization. We began by confirming that previously described differences in leukocyte integrin adhesive activity and membrane localization were also seen in K562 cell integrin transfectants. As expected, alpha 4beta 7 was able to initiate adhesion under flow and localized to microvilli, whereas alpha Lbeta 2 and alpha Mbeta 2 mediated adhesion only under static conditions and localized to the planar cell body. Two chimeras, alpha 4beta 7(alpha Lbeta 2c) and alpha 4beta 7(alpha Mbeta 2tc), were expressed to examine the roles of the extracellular, transmembrane, and cytoplasmic domains in adhesion and membrane localization. Both chimeras were as efficient in initiating adhesion under flow as the wild-type integrin alpha 4beta 7. The chimeras were found predominantly on microvilli, indicating that the extracellular domain (and not the transmembrane or cytoplasmic domains) determined membrane localization of alpha 4beta 7. We have so far been unable to determine whether beta 2 integrin localization to the cell body is also independent of the transmembrane and cytoplasmic domains. We attempted to address this issue by expressing a chimeric integrin composed of the extracellular domain of alpha Mbeta 2 and the transmembrane and cytoplasmic domains of alpha 4beta 7, but have not yet been successful in these experiments. In addition to alpha 4beta 7, several other integrins (alpha 4beta 1, alpha 5beta 1, alpha 6Abeta 1, alpha vbeta 3, and alpha Ebeta 7) were localized to microvilli. Transmembrane proteins known or suspected to interact with integrin extracellular domains, including tetraspan family members and CD47, were also found to be concentrated on microvilli.

The ability of leukocyte adhesion molecules to support initial adhesion under flow is influenced by several factors including affinity, avidity, and accessibility to endothelial ligands. Previous studies of nonintegrin adhesion molecules indicate that cytoplasmic domains can have dramatic effects upon initiating adhesion under flow without altering static adhesion. Experiments involving the use of L-selectin-CD44 chimeras suggest that cytoplasmic domains may influence adhesion under flow by targeting these receptors to the microvillous or cell body (see Introduction). However, it is clear that the cytoplasmic domain of L-selectin also has effects on initiation of adhesion that are independent of receptor positioning. A truncation mutant of L-selectin lacking the 11 COOH-terminal amino acid residues of the cytoplasmic domain was localized to microvilli and retained the ability to bind ligand, but was unable to support rolling on endothelium (Kansas et al., 1993; Pavalko et al., 1995). This mutant lost the ability to associate with the cytoskeletal proteins alpha -actinin and vinculin, suggesting that interactions between adhesion molecule cytoplasmic domains and cytoskeletal proteins can be important in regulation of initial adhesive interactions under flow. We found that replacement of the cytoplasmic and/or transmembrane domains of alpha 4beta 7 (which does support adhesion under flow) with the homologous domains of alpha Lbeta 2 or alpha Mbeta 2 (which do not) did not affect adhesion to the alpha 4beta 7 ligand MAdCAM-1 under either static or flow conditions. This indicates that the cytoplasmic domains cannot account for the differences in ability of these integrins to support adhesion under flow. We also found that truncation of the alpha 4 subunit after the conserved GFFKR motif had little if any effect on initiation of alpha 4beta 7-mediated adhesion to MAdCAM-1. Others have previously shown that the same truncation of alpha 4 did not affect the ability of alpha 4beta 1 to initiate adhesion to its ligand, VCAM-1 (Kassner et al., 1995). The alpha 4 truncation was reported to decrease the cell's resistance to detachment from VCAM-1 in the face of increasing shear force, suggesting that the alpha  subunit cytoplasmic domain plays a role in strengthening adhesion. We were unable to detect any difference in resistance to detachment between wild-type and chimeric alpha 4beta 7 integrins, suggesting that alpha 4beta 7, alpha Lbeta 2, and alpha Mbeta 2 integrin cytoplasmic domains are similar in their ability to mediate adhesion strengthening.

Many of the adhesion molecules that initiate adhesion to endothelium under flow are concentrated on leukocyte microvilli.These include L-selectin (Picker et al., 1991; Erlandsen et al., 1993; Pavalko et al., 1995), P-selectin glycoprotein ligand-1 (Moore et al., 1995; Bruehl et al., 1997), and the integrins alpha 4beta 7 and alpha 4beta 1 (Berlin et al., 1995; and this report). Other adhesion molecules, including CD44 and the integrins alpha Lbeta 2 and alpha Mbeta 2, are found predominantly on the cell body (Erlandsen et al., 1993; Berlin et al., 1995; von Andrian et al., 1995; and this report). Little information is available about the mechanisms that lead to the selective display of certain adhesion molecules on microvilli. It seems likely that interactions between the cytoplasmic domains of adhesion molecules and specific cytoskeletal elements can play an important role. In support of this concept, the localization of L-selectin-CD44 chimeras was shown to be determined by the cytoplasmic and/or transmembrane domains, and not by the extracellular domains (see Introduction). Concentration of integrins in other structures, such as focal adhesions and hemidesmosomes, is known to depend upon the beta  subunit cytoplasmic domain. We were surprised to find that our analysis of integrin chimeras did not demonstrate a role for the cytoplasmic or transmembrane domains of either the alpha  or beta  subunit in determining membrane localization. Replacement of the alpha 4beta 7 cytoplasmic and/or transmembrane domains with homologous domains of alpha Lbeta 2 or alpha Mbeta 2 did not interfere with microvillous localization. Put another way, replacement of the extracellular domain of the alpha Lbeta 2 or alpha Mbeta 2 integrins with the extracellular domain of alpha 4beta 7 resulted in a shift from cell body to microvillous localization. These results indicate an important role for the extracellular domain in directing localization of integrins to the microvillous versus the cell body.

Integrin extracellular domains can interact with other cell surface proteins. For example, the transmembrane protein CD47 interacts with alpha vbeta 3 and this interaction depends upon the extracellular domain of CD47 (Lindberg et al., 1996). Several members of the tetraspan family of transmembrane proteins, including CD53, CD63, CD81, and CD82, have been shown to coprecipitate with some integrins, including alpha 4beta 1, alpha 6beta 1, and alpha 4beta 7, but not with alpha Lbeta 2 or some other integrins (Berditchevski et al., 1996; Mannion et al., 1996). These interactions are likely to involve the integrin extracellular domain, since mutations of the alpha 4 subunit extracellular domain substantially reduce association whereas alterations of the alpha subunit cytoplasmic domain have no effect. We found that CD47, CD53, CD63, CD81, and CD82, all known to associate with integrins that we localized to microvilli, were themselves concentrated on microvilli. Our data are consistent with the hypothesis that interactions with tetraspans and CD47 help target certain integrins to microvilli. The widespread expression of tetraspans and CD47 on leukocytes and other cells makes this hypothesis difficult to test directly. At least one integrin that we localized to microvilli, alpha 5beta 1, apparently does not associate with tetraspans or CD47, suggesting that other interactions also are important (Berditchevski et al., 1996). This hypothesis assumes that alpha 4beta 7 and other microvillous integrins are actively concentrated on microvilli, but alpha Lbeta 2 and alpha Mbeta 2 are not. An alternative explanation of our results is that microvillous expression is the "default pathway" for integrins, and that the extracellular domains of alpha Lbeta 2 and alpha Mbeta 2 prevent these integrins from being displayed on microvilli. This could be mediated by interactions between beta 2 integrins and associated cell surface proteins. Although alpha Mbeta 2 has been shown to associate with CD32, the pattern of expression of CD32 (on both cell body and microvilli) suggests that this interaction is not responsible for the concentration of alpha Mbeta 2 on the cell body.

We found that many integrins and integrin-associated proteins were preferentially expressed on microvilli. Some of these integrins, including alpha 4beta 7 and alpha 4beta 1, play important roles in mediating leukocyte adhesion under flow. Other microvillous integrins, such as alpha 5beta 1 and alpha Ebeta 7, mediate adhesion to extracellular matrix proteins or epithelial cells, but have no known role in initiating leukocyte- endothelial interactions. This suggests that the assembly of multimolecular complexes containing integrins and integrin-associated proteins on microvilli may have other important roles in adhesion and signaling.


Footnotes

Received for publication 28 May 1997 and in revised form 24 July 1997.

   Address all correspondence to M. Abi Abitorabi, University of California, San Francisco, Box 0854, San Francisco, CA 94143-0854. Tel.: (415) 206-6649. Fax: (415) 206-4123.

We thank K.L. McDonald and P. Sicurello (Robert D. Ogg Electron Microscope Laboratory, University of California, Berkeley, CA) and G. Antipa and G. Lum (San Francisco State University, San Francisco, CA) for their expert advice and courtesy in allowing us to use their electron microscope facilities. We thank S. Wu for optimizing the static adhesion assay protocol and are grateful to R. Pytela and D. Sheppard for critically reviewing this manuscript.

This study was supported by National Institutes of Health grants HL50024 (to D.J. Erle) and HL03230 (to M. Tidswell). M. Abi Abitorabi was supported by National Institutes of Health training grant HL07155 and National Research Service Award 1F32HL09364.


Abbreviations used in this paper

ICAM-1, intercellular adhesion molecule-1; MAdCAM-1, mucosal addressin cell adhesion molecule-1; VCAM-1, vascular cell adhesion molecule-1.


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