E X P E R I M E N T A L C E L L R E S E A R C H 3 1 7 ( 2 0 1 1 ) 1 6 3 – 1 7 2

ava i l ab l e a t www.sc i enced i r ec t . com

www.e l sev i e r . com/ loca te /yexc r

Research Article

Intermediate monomer–dimer equilibrium structure of nativeICAM-1: Implication for enhanced cell adhesion

Hyun-Mee Oha, Min-Sung Kwona, Hyang-Jin Kima, Byeong-Hun Jeona, Hye-Ran Kima,Hyang-Ok Choia, Bo-Ra Naa, Soo-Hyun Eoma, Nam Woong Songb, Chang-Duk Juna,⁎aSchool of Life Sciences, and Immune Synapse Research Center, Gwangju Institute of Science and Technology, Gwangju, Republic of KoreabCenter for Nano-bio Convergence Research, Korea Research Institute of Standards and Science, Daejeon, Republic of Korea

A R T I C L E I N F O R M A T I O N

⁎ Corresponding author. Fax: +82 62 715 2546E-mail address: cdjun@gist.ac.kr (C.-D. Jun)Abbreviations: ICAM-1, intercellular adhes

glycosylphophatidylinositol; SDF-1α, stromal-d

0014-4827/$ – see front matter. Crown Copyrigdoi:10.1016/j.yexcr.2010.10.004

A B S T R A C T

Article Chronology:

Received 19 July 2010

Revised version received30 September 2010Accepted 1 October 2010Available online 15 October 2010

Dimeric intercellular adhesion molecule-1 (ICAM-1) has been known to more efficiently mediatecell adhesion than monomeric ICAM-1. Here, we found that truncation of the intracellular domain

of ICAM-1 significantly enhances surface dimerization based on the two criteria: 1) the bindingdegree of monomer-specific antibody CA-7 and 2) the ratio of dimer/monomer when a mutation(L42→C42) was introduced in the interface of domain 1. Mutation analysis revealed that thepositively charged amino acids, including very membrane-proximal 505R, are essential formaintaining the structural transition between the monomer and dimer. Despite a strong dimerpresentation, the ICAM-1 mutants lacking an intracellular domain (IC1ΔCTD) or containing R to Asubstitution in position 505 (505R/A) supported a lower degree of cell adhesion than did wild-typeICAM-1. Collectively, these results demonstrate that the native structure of surface ICAM-1 is not adimer, but is an intermediate monomer–dimer equilibrium structure by which the effectiveness ofICAM-1 can be fully achieved.

Crown Copyright © 2010 Published by Elsevier Inc. All rights reserved.

Keywords:

ICAM-1Immunoglobulin superfamilyDimerCell adhesionIntracellular domain

Introduction

Intercellular adhesion molecule-1 (ICAM-1; CD54) is a cell surfaceglycoprotein with five extracellular immunoglobulin (Ig)-likedomains, a hydrophobic transmembrane domain, and a shortintracellular domain. ICAM-1 is a ligand for at least two membersof the β2 family of leukocyte integrins, lymphocyte function-associated antigen-1 (LFA-1; αLβ2) and MAC-1 (αMβ2) [1,2].ICAM-1 is expressed basally on the surface of cells important inimmune responses, and is greatly upregulated by inflammatorymediators in response to immune challenge. Increased ICAM-1expression is important for granulocyte extravasations [3,4],

..ion molecule-1; Ig, immuerived factor-1α; FITC, fluo

ht © 2010 Published by El

lymphocyte-mediated cytotoxicity [5,6], and the development ofspecific immunologic responses involving cell–cell interactions[4,5,7,8].

Recombinant soluble ICAM-1 (sICAM-1), lacking the transmem-brane and intracellular domains, exists as a monomer in solution[9,10]. However, previous cross-linking studies have revealed thatICAM-1 appears to exist as a dimer and higher multimer in itsnative state on the cell surface [11,12]. Domain (D) 5 and/or thetransmembrane domain have been suggested to mediate dimeriza-tion [11,12]. Crystal structures of D1-D2 and D3-D5 fragments ofICAM-1 have been determined and also reveal two sites ofdimerization, in D1 and D4 [13,14]. A band-like one-dimensional

noglobulin; LFA-1, lymphocyte function-associated antigen-1; GPI,rescein isothiocyanate

sevier Inc. All rights reserved.

164 E X P E R I M E N T A L C E L L R E S E A R C H 3 1 7 ( 2 0 1 1 ) 1 6 3 – 1 7 2

ICAM-1 cluster, in conjunction with the complex structure of theICAM-1D1-D2/αLβ2 I domain [15], has been envisioned [14] and thisaccords with visualization of ICAM-1 tetramers in electron micros-copy [16]. Obviously, mutation analysis has corroborated thepresence of dimerization motifs in D1 and D4 [16,17].

Dimerization appears to enhance binding to LFA-1 [11,12].Indeed, LFA-1-expressing lymphoblasts bind to cells expressingwild-type ICAM-1, which is assumed to be dimeric, moreefficiently than cells expressing an equal amount of glycosylpho-phatidylinositol (GPI)-linked ICAM-1, which is largely monomeric[11]. In addition, engineered dimerization of sICAM-1 leads tosignificantly enhanced binding to LFA-1 compared with mono-meric sICAM-1 [16,18]. However, no direct evidence has beenreported on whether dimerization itself is a real single factor thatenhances cell adhesion in the context of native, i.e., cell surface-expressed, ICAM-1. Furthermore, whether or how dimerization isregulated on the cell surface is not fully delineated.

In the present work, we surprisingly found that truncation of theintracellular domain of ICAM-1 significantly increased surfacedimerization, as evidenced by the binding degree of the monomer-specific antibody CA-7, but decreased adhesion to LFA-1 expressingcells under static and flow conditions. These results demonstratethat, in contrast to the previous explanations regarding theeffectiveness of cell surface-expressed ICAM-1, the dimeric stateprobably does not have a direct impact on enhanced adhesion.Instead, it is likely that a structural transition betweenmonomer anddimer, hence architectural flexibility, is a key factor for the bettereffectiveness shown in native ICAM-1. Through mutation analysis,we identified that positively charged amino acids, including the verymembrane-proximal 505R, are important formaintaining the surfacedimeric state. Further, we identified that disruption of intracellulardomain associationwith cytoskeletal components doesnot affect thefunction of ICAM-1 in supporting leukocyte adhesion.

Materials and methods

Cells and antibodies

COS-7 (ATCC CRL-1651, Manassas, VA), 293T (ATCC CRL-1573),CHO-Lec1 (ATCC CRL-1735™), and Jurkat T (ATCC TIB-152™) celllines were maintained as previously described [19]. Immortalizedhuman umbilical vein endothelial cells (iHUVECs) were obtainedfrom Dr. R.-K. Park (Wonkwang University School of Medicine,Iksan, Korea) and confirmed that these cells express no endoge-nous ICAM-1 on resting state. Peripheral blood lymphocytes(PBLs) were prepared as described [20]. ICAM-1 mAbs R6.5[18,20], CA-7 [18], CBR-IC1/11 [18], and the LFA-1 activatingantibody LFA-1/2 [18,20] were prepared as described previously.

cDNA constructions

The human ICAM-1 cDNA constructs wt-IC1, IC1ΔCTD, IC1-GPI[9,17], IC1Δ521-532 [20], IC1ΔRKIKK [20], and IC1_GCN4 [16]were prepared as described previously. The mutant ICAM-1 cDNAsIC1ΔA1 (505RQ506), IC1ΔA2 (505RQRK508), IC1ΔA3 (505AQAA508),IC1ΔA4 (505RQRKIKK511), and IC1505R/A (505R/A) were constructedby PCR amplification using the human wild-type ICAM-1/pAPRM8as a template and the following primers: a sense primer for allmutants (5’-AGGATCCATGGCTCCCAGCAGC-3’) containing the

BamHI restriction site and antisense primers for IC1ΔA1 (5’-AAAGCGGCCGCTCACTGGCGGTTATAGAGGTACGT-3’), IC1ΔA2 (5’-AAAGCGGCCGCTCACTTCCGCTGGCGGTTATAGAG-3’), IC1ΔA3 (5’-AAAGCGGCCGCTCAGGCTGCCTGGGCGTTATAGAGGTACGTGCT-GAG-3 ’), and IC1ΔA4 (5’-AAAGCGGCCGCTCATTTCTTGATCTTCCGCTGGCG-3’) containing the NotI restriction site. ThecDNAs were then subcloned into the BamHI and NotI sites of thepEF1/V5-puro vector. For the ICAM-1 cDNAs with the mutation ofL42 to cysteine (C) in domain 1 and of R505 to alanine (A) incytoplasmic domain, site-directed mutagenesis was performed byusing the wild-type or mutant ICAM-1/pEF1/V5-puro vector as atemplate with the QuikChange kit (Stratagene, La Jolla, CA). Allconstructs were verified by DNA sequencing.

cDNA and siRNA transfections

COS-7, 293T, and CHO-Lec1 cells were transiently transfected withICAM-1 cDNAs or siRNA targeting ezrin and moesin using theLipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) or Dhar-maFECT reagent (Dharmacon, Chicago, IL), respectively, accordingto the manufacturer's instructions.

Western blot analysis

The expression of ezrin and moesin was determined by Westernblot analysis. Briefly, cell lysates were centrifuged at 14,000 rpmfor 20 min at 4 °C, and the protein samples were separatedthrough 12% SDS-PAGE gels and transferred onto a nylonmembrane by means of a Trans-Blot SD semidry transfer cell(Bio-Rad, Hercules, CA). The membrane was blocked in 5% skimmilk (1 h), rinsed, and incubated overnight at 4 °C with primaryantibodies (polyclonal anti-ERM, anti-ezrin, and anti-β-actin) inTBS containing 0.1% Tween 20 (TBS-T) and 3% skim milk. Excessprimary Ab was then removed by washing the membrane threetimes in TBS-T, and the membrane was incubated with 0.1 μg/mlhorseradish peroxidase-labeled secondary Ab (against goat, rabbit,or mouse) for 2 h. Following three washes in TBS-T, bands werevisualized by ECL Western blotting detection reagents andexposed to x-ray film.

Lentiviral infection and establishment of iHUVEC cell linesexpressing wild-type and mutant ICAM-1s

ICAM-1 cDNAs in pHJ-1 lentiviral vector were co-transfected withthe lentiviral packaging vectors (pHDM-Hgpm2, pRC/CMV-Rev1b,and pHDM.G) into 293T cells using the Lipofectamine 2000(Invitrogen). The culture supernatants were collected 48 h aftertransfection. The supernatants were pooled, concentrated byultracentrifugation. For viral infection, virus particles were treatedon iHUVECs (5×104/500 μl) in a 24 well plate in the presence of8 μg/ml polybrene. Infection efficiency was confirmed by immu-nocytochemistry and flow cytometry with Alexa 488-conjugatedICAM-1 mAb (CBR-IC1/11-Alexa 488) after 48 h of infection.

Immunoprecipitation

Immunoprecipitation was performed as previously described [20].Briefly, cell lysates were incubated with R6.5-bead conjugates at4 °C overnight with rotation. The immunoprecipitates wereanalyzed by SDS-PAGE (8–10% gel) and silver staining.

165E X P E R I M E N T A L C E L L R E S E A R C H 3 1 7 ( 2 0 1 1 ) 1 6 3 – 1 7 2

Flow cytometric analysis

Single-cell suspensions were incubated with ICAM-1 antibodies(R6.5 or CA-7) for 1 h on ice, and incubated with fluoresceinisothiocyanate (FITC)-conjugated anti-mouse IgG for 1 h on ice.

Fig. 1 – Intracellular domain of ICAM-1 determines cell surface ICAMimaging of COS-7 cells expressing wt-IC1, IC1ΔCTD or IC1_GPI. The(B) Cell surface dimerization of ICAM-1 was assessed by staining wantibody. The calculation of the CA-7/R6.5 ratio is described in Matcovalently dimerized ICAM-1s by formation of a disulfide bond in dand ΔCTD) and cysteine substitution mutants (L42C) were assessedand SDS-PAGE under nonreducing conditions. Molecular weight mamonomer (M) bands are shown on the right. The dimer/monomerUN-SCAN-IT gel™ image analysis software (Silk Scientific, Inc., Ore

Samples were then analyzed and quantified using the FACScan cellanalyzer (Becton Dickinson, San Jose, CA). Appropriate isotype-matched Ab controls were used in each experiment. The CA-7/R6.5ratio was calculated using the mean fluorescence intensity (MFI)with the same equation described elsewhere [17].

-1 dimerization. (A) Immunofluorescence staining and confocal3D view was generated by projecting the confocal Z-sections.ith CA-7 (monomer specific) or R6.5 (dimer+monomer)erials and methods. *P<0.05 vs. wt-IC1. (C) Determination ofomain 1. 293T cells expressing the indicated ICAM-1s (WT, GPI,for disulfide formation by immunoprecipitation with R6.5 mAbrkers are shown on the left, and the positions of dimer (D) andratio was calculated by the quantitation of the bands usingm, UT).

166 E X P E R I M E N T A L C E L L R E S E A R C H 3 1 7 ( 2 0 1 1 ) 1 6 3 – 1 7 2

Static and flow adhesion assays

Static and flow adhesion assays were performed as describedpreviously [20]. For static adhesion assays, iHUVECs expressingwt-IC1 or ICAM-1 mutants were grown to confluence in 96-microwell plates. Jurkat T cells were labeled with BCECF-AM,preincubated with CBR-LFA-1/2 antibody, and then allowed toadhere to iHUVECs for 30 min at 37 °C. The unbound T cells wereremoved and the fluorescence intensity was measured in amicroplate reader (FL500, Biotek,Winooski, VT). For flow adhesionassays, iHUVECs expressing wt-IC1 or ICAM-1 mutants weregrown on coverslips, and then stained with Cy3-conjugated anti-ICAM-1 Fab (CBR-IC1/11-Fab-Cy3) for 10 min before treatmentwith SDF-1α (100 ng/ml). PBLs were infused over the COS-7monolayer for 5 min at 0.2 dyn/cm2, and then subjected to shearregiment of 5 min each of 4, 10, 15, and 20 dyn/cm2. The number ofcells attached during shear stress was quantified at 20 dyn/cm2 bylive-cell time-lapse imaging using an FV1000 confocal microscope(Olympus Corporation, Japan).

Results

Intracellular domain of ICAM-1 does influence cell surfaceICAM-1 dimerization

The intracellular domain of ICAM-1 is critical for spatial anddynamic organization of ICAM-1 on the cell surface [20].Wild-typeICAM-1 (wt-IC1) is clustered on the microvilli-like protrusion,whereas truncation of the intracellular domain (IC1ΔCTD) resultsin uniform cell surface distribution [20]. Since an earlier report

Fig. 2 – (A) Putative dimerization motif in the transmembrane domintracellular domains of the ICAM family. (C) Schematic representaModel 1, a cis-repulsive force between two adjacent intracellular ddimeric state. Model 2, inter-spatial occupancy of binding proteinsthe surface dimeric presentation of ICAM-1.

demonstrated that GPI-linked ICAM-1 (IC1_GPI) is also uniformlydistributed on the cell membrane, we first examined the spatialdistribution patterns of these three ICAM-1s by confocal micros-copy. As expected, wt-IC1 staining was most prominent inmicrovillar projections (Fig. 1A). However, the majority of bothIC1ΔCTD and IC1_GPI were uniformly distributed on the cellsurface (Fig. 1A).

It has been demonstrated that IC1_GPI is monomeric because itbinds to the monomer-specific antibody CA-7 [11]. We thereforequestioned whether IC1ΔCTD is also monomeric on the cell surface.To this end, three ICAM-1 constructs were transfected in 293T cellsand the cell surface dimerizationwas assessed by stainingwith CA-7antibody, which binds to D5 [11], or staining with R6.5 antibody,which binds toD1-2 [16].Whereas approximately 30%ofwt-IC1wasmonomeric, approximately 95% of IC1_GPI was monomeric, and,surprisingly, only approximately 10% of IC1ΔCTD was monomeric,demonstrating that truncation of the intracellular domain enhancesthe dimeric state (Fig. 1B). We also examined in CHO-Lec1 cells, amutant clone that lacks GLcNAc glycosyl transferase so thatN-linkedcarbohydrates are blocked at the Man5-ClcNAC2-Asn intermediate,and found that CA-7 binding to the wt-IC1 was approximately 75%with R6.5, suggesting that N-linked carbohydrates hinder theaccessibility of CA-7. In contrast, only approximately 25% CA-7/R6.5 bound to the IC1ΔCTD, thereby unambiguously demonstratingthat the intracellular domain importantly contributes to the ICAM-1surface dimeric states (Fig. 1B).

The crystal structure of D1-D2 of ICAM-1 revealed a putativedimerization interface on the face containing β-strands B, E, and D(BED sheet) of D1 [13]. Along with this finding, introduction ofmutations in Leu-42 or Leu-43 residues to cysteine in the interfacevisualized in D1 in crystals sufficiently induced dimerization of

ain. (B) The sequence alignment of the transmembrane andtion of two models for determination of surface dimerization.omains of ICAM-1 contributes to the regulation of the surfacein the interface between two intracellular domains determines

167E X P E R I M E N T A L C E L L R E S E A R C H 3 1 7 ( 2 0 1 1 ) 1 6 3 – 1 7 2

native molecules on the cell surface [16]. To test whether thedimeric state in D5 also affects the dimerization in D1, thedisulfide-forming L42C cysteine substitutionwas introduced in thethree different ICAM-1s and these were expressed in 293T cells.The L42C mutation formed disulfide-linked dimers in all threetypes of ICAM-1s, as shown by the presence of an approximately150-kDa band in SDS-PAGE (Fig. 1C). To our surprise, however,whereas both wt-IC1 and IC1_GPI revealed similar dimer/mono-mer ratios (approximately 1:1), IC1ΔCTD showed a significantlyincreased dimeric state (approximately 8:1) (Fig. 1C). This resultdemonstrates that the intracellular domain influences the surfaceICAM-1 dimeric state both in D1 and D5.

Positively charged amino acids, including 505R, between twoidentical intracellular domains of ICAM-1, determine cellsurface dimerization

As shown in Fig. 2A, since the transmembrane domain of ICAM-1 hasglycine residues that cluster on one side of the predicted transmem-braneα-helix and form a hydrophilic patch in the inner leaflet of thebilayer, which is postulated to mediate dimerization [16], wehypothesized that the intracellular domain may have a role inregulating the outside dimeric state. At least five members of the

Fig. 3 – Positively charged amino acids between two identical intrac(A) Schematic representation of C-terminally truncated or mutatedassessed by CA-7/R6.5 binding as described in Materials andmethodby disulfide formation after cysteine substitution at domain 1 (L42

ICAM family have beenwell characterized [21]. Fig. 2B illustrates thesequence alignment of transmembrane and intracellular domains ofthe ICAM family. It was interesting to note that the intracellulardomainof ICAM-1 includesmorebasic amino acids than thatof otherICAM familymembers at themembrane-proximal region [21]. Thus,it can be deduced that a cis-repulsive force between two adjacentintracellular domains of ICAM-1 may contribute to the regulation ofthe surface dimeric state, as schematicallymodeled in Fig. 2C (model1). However, whereas ICAM-2 has a similar number of basic aminoacids as ICAM-1 in its intracellular domain, a cross-linking studyrevealed that ICAM-2 exists as a monomeric form [12]. Therefore,another possibility may affect surface dimerization. Previously, weand others reported that ERM molecules, including ezrin andmoesin, bind to the intracellular domain of ICAM-1 and are involvedin de novo microvilli formation [20,22]. Therefore, the inter-spatialoccupancy of these ERM proteins in the interface between twointracellular domains may also determine the surface dimericpresentation as modeled in Fig. 2C (model 2).

To test model 1, we designed a series of ICAM-1 constructs thatwere truncated or had mutated parts of the intracellular domain(Fig. 3A, a). As shown in Fig. 3B, no difference was found betweenwt-IC1 and IC1Δ521-532, in terms of the binding affinity to CA-7.This finding suggests that the C-terminal 12 amino acids were

ellular domains are critical for surface dimerization of ICAM-1.ICAM-1 cDNAs. (B) Dimerization of ICAM-1 at D4-D5 was

s. *P<0.05 vs. wt-IC1. (C and D) Dimerization at D1 was assessedC). Note, the results presented are from CHO-Lec1 cells.

168 E X P E R I M E N T A L C E L L R E S E A R C H 3 1 7 ( 2 0 1 1 ) 1 6 3 – 1 7 2

dispensable. Interestingly, IC1_GCN4, which makes a stabledimeric α-helical coiled-coil due to the GCN4 peptide fusion tothe end of transmembrane domain of ICAM-1 [17], showed abinding affinity to CA-7 similar to that of IC1ΔCTD in both 293Tcells and CHO-Lec1 cells. This result suggests that the transmem-brane domain alone is enough to drive surface dimerization(Fig. 3B). In contrast, while having less affinity to CA-7 thanwt-IC1,IC1ΔRKIKK, a 507RKIKK511 deletion mutant, had significantly moreaffinity to CA-7 than IC1ΔCTD, suggesting that the RKIKK motif isrequired but not critical for maintaining surface dimerization. Thisresult further suggests that othermembrane-proximal amino acidsmay also participate in the surface dimeric condition of ICAM-1.We therefore designedmore ICAM-1mutant constructs, which aredepicted in Fig. 3A, b. To our surprise, we found that mutantIC1ΔA1 (505RQ506) shows a similar affinity to CA-7 as IC1ΔA2(505RQRK508) and IC1ΔA4 (505RQRKIKK511), though the bindingaffinity was lower than that of wt-IC1 (Fig. 3B). In addition, thelarge difference between IC1ΔA1 (505RQ506) and IC1ΔA3(505AQAA508) in affinity to CA-7 demonstrates that 505R is a criticalamino acid residue for determining surface dimerization. Similarpatterns were also observed in domain 1 dimerization when L42Cwas introduced. As shown in Fig. 3C, the dimer/monomer ratio ofIC1ΔA1 was not significantly different from IC1ΔA4. Finally, anICAM-1 construct containing only 505R to A substitution (505R/A)showed increased domain 1 dimerization as compared to wt-IC1(Fig. 3D). Taken together, these results suggest that the positivelycharged amino acids, including the very membrane-proximal 505R,in the intracellular domain create repulsive dimerization force

Fig. 4 – Silencing of ezrin and moesin does not increase surface dimtransfected with siRNA against ezrin and moesin and analyzed for ecells were co-transfected with siRNA against ezrin and ICAM-1 cDNwere observed by confocal microscopy (Olympus). Bar=1 μm. (C aD4-D5 (C) and disulfide formation at D1 (D) were also determined

between two adjacent ICAM-1s and therefore induce intermediatemonomer–dimer equilibrium structure in native ICAM-1.

To test model 2, we next examined the involvement of ICAM-1binding proteins, such as ezrin and moesin, in surface dimeriza-tion. siRNA targeting each of the candidate genes significantlyreduced the endogenous protein expression (Fig. 4A). In addition,it significantly reduced ICAM-1-induced microvilli elongation inCOS-7 cells (Fig. 4B). However, knock-down of both ezrin andmoesin did not significantly increase surface dimerization indomain 1 (dimer/monomer ratio) and domain 5 (CA-7/R6.5binding ratio) (Fig. 4C and D). Together, these results demonstratethat ICAM-1 binding proteins such as ezrin and moesin are notcritically involved in regulation of surface dimerization.

Strong dimeric presentation reduces LFA-1-mediatedleukocyte adhesion in both static and flow condition

Next, we questioned whether strong dimeric presentation, asshown in IC1ΔCTD or 505R/A, is correlated with the ability of celladhesion under the both static and flow condition. To this end, weestablished immortalized HUVEC cell lines that ectopically expressexogenous wt-IC1, IC1ΔCTD, IC1_GPI, and IC1505R/A by means oflentiviral transfection. Confocal microscopy and flow cytometricanalysis revealed that over 95% cells were positive for wild-typeand mutant ICAM-1s (Fig. 5A). We also employed CBR-LFA-1/2mAb, as this antibody could fully activate LFA-1 without affectingother cellular responses [20]. As shown in Fig. 5B, interestingly, theability of iHUVEC IC1ΔCTD to support leukocyte adhesion was less

erization in domain 1 and domain 5. (A) 293T cells werezrin and moesin expression by Western blot analysis. (B) COS-7As. Then, the expression and localization of indicated ICAM-1snd D) 293T cells were transfected as above (B). CA-7 binding toas described in Fig. 1B and C.

169E X P E R I M E N T A L C E L L R E S E A R C H 3 1 7 ( 2 0 1 1 ) 1 6 3 – 1 7 2

than that of iHUVEC wt-IC1 under static condition and comparableto that of iHUVEC IC1_GPI which revealed a weak adhesion to theLFA-1/2 mAb-activated Jurkat T cells (Fig. 5B). Thus, this evidenceargues the idea that dimeric presentation of membrane ICAM-1enhances leukocyte adhesion [11,12]. More interestingly, iHUVEC505R/A also revealed slightly but significantly less ability to supportleukocyte adhesion than iHUVEC wt-IC1 (Fig. 5B). These resultssuggest that an intermediate monomer–dimer equilibrium struc-ture seen in wild-type ICAM-1 can be critical for the effectivenessof ICAM-1.

To more preciously evaluate the effect of strong dimericpresentation, we next performed the adhesion assay under shearflow conditions. For this experiment, we employed stromal-derived factor-1α (SDF-1α) to mimic better physiologic condi-tions. Little change of leukocyte adhesion was observed under ashear force of 4 dyn/cm2. However, there was a significantreduction of leukocyte adhesion on the monolayers of iHUVECIC1ΔCTD and IC1_GPI under a shear force of 20 dyn/cm2 (Fig. 5C).Leukocyte adhesion was also reduced on the monolayers ofiHUVEC 505R/A (Fig. 5C). Since 505R/A has an intact intracellulardomain except R to A substitution in position 505, this resultimplies that cytoskeletal association with intracellular domainmay minimally influence the cell adhesion in wild-type ICAM-1. Insupport of these findings, we observed that disruption of actincytoskeleton by cytochalasin B and latrunculin A did not alter theability of ICAM-1 to support leukocyte adhesion (Fig. 5D). Overall,the present results demonstrate that the native structure of surface

Fig. 5 – Strong dimeric presentation of ICAM-1 reduces leukocyte adhmutant ICAM-1s. Cells were stainedwith anti-ICAM-1 (CBR-IC1/11-Aleconstruct was determined by confocal microscopy (left; wt-IC1) andunder static (B) and flow(C) conditionswereperformedusing iHUVECwere treated with cytochalasin B (CB; 1 μg/ml) or latrunculin A (Lat Adescribed above B. Bar graphs show the mean±SD of three independ

ICAM-1 is not a dimer, but is an intermediate monomer–dimerequilibrium structure by which the effectiveness of ICAM-1 can befully achieved.

Discussion

It has been predicted that the transmembrane domain of ICAM-1contributes to dimerization at D4 and D5 [11]. However, little isknown about whether the intracellular domain influences thedimeric state at D4 and D5. In addition, whether dimerization atD4 and D5 also reflects the dimerization at D1 has not beendetermined. By creating engineered mutants, we unambiguouslydemonstrated that a cis-repulsive force between two adjacentintracellular domains of ICAM-1 has a critical function in cellsurface dimerization not only at D4-D5 but also at D1. We alsodetermined that positively charged amino acids, including 505R atthe very membrane-proximal region, are essential in inducing cis-repulsive force. Although dimeric ICAM-1 has been suggested tomore efficiently mediate cell adhesion than the monomeric form,we claim that the effectiveness of ICAM-1 is not solely dependenton the dimeric state.

Engineered dimerization of sICAM-1 has been known to lead toenhanced binding to LFA-1 compared with monomeric sICAM-1[12,16,18]. Additionally, Miller et al. demonstrated that dimericICAM-1 mediates better adhesion than monomeric ICAM-1, asLFA-1-expressing lymphoblasts bind to cells expressing wild-type

esion. (A) Establishment of iHUVECs expressing wild-type andxa 488)mAbs (10 μg/ml) and then the expression of each ICAM-1flow cytometer (right). Bar=10 μm. (B and C) Adhesion assayss expressing indicated ICAM-1s. (D) iHUVECs expressingwild-type; 0.1 μM) for 30 min. Then, adhesion assay was performed asent experiments. *P<0.05, **P<0.001 vs. wt-IC1.

170 E X P E R I M E N T A L C E L L R E S E A R C H 3 1 7 ( 2 0 1 1 ) 1 6 3 – 1 7 2

ICAM-1, which is assumed to be dimeric, more efficiently than cellsexpressing an equal amount of GPI-linked ICAM-1, which is largelymonomeric [11]. Strictly speaking, however, the authors’ inter-pretation may not have been correct because we show that thenative form of ICAM-1 is not a real dimer based on at least thefollowing two criteria. First, truncation of the intracellular domaininduces better dimerization than wild-type ICAM-1 in terms ofbinding affinity to the monomer-specific antibody CA-7 and theincreased dimer/monomer ratio at D1. Second, deletion ormutation of basic amino acids, including 505R, at the membrane-proximal region, induces strong dimerization on the cell surface.Therefore, factors other than dimerization must explain thefinding by Miller et al. that the native form of ICAM-1 has astronger avidity than GPI-linked ICAM-1 in a cell-based system. Ifdimerization is not likely to directly enhance adhesion, what otherfactors influence ICAM-1 avidity in its native form? One potentialfactor is structural plasticity, which is intrinsic in the ecto-domainof ICAM-1. A recent report suggested that the monomer–dimertransition is associated with a large, physiologically relevant, IgSFdomain rearrangement [17]. Through a comparison of themonomeric structure of D3-D5 with the dimeric ICAM-1 struc-ture [14,17], the authors defined two states that appear to coexistin equilibrium with each other and dynamically interconvert on

Fig. 6 – Proposed model of surface ICAM-1 architecture. In this mointermediate state between monomer and dimer. We suggest thatcritically involved in the regulation of the surface dimeric state.

the cell surface. Another factor is the architectural flexibility,which can be influenced by the other domains includingintracellular domain. The fact that truncation or mutation ofbasic amino acids in the intracellular domain increases surfacedimerization suggests a pivotal role for the intracellular domain inmaintaining such architectural flexibility. In this regard, thesimultaneous increase in dimerization at both D4-D5 and D1may reflect an increase in the architectural rigidity of ICAM-1 onthe cell surface. Thus, this architectural rigidity may result inreduced leukocyte–endothelium interaction as shown in Fig. 5.One more factor is spatial organization of surface ICAM-1s, whichis influenced by several inside factors including intracellularbinding proteins such as α-actinin, ezrin, and moesin [20,23,24].We have previously reported that ICAM-1 is localized at themicrovillar membrane protrusion. Moreover, ICAM-1 by itselfdirectly regulated the de novo elongation of microvilli and this wassignificantly reduced by siRNA targeted to the ezrin [20]. Inaddition, we found that microvillar localization of ICAM-1 is linkedto the functional consequences in terms of transmigration ofleukocytes under the physiological shear flow condition andformation of ring-shaped membrane projection at the leukocyte-endothelial junctional interface [20]. From the point of this view,one could deduce that reduced degree of leukocyte adhesion is due

del, the native form of ICAM-1 is not a dimer but is in thethe transmembrane domain and intracellular domain are

171E X P E R I M E N T A L C E L L R E S E A R C H 3 1 7 ( 2 0 1 1 ) 1 6 3 – 1 7 2

to the loss of microvilli structure rather than the loss of themonomer–dimer dynamic balance. However, as 505R/A has anintact intracellular domain except R to A substitution in position505 and shows no alteration of microvilli structure (data notshown), cytoskeletal association of intracellular domain mayminimally affect the cell adhesion. On the other hand, it may berelated to the cytoskeleton-driven active movement on the cellsurface, e.g., the ring-liked clusters formed after engagement withthe LFA-1-bearing cells [22,25,26] and the trafficking of ICAM-1 tothe immunological synapse [27]. This conclusion is also supportedby the present observation that actin disruption does not alter theability of ICAM-1 to support leukocyte adhesion.

In the current study, we found that the ezrin and moesin arenot involved in ICAM-1 dimerization, but did not address apotential role forα-actinin, a protein that binds to the ICAM familymembers including ICAM-1, -2, and -5 through the basic aminoacids in the cytoplasmic region [23,28]. Since other ICAMsincluding ICAM-2 also have similar basic amino acids but stillmay be more in monomeric form, it could be speculated thatmonomer–dimer balance is mediated not by simple cis-repulsiveforce but by specific interaction with α-actinin. However, thisspeculation is not likely based on at least the following twoaspects. First, the present results demonstrate that half of nativeICAM-1 has dimeric nature at D1 and D5 (Fig. 1B and C) [13], butthis is not observed in ICAM-2 [29]. If association of intracellulardomain with α-actinin determines the dimer–monomer balance,the ecto-domain of native ICAM-1 should be a monomer. Second,α-actinin binding motif (507RKIKK511) in ICAM-1 is minimallyrequired but not essential in opposing dimerization force createdby transmembrane domain. Indeed, the very membrane-proximal505R is most essential which induces cis-repulsive force betweentwo adjacent ICAM-1s. However, this amino acid is found only 5amino acids apart from the start region of intracellular domain inICAM-2.

Based on the CA-7 antibody staining, only approximately 30% ofwild-type ICAM-1 is monomeric while greater than 90% of GPI-linked ICAM-1 is monomeric. This result demonstrates that thelevel of dimerization at D1 is not always correlated with the levelof dimerization at D4-D5 and further implies an important role forthe intracellular domain in controlling the extracellular architec-ture. In spite of significant difference in terms of dimerization atD4-D5, these findings raise the question of why wild-type ICAM-1and GPI-linked ICAM-1 have a similar level of dimerization at D1.The answer may give us a clue to why wild-type ICAM-1 showsbetter adhesion than monomeric ICAM-1 (IC1_GPI) or dimericICAM-1 (IC1ΔCTD and 505R/A).

In summary, the current findings show that the native form ofICAM-1 is not a dimer but is in the intermediate state betweenmonomer and dimer. We suggest that spatial and architecturalflexibility shown in wild-type ICAM-1 may be critically involved inthe regulation of cell adhesion. Since the intracellular domain,especially the very membrane-proximal region, is spaced by a cis-repulsive forcegeneratedbybasic amino acids, extracellular domainscan bemaintained in amonomer–dimer equilibrium states as shownin Fig. 6(a) and (b). Swapping of transmembrane domain to the GPIresults in loss of the equilibrium and induces a monomeric state atD4-D5. Interestingly, this condition does not change dimerization atD1, as shown in Fig. 6(c). However, truncation of the intracellulardomainormutationof positively charged aminoacids, including505R,induces strong dimerization both at D4-D5 and at D1 [Fig. 6(d) and

(e)]. In this condition, ICAM-1 may lose spatial and architecturalflexibility on the surface membrane.

Acknowledgments

This workwas supported by theMid-career Researcher Program ofthe National Research Foundation of Korea/Ministry of Education,Science and Technology [2010-0027671], Korea Research Founda-tion Grant funded by the Korean government [KRF-2008-314-C00265], Cell Dynamics Research Center [2010-0001621], andBioImaging Research Center at GIST.

R E F E R E N C E S

[1] T.A. Springer, Adhesion receptors of the immune system, Nature346 (1990) 425–434.

[2] T.A. Springer, Traffic signals for lymphocyte recirculation andleukocyte emigration: the multistep paradigm, Cell 76 (1994)301–314.

[3] C.W. Smith, R. Rothlein, B.J. Hughes, M.M. Mariscalco, H.E. Rudloff,F.C. Schmalstieg, D.C. Anderson, Recognition of an endothelialdeterminant for CD 18-dependent humanneutrophil adherence andtransendothelial migration, J. Clin. Invest. 82 (1988) 1746–1756.

[4] H. Xu, J.A. Gonzalo, Y. St Pierre, I.R. Williams, T.S. Kupper, R.S.Cotran, T.A. Springer, J.C. Gutierrez-Ramos, Leukocytosis andresistance to septic shock in intercellular adhesion molecule1-deficient mice, J. Exp. Med. 180 (1994) 95–109.

[5] M.W. Makgoba, M.E. Sanders, G.E. Ginther Luce, E.A. Gugel, M.L.Dustin, T.A. Springer, S. Shaw, Functional evidence thatintercellular adhesion molecule-1 (ICAM-1) is a ligand forLFA-1-dependent adhesion in T cell-mediated cytotoxicity, Eur. J.Immunol. 18 (1988) 637–640.

[6] S.J. Mentzer, R. Rothlein, T.A. Springer, D.V. Faller, Intercellularadhesion molecule-1 (ICAM-1) is involved in the cytolytic Tlymphocyte interaction with a human synovial cell line, J. Cell.Physiol. 137 (1988) 173–178.

[7] J.E. Sligh Jr., C.M. Ballantyne, S.S. Rich, H.K. Hawkins, C.W. Smith,A. Bradley, A.L. Beaudet, Inflammatory and immune responses areimpaired in mice deficient in intercellular adhesion molecule 1,Proc. Natl Acad. Sci. USA 90 (1993) 8529–8533.

[8] D.M. Altmann, N. Hogg, J. Trowsdale, D. Wilkinson, Cotransfectionof ICAM-1 and HLA-DR reconstitutes human antigen- presentingcell function in mouse L cells, Nature 338 (1989) 512–514.

[9] D.E. Staunton, M.L. Dustin, H.P. Erickson, T.A. Springer, Thearrangement of the immunoglobulin-like domains of ICAM-1 andthe binding sites for LFA-1 and rhinovirus, Cell 61 (1990) 243–254.

[10] R. Rothlein, E.A. Mainolfi, M. Czajkowski, S.D. Marlin, A form ofcirculating ICAM-1 in human serum, J. Immunol. 147 (1991)3788–3793.

[11] J. Miller, R. Knorr, M. Ferrone, R. Houdei, C.P. Carron, M.L. Dustin,Intercellular adhesion molecule-1 dimerization and itsconsequences for adhesion mediated by lymphocyte functionassociated-1, J. Exp. Med. 182 (1995) 1231–1241.

[12] P.L. Reilly, J.R.Woska Jr., D.D. Jeanfavre, E. McNally, R. Rothlein, B.J.Bormann, The native structure of intercellular adhesionmolecule-1 (ICAM-1) is a dimer. Correlation with binding toLFA-1, J. Immunol. 155 (1995) 529–532.

[13] J.M. Casasnovas, T. Stehle, J.H. Liu, J.H. Wang, T.A. Springer, Adimeric crystal structure for the N-terminal two domains ofintercellular adhesion molecule-1, Proc. Natl Acad. Sci. USA 95(1998) 4134–4139.

[14] Y. Yang, C.D. Jun, J.H. Liu, R. Zhang, A. Joachimiak, T.A. Springer,J.H. Wang, Structural basis for dimerization of ICAM-1 on the cellsurface, Mol. Cell 14 (2004) 269–276.

172 E X P E R I M E N T A L C E L L R E S E A R C H 3 1 7 ( 2 0 1 1 ) 1 6 3 – 1 7 2

[15] M. Shimaoka, T. Xiao, J.H. Liu, Y. Yang, Y. Dong, C.D. Jun, A.McCormack, R. Zhang, A. Joachimiak, J. Takagi, J.H. Wang, T.A.Springer, Structures of the alpha L I domain and its complex withICAM-1 reveal a shape-shifting pathway for integrin regulation,Cell 112 (2003) 99–111.

[16] C.D. Jun, C.V. Carman, S.D. Redick, M. Shimaoka, H.P. Erickson, T.A.Springer, Ultrastructure and function of dimeric, solubleintercellular adhesion molecule-1 (ICAM-1), J. Biol. Chem. 276(2001) 29019–29027.

[17] X. Chen, T.D. Kim, C.V. Carman, L.Z. Mi, G. Song, T.A. Springer,Structural plasticity in Ig superfamily domain 4 of ICAM-1mediates cell surface dimerization, Proc. Natl Acad. Sci. USA 104(2007) 15358–15363.

[18] C.D. Jun, M. Shimaoka, C.V. Carman, J. Takagi, T.A. Springer,Dimerization and the effectiveness of ICAM-1 in mediatingLFA-1- dependent adhesion, Proc. Natl Acad. Sci. USA 98 (2001)6830–6835.

[19] M.C. Montoya, D. Sancho, G. Bonello, Y. Collette, C. Langlet, H.T.He, P. Aparicio, A. Alcover, D. Olive, F. Sanchez-Madrid, Role ofICAM-3 in the initial interaction of T lymphocytes and APCs, Nat.Immunol. 3 (2002) 159–168.

[20] H.M. Oh, S. Lee, B.R. Na, H. Wee, S.H. Kim, S.C. Choi, K.M. Lee, C.D.Jun, RKIKK motif in the intracellular domain is critical for spatialand dynamic organization of ICAM-1: functional implication forthe leukocyte adhesion and transmigration, Mol. Biol. Cell 18(2007) 2322–2335.

[21] C.G. Gahmberg, Leukocyte adhesion: CD11/CD18 integrins andintercellular adhesion molecules, Curr. Opin. Cell Biol. 9 (1997)643–650.

[22] O. Barreiro, M. Yanez-Mo, J.M. Serrador, M.C. Montoya, M.Vicente-Manzanares, R. Tejedor, H. Furthmayr,

F. Sanchez-Madrid, Dynamic interaction of VCAM-1 andICAM-1 with moesin and ezrin in a novel endothelial dockingstructure for adherent leukocytes, J. Cell Biol. 157 (2002)1233–1245.

[23] O. Carpen, P. Pallai, D.E. Staunton, T.A. Springer, Association ofintercellular adhesion molecule-1 (ICAM-1) withactin-containing cytoskeleton and alpha-actinin, J. Cell Biol. 118(1992) 1223–1234.

[24] L. Heiska, K. Alfthan, M. Gronholm, P. Vilja, A. Vaheri, O.Carpen, Association of ezrin with intercellular adhesionmolecule-1 and -2 (ICAM-1 and ICAM-2). Regulation byphosphatidylinositol 4,5-bisphosphate, J. Biol. Chem. 273 (1998) 21893–21900.

[25] C.V. Carman, C.D. Jun, A. Salas, T.A. Springer, Endothelial cellsproactively form microvilli-like membrane projections uponintercellular adhesion molecule 1 engagement of leukocyteLFA-1, J. Immunol. 171 (2003) 6135–6144.

[26] C.V. Carman, T.A. Springer, A transmigratory cup in leukocytediapedesis both through individual vascular endothelial cells andbetween them, J. Cell Biol. 167 (2004) 377–388.

[27] A. Grakoui, S.K. Bromley, C. Sumen, M.M. Davis, A.S. Shaw, P.M.Allen, M.L. Dustin, The immunological synapse: a molecularmachine controlling T cell activation, Science 285 (1999)221–227.

[28] H. Nyman-Huttunen, L. Tian, L. Ning, C.G. Gahmberg,alpha-Actinin-dependent cytoskeletal anchorage is important forICAM-5-mediated neuritic outgrowth, J. Cell Sci. 119 (2006)3057–3066.

[29] J.M. Casasnovas, T.A. Springer, J.H. Liu, S.C. Harrison, J.H. Wang,Crystal structure of ICAM-2 reveals a distinctive integrinrecognition surface, Nature 387 (1997) 312–315.