Structure/Function Studies on Vascular Cell Adhesion Molecule- 1”

Vascular cell adhesion molecule-1 (VCAMl) is a member of the immunoglobulin (Ig) superfamily which interacts with the integrin very late antigen-4 (VLA4). The VCAMlIVLA4 interaction mediates both adhesion and signal transduction and is thought to play an im- portant role in inflammatory and immune responses in vivo. The major form of human VCAMl contains seven extracellular Ig-like domains, with domain 1 designated aa the most N-terminal. We have examined the relationship between human VCAMl structure and function using a combination of domain truncation mutants and proteolytic fragmentation of recombinant soluble VCAM1. We have characterized two regions of VCAM1, localized to domains 4 and 5, which are highly sensitive to proteolytic cleavage, localized the epitope of the blocking monoclonal antibody 4B9 to domain 1, and found that domains 1-3 are sufficient for both its adhesive function and its ability to initiate T cell activation.

VCAMl is a member of the immunoglobulin superfamily (1). Alternative splicing is observed in human umbilical vein endothelial cells, generating two forms of VCAMl with either six or seven extracellular Ig-like domains, with the longer form predominant (16)(17)(18). We have begun to examine the * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Construction and Expression of Deletion Mutants-Truncated forms of VCAMl were constructed from either the full-length sevendomain VCAMl pCDM8 clone l E l l (17) or the full-length sixdomain VCAMl pCDM8 clone 41 (1). Seven-domain-(7D) soluble VCAMl was produced by truncation of full-length 7D VCAMl at nt 2193 by digestion with AluI and ligation of a stop codon adaptor. This construct encodes the VCAMl signal sequence and the extracellular first 674 amino acids of VCAMl (17). Six-domain-(6D) soluble VCAM was produced by truncation of full-length 6D VCAMl at nt 1924 by digestion with AluI and ligation of a stop codon adaptor as above. This construct encodes the VCAMl signal sequence and the extracellular first 582 amino acids of the 6D VCAMl sequence (1). Truncated VCAM constructs were obtained by polymerase chain reaction amplification from the appropriate NotI-digested VCAM1-pCDM8 plasmids and a forward-reverse primer pair in which the reverse primer encodes a stop codon. For constructs Dl, D1+2, D1+2+3, and D1+5+6, the polymerase chain reaction products were digested with ApaLI, and the appropriate fragments were purified from agarose gels. These were ligated to a pCDM8 VCAMl vector digested with ApaLI and retaining nt 1-388 of VCAM1, encoding the VCAMl signal sequence and part of domain 1. To obtain D2+3, the polymerase chain reaction product was ligated to a pCDM8 VCAMl vector fragment encoding the VCAMl signal sequence nt 1-178. Further restriction digests and manipulations used to generate the appropriate pCDM8 vector pieces followed standard protocols and are not described here. The forward primers used were as follows: P1, into T75 flasks at about 1.5 X lo6 cells/flask in Dulbecco's modified Eagle's medium, 10% fetal bovine serum. 48-h post-transfection, monolayers were washed twice with Hank's balanced salt solution, incubated with 5 ml/flask of cysteine/methionine-free RPMI containing 2% dialyzed fetal bovine serum, 4 mM Gln (Cys/Met-RPMI/ 2) for 1-2 h at 37 "C, washed twice with Hank's balanced salt solution, and proteins labeled with 5 ml/flask of Cys/Met-RPMI/2 containing 150 pCi each of [R5S]cysteine and methionine (Du Pont-New England Nuclear) for 4 h at 37 "C. Conditioned medium was collected and cells removed by centrifugation. Adherent COS cells were harvested with 5 ml/flask of PBS containing 5 mM EDTA, washed once with PBS, and lysed for 40 min on ice with 1 ml of 0.05 M Tris-HCl, 0.15 M NaC1, pH 7.0, containing 2% Triton X-100, 0.1% aprotinin, and 2 mM phenylmethylsulfonyl fluoride. Cell debris was removed by centrifugation (Eppendorf G414 microfuge, 5 min) and the resulting cell lysates used at 250 pl/immunoprecipitation (IP), which was performed as follows. To generate a preclearing reagent, protein G-Sepharose (Pharmacia LKB Biotechnology Inc.) was coated with anti-ELAM1 mAb BBll(20) at 2 pg/pl of beads, in 0.5 ml of IP wash buffer (PBS, pH 7.2, containing 0.5% Tween 20, 0.05% SDS, 0.1% bovine serum albumin, and 0.02% sodium azide) for a minimum of 2 h and unbound mAb removed with five washes with I P wash buffer. 0.5 ml of labeled conditioned medium or 0.25 ml of cell lysate was precleared over 10 pl of BBll/protein G beads for a t least 2 h a t 4 "C with rocking. Protein G beads were coated either with 4B9 exactly as described for B B l l or with polyclonal anti-VCAM1 murine serum a t a dilution of 1:20, and the precleared supernatants were then incubated with 10 pl of beads for at least 2 h at 4 "C with rocking, and the beads washed 5 times with 1 ml each of I P wash buffer. Beads were boiled for 3 min in 50 pl of SDS-PAGE reducing sample buffer, eluted proteins run on a 4-20% Daiichi gradient gel, and visualized by radioautography.
Adhesion Assays-The ability of truncated soluble forms of VCAMl to support adhesion was assessed after immunoaffinity chromatography on mAb 4B9 and immobilization on plastic essentially as described (21). Briefly, COS cell-conditioned medium generated as described above and containing truncated soluble forms of VCAMl was concentrated 10-fold by ultrafiltration and run over a 1-ml 4B9 immunoaffinity resin. After elution a t pH 3.0, column fractions were immediately neutralized, diluted 1:5 into a bicarbonate binding buffer, pH 9.2, and incubated in polystyrene 96-well plates overnight. Eluted material was not quantified. After blocking with bovine serum albumin, adhesion of Ramos cells to the plates was assessed as described (21).
Proteolytic Digestion of VCAMl-rsVCAM1, purified as described (21), was concentrated to 2.6 mg/ml (Amicon YM-30) and stored frozen at -70 "C until use. For digestion with endoproteinase Glu-C, 5.4 mg of rsVCAM1 in 10 ml of 0.07 M sodium phosphate, pH 7.7, was treated with 50 pg of endo-Glu-C (Protease Staphylococcus aureus, Worthington) at 50 pg/ml, and incubated a t 37 "C for 1 h. Bovine a2-macroglobulin (Boehringer Mannheim) was added to 0.265 mg/ml and the sample further incubated for 10 min a t 23 "C. The digest was batch loaded onto 5 ml of 4B9 affinity resin (21) for 2 h a t 4 "C. The resin was collected in a 1-cm diameter column, washed sequentially with PBS, PBS with 0.3 M NaCl, and PBS with 0.5 M NaCI, and then eluted with 0.1 M glycine, pH 3.0. The eluate was neutralized with HEPES, pH 7.5, and loaded onto a Superdex 75 fast protein liquid chromatography column equilibrated with PBS and run at 7 ml/h. 0.35-ml fractions were collected and monitored by Am and SDS-PAGE. Peak fractions were stored a t -70 "C. For papain digestion, rsVCAMl (210 pg) in 0.47 ml of 50 mM sodium phosphate, pH 7.0,20 mM glutathione, 0.25 mM EDTA, and 10 pg of papain were incubated a t 37 "C for 80 min. Dimethylformamide was added to 15% (v/v), and the sample was further incubated for 2 h (22). The papain inhibitor E64 (Boehringer Mannheim) was added to 20 pglml. The digest was fractionated on a Superdex 75 column in PBS, 7 ml/h, and selected fractions assayed for adhesion as described above. Samples both before addition of dimethylformamide (predominantly 35-kDa cleavage product) and after addition of dimethylformamide (predominantly 28-kDa cleavage products) were submitted for N-terminal sequence analysis.
T Cell Actiuation-T cell activation assays were performed as described (15). Briefly, mAb OKT3 was incubated in microtiter wells overnight at 4 "C, excess removed, and rsVCAMl or VCAMl(1-292) then added for 2-3 h a t room temperature. Plates were washed, incubated with medium, purified T cells added, and their proliferation measured at 3 days as described (15).
N-terminal Sequence Analysis-Samples were subjected to N-ter-minal sequence analysis in an Applied Biosystems 470 sequenator. Phenylthiohydantoin-amino acids were analyzed on line in a 120A Phenylthiohydantoin amino acid analyzer. Samples derived from HPLC column fractions were loaded on polybrene-treated discs.
Other selected products were sequenced using the procedure of Matsudaira (23). Samples were subjected to SDS-PAGE, transferred to polyvinylidene difluoride membranes (Immobilon, Millipore), stained with Coomassie Blue, and sequenced directly.

Studies with Secreted Truncated F o r m of VCAMl-
The ability of the VCAM1-directed blocking mAb 4B9 (19), and a murine polyclonal anti-VCAM1 serum, to immunoprecipitate secreted truncated forms of VCAMl was examined (Fig. LA). The polyclonal anti-VCAM1 antiserum was able to immunoprecipitate proteins encoded by constructs containing domains l and 2 (D1+2), domains 2 and 3 (D2+3), and domains 1, 2, and 3 (D1+2+3), but not domain 1 alone. The results show that the truncated two-or three-domain forms of VCAM1, but not the single domain construct, were well secreted by transiently transfected COS cells. When mAb 4B9 was used, the proteins encoded by D1+2 and D1+2+3, but not D2+3, were immunoprecipitated ( . Domain constructs are indicated at the top. Apparent differences in kDa are consistent with differential glycosylation of VCAM1-derived fragments, since potential N-glycosylation sites occur in domains 3,4, 5 (two sites), and 6 (two sites) in VCAMl (1,17). Molecular weight markers (X lo-') are indicated at the left. Panel B, adhesion of Ramos cells to immobilized soluble forms of VCAM1. Truncated soluble forms of VCAMl were transiently expressed in COS cells, purified from conditioned medium by 4B9 immunoaffinity chromatography, immobilized on plastic, and their ability to support Ramos cell adhesion assessed. Ramos cell adhesion to VCAMl containing seven domains, six domains, D1+2+3, and D1+2, either in the presence or absence of blocking mAb 4B9, is shown. the apparent molecular weights by SDS-PAGE of domain constructs of the same length likely reflect the presence of Nglycosylation sites in domains 3 through 6 (Fig. LA). These results suggested that mAb 4B9 binds to either domain 1 or the junction formed by domains 1 and 2. A construct containing domains 1, 5, and 6 was also examined (Fig. lA). mAb 4B9 immunoprecipitates the protein encoded by this construct also, supporting the conclusion that the 4B9 epitope lies within the most N-terminal domain of VCAM1, domain 1.
The ability of soluble truncated forms of VCAMl to support cell adhesion was next examined (Fig. 1B). The medium conditioned by COS cells transfected with constructs for fulllength soluble seven-domain, six-domain, D1+2+3, and D1+2 forms of VCAMl was passed over a 4B9 immunoaffinity resin (see "Experimental Procedures"), and the eluate immobilized on plastic and examined for its ability to support adhesion. Both the seven-and six-domain forms of VCAMl support Ramos cell adhesion, which is completely blocked by mAb 4B9, consistent with the behavior of both forms expressed as membrane proteins in COS cells (17,24). We found that both D1+2+3 and D1+2 also supported Ramos cell adhesion (Fig.  1B). Interestingly, mAb 4B9 blocked only the three-domain form of VCAM1. In separate experiments, the anti-VLA4 mAb HP2/1 blocked Ramos cell adhesion to all the truncated forms completely (not shown). The data show that the first two N-terminal domains of VCAMl are sufficient to support VLA4-dependent cell adhesion. However, since no attempt was made to quantitate amounts of truncated forms eluted from the affinity resin, quantitative conclusions regarding comparative adhesive ability could not be drawn.
Proteolytic Fragmentation of VCAMl-We have recently described the purification and functional characterization of a soluble form of VCAMl containing seven extracellular Iglike domains (rsVCAMl), stably secreted from CHO cells (21). CHO cell-derived 7D rsVCAMl retains its adhesive function when immobilized on plastic, as does the COS cellderived 7D material (Fig. 1B). We examined the susceptibility of CHO cell-derived seven-domain rsVCAM1 to proteolysis (Fig. 2) and found that rsVCAMl is readily degraded into large fragments which then are quite resistant to further degradation. For example, endoprotease Glu-C cleaves rs-VCAMl into major fragments of about 50 and 35 kDa ( To determine cleavage sites, the proteolysis of rsVCAMl with endoprotease Glu-C was examined in more detail. First, the 50-and 35-kDa fragments observed by SDS-PAGE (Fig.  2) were sequenced directly after Western blotting. The 50-kDa fragment gave the same N-terminal sequence as rs-VCAMl itself, while the 35-kDa fragment gave two sequences, indicating that it consisted in fact of two fragments of similar mass. One sequence was that of the N-terminal, while the second began at Met-444 of the mature seven-domain sequence, in domain 5 (1,17). The results show that rsVCAMl is cleaved C-terminal to Glu-443, generating 50-and 35-kDa fragments, and that another highly sensitive cleavage site exists for endoprotease Glu-C, generating a shorter 35-kDa N-terminal fragment. Cleavage at Glu-443 occurs between the two highly conserved cysteines (Cys-400 and Cys-459) found in domain 5 of VCAMl and characteristic of the majority of Ig-superfamily domains (25, 26). The 50-and 35-kDa fragments were observed upon SDS-PAGE whether reducing or non-reducing sample buffer was used (not shown), suggesting that these two cysteines are not disulfide-linked (see below).
Another endoproteinase Glu-C digest containing the 50-and 35-kDa fragments was passed over a 4B9 immunoaffinity resin. As expected from the specificity of 4B9 for domain 1, as defined above, and the sequence information obtained on the fragments, the 4B9 resin bound 50-and 35-kDa fragments (Fig. 3, lanes f-h), which both gave the expected N-terminal sequence. Material not binding to the 4B9 resin contained a diffuse 35-kDa band which we presume to be the C-terminal fragment (Fig. 3, lanes c and d ) . The two N-terminal fragments could be readily separated by gel filtration chromatography (Fig. 3) and isolated in highly purified form for further analysis. The isolation by 4B9 affinity chromatography of only the N-terminal50-kDa fragment of VCAM1, but not its complementary 35-kDa C-terminal fragment, following cleavage a t Glu-443 not only confirms that the 2 cysteines in domain 5 are not disulfide-linked but also shows that the two fragments do not remain associated following cleavage.
Characterization of the endo-Glu-C cleavage site which generates the 35-kDa N-terminal fragment required determination of the sequence of its C terminus. To do this, the purified 35-kDa fragment was digested with trypsin and the resultant mixture of peptides subjected to anhydrotrypsin affinity chromatography. Peptides other than the C-terminal peptide contain a C-terminal arginine or lysine and are retained by the column. In contrast, since the most C-terminal peptide is derived from an endo-Glu-C digest and has a Cterminal Glu, it will drop through the column. Following anhydrotrypsin affinity chromatography, a single peptide was purified by reversed-phase HPLC (Fig. 4). The peptide (about 150 pmol) was subjected to N-terminal sequence analysis. Its sequence (EVELIVQEKPFTVE) identifies it as Glu-279 to Glu-292 of the mature sequence of VCAM1. Thus, the second cleavage site for endoprotease Glu-C is Glu-292, six amino acids into the fourth domain of VCAM1, as defined by the intron/exon boundary at . Therefore, this cleavage site is specific for the alternately spliced seven-domain form of VCAM1. Whether the link between domain 3 and either domain 4 or 5 is generally protease sensitive, or whether this sensitivity is limited to 7D VCAM1, remains to be determined.
The 50-kDa N-terminal fragment could also be readily purified (Fig. 3). We examined whether the purified 50-kDa fragment, when incubated with endo-Glu-C, could be cleaved at Glu-292 to generate the 35-kDa fragment. In fact, under conditions where intact rsVCAMl was readily cleaved to 50and 35-kDa fragments, the purified 50-kDa fragment was resistant to cleavage (Fig. 5). This observation argues that the cleavages at the two sites occur independently and that cleavage a t Glu-443 results in a conformational change which renders the Glu-292/Ile-293 bond less susceptible to cleavage.
We have also examined the N-terminal sequences of papain-derived VCAMl fragments. The 35-kDa fragment (Fig.  2)) was sequenced following SDS-PAGE and Western blotting, and found to be derived from the N-terminal of rs-VCAM1. Fragments of about 28 kDa, derived from further cleavage observed in DMF (22)) were derived from this Nterminal 35-kDa fragment (not shown). These fragments had N termini of either Ala-73 or Gly-83, indicating that they  (1 mg) was digested with endo-Glu-C as described under "Experimental Procedures," and 35-and 50-kDa fragments isolated by 4B9 affinity chromatography as described in Fig. 3. The fragments were dialyzed against 0.1% SDS, 10 mM Tris, pH 6.8, concentrated to 1 mg/ml in a Speed Vac concentrator (Savant), and subjected to SDS-PAGE on a 10-20% gradient gel (Integrated Separation Systems). The fragments were electroblotted to nitrocellulose using 10 mM CAPS, pH 11.25, 10% methanol buffer (1 h, 200 mA in a miniblot apparatus), and visualized by staining with Ponceau S, 0.5% in 1% acetic acid. The 35-kDa band was excised with a razor blade, and the immobilized products (-20 pg/sample) were digested with 2 pg of trypsin (40). 20% of the digest was analyzed directly by reversed-phase HPLC on a C,S column (Vydac, 2.1 x 250 mm) developed with a 90-min gradient of 0-60% acetonitrile in 0.1% trifluoroacetic acid a t 0.3 ml/min. The effluent was monitored at both 214 and 280 nm, and 0.5-min fractions were collected (panel A ) . The remaining 80% of the sample was dried (Speed Vac) and subjected to C-terminal fragment isolation on an anhydrotrypsin column (Pierce) using the supplier's protocol. The unbound fraction was analyzed by HPLC as described above and showed a single major peak at 44 min  -h) in 0.1 M sodium phosphate, pH 7.7, were treated with endo-Glu-C for 1 h at 37 "C, subjected to SDS-PAGE, and the cleavage products visualized by staining with Coomassie Brilliant Blue. Lanes a-c correspond to treatments with 5, 10, and 20 pg/ml protease, respectively. Lanes dh correspond to treatments with 0, l , 5, 10, and 20 pg/ml protease, respectively. Molecular weight markers from Bethesda Research Laboratories are shown at the left.
proteolytically generate convenient quantities of a 292-aminoacid fragment containing D1+2+3 for further functional analysis. To this end, the 35-kDa N-terminal fragment was

Structure and Function
of VCAMl purified to homogeneity as shown above (Fig. 3) and examined for function. Consistent with the results of Fig. lB, when immobilized on plastic VCAM( 1-292) supports VLA4-dependent adhesive function (Fig. 6A). The papain-derived Nterminal fragments were also examined. The 28-kDa fragments containing D2 and D3 could neither bind mAb 4B9 nor support adhesion of Ramos cells, while the 35-kDa fragment containing D l through D3 did both (not shown).
We have recently shown that 7D rsVCAMl can stimulate T cell proliferation when coimmobilized with T cell receptordirected mAbs (15). When VCAM(1-292) was examined, we found that it also could stimulate T cell proliferation as well as full-length rsVCAM1, and this was blocked by mAb 4B9 (Fig. 6B). The results show that the first three domains of VCAMl (and probably only the first two, see Fig. 1B) are sufficient for both of the functions of VCAMl described to date, namely VLA4-dependent adhesion and activation. Fig. 7 summarizes the fragments of VCAMl that were generated through both molecular biological and biochemical techniques, and their ability to support VLA4-dependent adhesion and/or activation, and to bind mAb 4B9.

DISCUSSION
VCAMl is a member of the immunoglobulin superfamily which serves as an adhesion molecule and signal transducer to leukocytes expressing its counter-receptor VLA4 (3,4,(13)(14)(15). As part of our efforts to probe the relationship between VCAMl structure and function, as well as to define other putative functions of the molecule, we have begun to examine VCAMl fragments for functional activity. Examination of both truncated recombinant soluble forms of VCAMl and   coimmobilized with OKT3 stimulated as effectively as rsVCAMl and OKT3, and this stimulation was blocked by mAb 4B9, but not by control mAb MOPC21. proteolytic fragments shows that the first three domains of VCAMl are sufficient for both VLA4-dependent adhesion and T cell activation. In addition, immunoprecipitation studies of truncated VCAMl constructs show that the blocking mAb 4B9 (19) binds to domain 1 of VCAM1. Recent studies with human VCAMl/ICAMl chimeric proteins expressed in COS cells (24), and human/murine VCAMl chimeric proteins expressed on phage' confirm these results. Finally, studies with D1+2 suggest that the first two domains are sufficient for adhesion. In the course of these studies, both Damle and Aruffo (13) and Taichmann et al. (27) have generated VCAM1-Ig fusion proteins containing the first three domains of VCAM1. These fusion proteins, which are bifunctional homodimers, can support VLA4-dependent adhesion and, in one instance (13), stimulate CD4+ T cells. Our results confirm and extend these data since they show that monomeric VCAMl fragments are sufficient for VLA4-dependent adhesion and activation.
The structure of seven-domain VCAMl consists of two internal repeat units of three Ig-like units, domains 1 and 4, 2 and 5, and 3 and 6, respectively, being highly homologous, and suggesting an intergenic duplication event in the evolutionary history of the gene (17,18). As the first three domains of VCAMl are alone sufficient for VLA4-dependent adhesion, the role of the remainder of the molecule remains to be fully defined. We have recently cloned cDNAs for both murine and rat VCAMl (28). Both cDNAs encode seven-domain forms of VCAM1, which are both about 76% identical to human VCAM1, this degree of identity being maintained throughout all seven of the extracellular Ig-like domains. By way of comparison, the adhesion molecule ICAM1, an Ig superfamily member which shares many similarities with VCAMl (29), shows about 50% identity between murine and human sequences (30). The very high degree of sequence homology across species and throughout VCAMl strongly argues that the remainder of VCAMl plays a critical functional role. The proteolysis experiments described above suggest that the more C-terminal Ig-like domains of VCAMl have unusual structural features, which may be relevant to VCAMl function. First, VCAMl possesses two regions which are very protease sensitive, one between domains 3 and 4, and one in domain 5. The reasons for this are unclear, but release of other cell membrane-associated adhesion molecules from the cell surface, including L-selectin and ICAM1, is well established (31,32). It is possible that proteolytic degradation of VCAMl occurs in vivo and that this may play a role in its regulation.
Second, the protease-sensitive region in domain 5 occurs between the highly conserved cysteines in this domain, arguing that there is no disulfide bond in domain 5. Although Ig domains without a disulfide bond have been described (25), they are unusual. Third, cleavage within domain 5 generates an N-terminal fragment of about 50 kDa which is no longer as sensitive to cleavage between domains 3 and 4. The data from rsVCAMl argue that the conformation of the intact molecule is strongly dependent upon its C-terminal half, and we can now examine the proteolytic sensitivity of cell-associated full-length VCAMl to confirm and extend these results.
Recent results also illustrate the importance of domains other than the most N-terminal three domains of VCAM1. A comparison of COS cells expressing the same amounts of either the six-or seven-domain forms of VCAM1, both of which contain D l through D3, shows that the seven-domain form binds about twice as many VLA4-expressing cells (17,24). To examine the implications of this result, Osborn et al. (24) generated a series of VCAMl/ICAMl chimeric con-* C. Hession (33). Thus, VCAMl appears to be a cell surface receptor with two binding sites for VLA4, and the results clearly reinforce the importance of VCAMl structure beyond the first three domains? Although D4 is highly homologous to Dl, and in fact can replace it (24), the studies of Osborn et al. (24) show that mAb 4B9 binds only to Dl, and not to D4. These studies also raise the possibility that D4-6, homologous to Dl-3, might support VLA4-dependent cellular activation. The proteolysis studies described above show that D4-7 are much more readily degraded than Dl-3, and therefore fragments containing D4-D6 were not readily generated. Further studies are ongoing to answer this question.
VCAMl is related to Ig superfamily member ICAM1, which contains five extracellular Ig-like domains (29). It is interesting to note that analyses of ICAMl domain deletion mutants, and of mutants containing single amino acid substitutions within the first three domains, show that the binding site for its integrin ligand LFAl is located in the first two most Nterminal domains (34). Consistent with this result, all mAbs to ICAMl which block LFAl-dependent adhesion bind to either the first or second domain (34)(35)(36). Thus, the two most N-terminal domains of ICAMl appear to be sufficient for LFA1-dependent adhesion. ICAMl has other functions which may give clues as to the role of other portions of the VCAMl molecule. For example, ICAMl serves as an adhesion molecule not only for LFA1, but also for a second integrin, Macl (37). Importantly, Macl interacts with ICAMl via its third Ig-like domain (35), defining an important functional role for another region of the ICAMl molecule. In addition, glycosylation of ICAMl regulates its interaction with Macl (35). It is possible that differential glycosylation of VCAM1, which can only Because we could not quantitate the amount of immobilized VCAMl fragments used in our functional assays (e.g. in Figs. 1 and 6, see "Results"), the fact that Dl-3 are sufficient to support VCAM function is not in conflict with these conclusions. BudnQ + + + + + + + occur in D3 through D6, may affect VCAMl functions, or that other domains of VCAMl may also serve an alternate adhesive function. In fact, recent studies (38,39) show that the integrin a4//37, in which the a chain of VLA4 is associated with an alternate /3 chain, can serve as a counter-receptor for both VCAMl and the CS1 region of fibronectin. However, the interaction of a4B7 with VCAMl is much weaker than that of a4/3l, requiring activation, e.g. with phorbol esters, and is dominated by a4pl when both are present in the same cell (38). Whether a407 interacts weakly with the same region of VCAMl as a4/31, or with other domains, remains to be seen.
In summary, recent studies show that while the N-terminal domains of VCAMl may be sufficient for its VLA4-dependent adhesive and activating functions (13, 27, this study), the other domains of VCAMl are clearly important for VCAMl function. Their sequence is very highly conserved across species (28), the fourth extracellular Ig-like domain of VCAMl can in fact support VLA4-dependent adhesion (24,33), and the proteolysis experiments described above indicate a central role for this portion of VCAMl in its overall conformation in solution. Taken jointly, the results suggest a complex relationship between VCAMl structure and function.