Divalent Cations and Ligands Induce Conformational Changes That Are Highly Divergent among beta 1 Integrins*

Gianfranco BazzoniDagger , Lan Ma§, Marie-Luise Blue§, and Martin E. HemlerDagger

From the Dagger  Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115 and the § Bayer Research Center, West Haven, Connecticut 06516

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Here we show striking differences in conformational regulation among beta 1 integrins. Upon manganese stimulation, a beta 1 epitope defined by monoclonal antibody (mAb) 9EG7 was induced strongly (on alpha 4beta 1), moderately (on alpha 5beta 1), weakly (on alpha 2beta 1), or was scarcely detectable (on alpha 6beta 1 and alpha 3beta 1). Comparable results were seen for the beta 1 epitope defined by mAb 15/7. Likewise, soluble ligands caused strong (alpha 4beta 1), moderate (alpha 5beta 1), weak (alpha 2beta 1, alpha 6beta 1), or minimal (alpha 3beta 1) induction of the 9EG7 epitope. Exchange or deletion of alpha  chain cytoplasmic tails did not alter Mn2+-induced 9EG7 epitope levels. Upon removal of calcium by EGTA or EDTA, the hierarchy of 9EG7 epitope induction was similar (alpha 5beta 1 alpha 2beta 1 > alpha 6beta 1 > alpha 3beta 1), except that EGTA reduced rather than induced 9EG7 expression on alpha 4beta 1. Thus in contrast to other beta 1 integrins, calcium uniquely supports constitutive expression of the 9EG7 epitope on alpha 4beta 1. Likewise, calcium supported vascular cell adhesion molecule-stimulated 9EG7 appearance on alpha 4beta 1, whereas calcium inhibited ligand-induced 9EG7 epitope on other integrins. Constitutive expression of 9EG7 on alpha 4beta 1 was eliminated by a D698E mutation in alpha 4, suggesting that Asp-698 may play a key role in maintaining atypical alpha 4beta 1 response to calcium. In conclusion, our results (i) demonstrate that mAb such as 9EG7 and 15/7 have limited diagnostic utility as reporters of ligand or Mn2+ occupancy for beta 1 integrins, (ii) indicate pronounced differences in conformational flexibilities (alpha 4beta 1 > alpha 5beta 1 > alpha 2beta 1 > alpha 6beta 1 > alpha 3beta 1), (iii) allow us to hypothesize that beta 1 integrins may differ markedly in conformation-dependent inside-out signaling, and (iv) have uncovered an atypical alpha 4beta 1 response to calcium that requires alpha 4 Asp-698.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Cell adhesion mediated by cell-surface receptors in the integrin family regulates a host of cellular events including cell migration, spreading, proliferation, apoptosis, and gene induction (1-4). By inside-out signaling, many cellular agonists "activate" integrins, i.e. they trigger increased ligand binding and cell adhesion capability. In some cases, "activation" of these integrins is accompanied by changes in their affinity for ligands and/or for ligand mimetic monoclonal antibodies (5). Then upon ligand binding, integrins undergo pronounced conformational changes, resulting in the appearance of Ligand-Induced Binding Sites (LIBS)1 on beta 1 (6-8), beta 2 (9), and beta 3 (10-13) integrins as detected by specific monoclonal antibodies. Integrin-mediated ligand binding requires divalent cations, and Ca2+, Mg2+, and especially Mn2+ can themselves cause pronounced integrin conformational changes. Notably, many "LIBS" epitopes regulated by ligand binding are also regulated positively or negatively by divalent cations, thus leading to renaming (6) as "CLIBS" (Cation and Ligand-Influenced Binding Sites). At least six non-overlapping CLIBS epitopes have been defined, with three sites located in beta 1 (6-8, 14-17) and three other sites in the beta 3 subunit (18, 19). Integrin alpha  subunit sites regulated by ligands and divalent cations have also been described (9, 12, 20, 21). Thus, major changes occur throughout the integrin molecule upon ligand and/or divalent cation binding. Displacement of divalent cations may itself be a key feature of ligand binding (22). Integrin beta 1 CLIBS epitopes, such as those defined by mAb 15/7 (7) and 9EG7 (23), were suggested to be "activation epitopes," but subsequent reports showed very poor correlation with integrin activation (6, 16).2 The mAb 9EG7 and/or 15/7 epitopes can be induced not only by ligand or Mn2+ but also by functionally disruptive cytoplasmic tail deletion or integrin denaturation (7, 16, 23). Thus, CLIBS epitopes may in general be reporters of integrin unfolding (25). Because epitopes recognized by mAb 9EG7 and 15/7 are induced upon ligand occupancy of alpha 4beta 1 and alpha 5beta 1, it has been widely assumed that these epitopes would be diagnostic for ligand occupancy of all beta 1 integrins. Here we show a wide variation in CLIBS epitope induction on different beta 1 integrins, indicating that CLIBS epitopes are not useful for determining the extent of Mn2+ or ligand binding for several beta 1 integrins. However, our CLIBS epitope results do suggest that beta 1 integrins differ widely in the extent to which alpha -beta chain unfolding accompanies occupancy by ligand or Mn2+. In addition, we have used the 9EG7 CLIBS epitope to investigate anomalous regulation of alpha 4beta 1 by calcium.

    EXPERIMENTAL PROCEDURES
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Results
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Antibodies, Ligands, and Cells-- The anti-beta 1 monoclonal antibodies used were as follows: 9EG7, rat anti-mouse, with cross-reactivity for human and hamster beta 1 (23); mAb 13, rat anti-human (26); and the mouse anti-human antibodies 15/7 (27) and A-1A5 (28). Other antibodies (all mouse anti-human) include: A2-IIE10, anti-alpha 2 (29); A3-IIF5, anti-alpha 3 (30); B5G10, anti-alpha 4 (31); A5-PUJ2, anti-alpha 5 (32); A6-ELE, anti-alpha 6 (33); and negative control antibodies P3 (34) and rat anti-E-selectin (from Dr. Pnina Brodt, McGill University).

The GRGDSP peptide was purchased from Life Technologies, Inc. Recombinant soluble VCAM-1 (35) was a gift from Dr. Roy Lobb (Biogen, Cambridge, MA). Rat collagen type I was obtained from Collaborative Biomedical Products (Bedford, MA). Laminin-1, from mouse Engelbreth-Holm-Swarm sarcoma, was from Life Technologies, Inc., and purified laminin-5 was from Dr. R. Burgeson. K562 cells were cultured in RPMI 1640 with 10% fetal bovine serum and transfected by electroporation with alpha 2, alpha 3, or alpha 4 cDNA in the pFneo vector or with alpha 6 cDNA in the pRc/CMV vector as described previously (36, 37).

Flow Cytometry-- Cells were sequentially washed with phosphate-buffered saline and Tris-buffered saline (24 mM Tris-HCl, pH 7.4, 137 mM NaCl, 2.7 mM KCl) and resuspended with Tris-buffered saline containing 5% bovine serum albumin and 0.02% sodium azide. Cells were then incubated with agonists (MnCl2, ligands, EDTA, or EGTA) for 15-30 min at 37 °C. Aliquots of 2 × 105 cells were then incubated for 45 min on ice with 9EG7 or other primary antibodies (2 µg/ml). Cells were washed three times with Tris-buffered saline containing 1% bovine serum albumin and 0.02% sodium azide and incubated for 45 min on ice with fluorescein isothiocyanate-conjugated goat anti-mouse or anti-rat IgG (Sigma). At least 3000 cells were then analyzed using a FACScan machine (Becton Dickinson, Oxnard, CA) to obtain mean fluorescence intensity values. To calculate percentages of alpha 2beta 1, alpha 3beta 1, alpha 4beta 1, and alpha 6beta 1 bearing the 9EG7 epitope in K562 cells, we solved for 9EG7% of alpha z according to Equation 1.
<UP>9EG7 total = </UP>(<UP>9EG7 % of &agr;</UP><SUB><UP>Z</UP></SUB>)(<UP>&agr;</UP><SUB><UP>Z</UP></SUB>)<UP> + </UP>(<UP>9EG7 % of &agr;</UP><SUB><UP>5</UP></SUB>)(<UP>&agr;</UP><SUB><UP>5</UP></SUB>) (Eq. 1)
To obtain the percentage of alpha 5beta 1 expressing 9EG7 (9EG 7% of alpha 5), we determined 9EG7 levels relative to total beta 1 (measured using mAb 13) in a cell line (K562) that only expresses alpha 5beta 1. alpha Z represents the mean fluorescence intensity value for alpha 2, alpha 3, alpha 4, or alpha 6 in each K562 transfectant. Levels of 9EG7 were measured after the addition of MnCl2, ligands, EDTA, or EGTA. However, levels of each alpha  chain and total beta 1 were determined using saturating amounts of the appropriate antibodies, added to cells untreated with any ligands, chelators, or divalent cations. In the transfectants, expression of alpha Z was typically 3-5-fold greater than alpha 5 and was always at least 2-fold greater.

Construction of Chimeric Molecules-- Cloning of human alpha 2 (38), alpha 3 (39), and alpha 4 (40) cDNA, as well as the generation of the chimeric constructs X2C4 and X2C5 (36), have been previously described. Construction of chimeric X2C3 was carried out by overlap extension (41). Two segments were generated in separate PCR reactions, each containing one set of outer and inner primers and either alpha 2 or alpha 3 cDNA. For alpha 2 cDNA we used the oligonucleotide primers 5'-CAG-TTG-TAA-TGC-AGA-TAT-CAA-TCC-3' (corresponding to nucleotides 3099-3122 of alpha 2 and containing the underlined EcoRV restriction site) and 5'-GAA-GCC-GCA-CTT-CCA-TAA-AAT-TGC-AAC-3' (corresponding to a cohesive end for nucleotides 3127-3119 of alpha 3 and 3513-3496 of alpha 2). For alpha 3 cDNA we used the primers 5'-GCA-ATT-TTA-TGG-AAG-TGC-GGC-TTC-TTC-3' (corresponding to a cohesive end for nucleotides 3499-3507 of alpha 2 and 3113-3130 of alpha 3) and 5'-GGT-CTA-GAA-GTC-ACA-CCA-GGT-GGG-3' (corresponding to nucleotides 3270-3252 of alpha 3 and introducing an XbaI site). A second PCR reaction was performed using these two segments and the two outer primers. The resulting alpha 2/alpha 3 chimeric product was digested with EcoRV and XbaI, ligated to alpha 2 cDNA, and finally cloned in pFneo (42). The X2C6 was prepared similarly by overlap extension PCR, starting with an alpha 2 cDNA clone (38) and alpha 6 cDNA from reverse transcriptase-PCR of mRNA from HT1080 cells.

To make the X3C0 construct, the alpha 3 subunit cytoplasmic domain was truncated just after the GFFKR sequence by creating a termination codon at position 3130 of alpha 3 cDNA. The stop codon (underlined) was introduced into the antisense primer 5'-G-AGT-GCG-GGC-TCA-TCG-CTT-GAA-GAA-GCC-GC-3' corresponding to nucleotides 3146-3120. The upstream primer (5'-GC-AGG-CGC-CGA-CAG-CTG-GAT-CCA-GGG-3') corresponds to nucleotides 2688-2713. The resulting PCR product was inserted into full-length alpha 3 cDNA in the pFneo vector. The construction of alpha 4 mutants D408E, N283E, D346E, D489E, D698E, D811E, and the combined D489E/D698E/D811E mutant (LEV) have been detailed elsewhere (43, 44).

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Manganese-dependent Induction of the 9EG7 Epitope Differs among beta 1 Integrins-- In K562 cells, Mn2+ induces the 9EG7 epitope on the beta 1 subunit of the alpha 5beta 1 integrin (6). To evaluate 9EG7 epitope expression on other beta 1 integrins, we compared K562 cells (which express only alpha 5beta 1) with K562-alpha 2, K562-alpha 3, and K562-alpha 4 transfectants. In the absence of added divalent cations, relatively low levels of the 9EG7 epitope were detected in K562-alpha 2, K562-alpha 3, or K562 cells (Fig. 1A, left panels), whereas moderate constitutive levels were seen in K562-alpha 4 cells. However, in the presence of 5 mM Mn2+, the 9EG7 epitope was increased greatly on K562-alpha 4 cells, moderately on K562 and K562-alpha 2 cells, but hardly at all on K562-alpha 3 cells. A Mn2+-induced conformational change was also detected using mAb 15/7, and again it was most obvious for K562-alpha 4 cells, less obvious for K562 and K562-alpha 2 cells, and not at all obvious for K562-alpha 3 cells (Fig. 1B).


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Fig. 1.   Differences among beta 1 integrins in 9EG7 and 15/7 epitope expression. K562 cells transfected with alpha 2, alpha 3, or alpha 4 integrin cDNA or control vector (K562) were incubated in the absence of added cations (Control) or in the presence of 5 mM MnCl2. Then cells were analyzed by flow cytometry for surface staining with mAb 9EG7 (dotted line) or rat anti-beta 1 mAb 13 (broken line) (A) and with 15/7 (dotted line) or mouse anti-beta 1 A-1A5 (broken line) (B). Negative control staining (solid lines) was with rat anti-E-selectin (A) or mouse P3 antibody (B).

For K562-alpha 2, K562-alpha 3, and K562-alpha 4 transfectants analyzed in Fig. 1, induction of the 9EG7 and 15/7 epitopes is partly due to a small contribution of endogenous alpha 5beta 1. Therefore, to obtain a more precise assessment of epitope induction on individual beta 1 integrins in transfected cells, we subtracted the contribution of endogenous alpha 5beta 1 in subsequent experiments. A more detailed analysis revealed that stimulation of the 9EG7 epitope by Mn2+ (up to 10 mM) was dose-dependent, and at all concentrations tested, induction was highest for alpha 4beta 1, intermediate for alpha 5beta 1, low for alpha 2beta 1, and essentially undetectable on alpha 6beta 1 or alpha 3beta 1 (Fig. 2A). A very similar hierarchy of Mn2+-dependent 9EG7 expression was found upon comparing Chinese hamster ovary (CHO) cells that constitutively express alpha 5beta 1 with CHO-alpha 2, -alpha 3, and -alpha 4 transfectants (not shown).


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Fig. 2.   Effects of Mn2+ on 9EG7 expression and cell adhesion. A, cells were stimulated with manganese and analyzed for 9EG7 expression by flow cytometry. Levels of 9EG7 (% alpha ) are presented relative to specific levels of alpha 2beta 1, alpha 3beta 1, alpha 4beta 1, or alpha 6beta 1, after subtraction of the contribution of background alpha 5beta 1 (see "Experimental Procedures"). B, manganese stimulates alpha 3beta 1 adhesive function. Adhesion of K562-alpha 3 cells to a laminin-5-containing matrix (deposited by A431 cells as described (30)) was determined at varying Mn2+ levels. Also, K562-alpha 3 cells (and control K562 cells) in 1 mM Mn2+ were tested in an adhesion assay (6) on purified laminin-5 (coated at 5 µg/ml).

Although Mn2+ stimulated little or no 9EG7 epitope expression on alpha 2beta 1, alpha 6beta 1, and alpha 3beta 1, the cation did efficiently stimulate cell adhesion mediated by these integrins. For example, adhesion of K562-alpha 3 cells to a laminin-5 containing matrix (made by A431 cells) was dose-dependently induced by Mn2+, with maximal stimulation at >= 0.1 mM (Fig. 2B). Also, Mn2+ (1 mM) strongly stimulated adhesion of K562-alpha 3 cells, but not control K562 cells, to purified laminin-5 (Fig. 2B). Elsewhere, 1 mM Mn2+ stimulated a >22-fold increase in affinity of purified soluble alpha 3beta 1 for laminin-5.3 In other experiments 0.1-1 mM Mn2+ stimulated to a similar extent K562-alpha 2 cell adhesion to collagen, K562-alpha 4 adhesion to VCAM-1, K562-alpha 6 adhesion to laminin-1, and untransfected K562 cell adhesion to fibronectin (not shown). Thus, failure to induce the 9EG7 epitope (Fig. 2A) is not due to lack of integrin responsiveness to Mn2+.

Immunoprecipitation results also showed preferential recognition of specific Mn2+-treated alpha beta 1 combinations by mAb 9EG7. Previously it was shown that 9EG7 immunoprecipitated a major fraction of alpha 4beta 1 from a Mn2+-treated lysate (23). In the presence of 1 mM Mn2+, the 9EG7 mAb precipitated a moderate amount (~10% of total alpha 5beta 1 available) of alpha 5beta 1 from a lysate of 125I-labeled K562 cells (not shown). In sharp contrast, the 9EG7 mAb precipitated little if any alpha 3beta 1 from K562-alpha 3 cell lysate in 1 mM Mn2+ (not shown).

Influence of Integrin alpha  Chain Cytoplasmic Domains on Mn2+-induced 9EG7 Expression-- Exchange of integrin alpha  chain cytoplasmic domains may alter integrin function and presumably also conformation (46). Thus, we hypothesized that variable induction of 9EG7 might be due to variations among alpha  chain cytoplasmic domains. However, exchange of the integrin alpha 2 cytoplasmic domain with the alpha 3 or alpha 6 tail did not cause 9EG7 expression to decrease (see K562-X2C3 and K562-X2C6 in Table I, top). Conversely, exchange with the alpha 4 or alpha 5 tails (K562-X2C4 and -X2C5) did not cause a marked increase in 9EG7 epitope expression. Similarly, deletion of the alpha 3 tail (K562-X3C0) did not prevent the 9EG7 epitope on alpha 3beta 1 from being largely unresponsive to Mn2+ (Table I, bottom).

                              
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Table I
9EG7 expression is not markedly influenced by alpha  chain cytoplasmic domains

Differential Induction of the 9EG7 Epitope by Soluble Ligands-- The 9EG7 epitope is also induced by soluble ligand (6), thus providing an opportunity for further comparisons among beta 1 integrins. In the presence of 5 mM Mg2+, which by itself has no effect on 9EG7 expression, CS1 peptide (DELPQLVTLPHPNLHGPEILDVPST, not shown) or VCAM-1 (Fig. 3) induced a high level of 9EG7 on alpha 4beta 1. Similarly, fibronectin induced a moderate level of 9EG7 on alpha 5beta 1, and collagen I induced a low 9EG7 level on alpha 2beta 1. However, soluble laminin-1 and laminin-5 induced little, if any, 9EG7 epitope on alpha 6beta 1 and alpha 3beta 1, respectively (Fig. 3). Also, the GD6 laminin peptide (at 0.5 mg/ml) that was described as a ligand for alpha 3beta 1 (47) induced no 9EG7 expression on K562-alpha 3 cells (not shown). In control experiments, mock-transfected K562 cells showed no induction of 9EG7 in response to maximal doses of soluble VCAM-1, collagen I, laminin-1, or laminin-5 (not shown).


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Fig. 3.   Induction of the 9EG7 epitope by soluble ligands. K562-alpha 4, K562, K562-alpha 2, K562-alpha 6, and K562-alpha 3 cells were preincubated with soluble VCAM-1, fibronectin, collagen I, laminin-1, or laminin-5, respectively, in 5 mM Mg2+ for 10 min at 37 °C. The mAb 9EG7 (2 µg/ml) was then added, and cell suspensions were incubated for an additional 45 min on ice, stained, and analyzed by flow cytometry.

Failure to induce 9EG7 on alpha 6beta 1 or alpha 3beta 1 was not due to inactivity of the respective integrins. Under experimental conditions similar to those used in Fig. 3 (i.e. 5 mM Mg2+, 2 µg/ml 9EG7), K562-alpha 6 and K562-alpha 3 cells displayed strong cell adhesion to laminin-1 and laminin-5, respectively (not shown). This adhesion was specifically mediated by alpha 6beta 1 or alpha 3beta 1 since it was not seen in control K562 cells (not shown). Furthermore, we show elsewhere that alpha 3beta 1 binds to laminin-5 in the presence of mAb 9EG7, with a Kd of ~0.088 µM.3 Thus at 0.3 µM laminin-5 (as used in Fig. 3), the majority of alpha 3beta 1 should be occupied by ligand.

As an extension of results showing 9EG7 induction by Mn2+ (Fig. 2A) or by ligand (Fig. 3), we next analyzed effects of Mn2+ and ligand (at 0.3 µM) added together. Stimulation of 9EG7 was already maximal (near 60%) when 0.3 µM VCAM-1 was added to K562-alpha 4 cells in the presence of 0.05 mM Mn2+ (Fig. 4). In comparison, a 20-fold greater level of Mn2+ (1.0 mM) was required to support comparable levels of 9EG7 (50-60%) on alpha 5beta 1, alpha 2beta 1, and alpha 6beta 1 when stimulated with 0.3 µM fibronectin, collagen, or laminin-1, respectively. In contrast, alpha 3beta 1 still showed minimal 9EG7 expression even in the presence of 1.0 mM Mn2+ plus 0.3 µM laminin-5. To summarize, results of 9EG7 induction by Mn2+ (Fig. 2A), ligand (Fig. 3), or both together (Fig. 4) show a generally consistent hierarchy among the different beta 1 integrins.


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Fig. 4.   Induction of the 9EG7 epitope by ligand plus manganese. K562 transfectants were preincubated (10 min at 37 °C) with Mn2+ and either 0.3 µM ligand () or control buffer (square ). The mAb 9EG7 was then added (as in Fig. 3), and flow cytometry was carried out.

Divalent Cation Chelating Agents Variably Affect 9EG7 Expression on beta 1 Integrins-- Major differences in 9EG7 expression among beta 1 integrins were also observed upon incubation with EDTA or EGTA. Consistent with previous results (6), 9EG7 expression was moderately induced on alpha 5beta 1 by EDTA (Fig. 5A) or EGTA (Fig. 5B). In comparison, the 9EG7 epitope was induced to a lower extent on alpha 2beta 1 and hardly at all on alpha 6beta 1 and alpha 3beta 1 (Fig. 5, A and B). On alpha 4beta 1, the 9EG7 epitope was diminished, rather than induced, upon the removal of divalent cations. For all integrins tested, similar effects were seen with EDTA and EGTA (Fig. 5, A and B), indicating that calcium, rather than magnesium or other divalent cations, is playing a key regulatory role.


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Fig. 5.   Effect of divalent cation chelation on 9EG7 expression. The various K562 cell transfectants were pretreated (30 min, 37 °C) with EDTA (A) or EGTA (B) and then analyzed for 9EG7 expression.

Further Exploration of Unique Properties of alpha 4beta 1-- Consistent with results in Figs. 2 and 5B, alpha 5beta 1 in K562 cells showed a moderate increase in the 9EG7 epitope upon Mn2+ or EGTA stimulation (Fig. 6A). Also, alpha 4beta 1 in Molt-4 cells showed a much stronger response to Mn2+ but showed a decrease rather than increase in response to EGTA (Fig. 6C). These results confirm for alpha 4beta 1 in Molt-4 cells (where it is the only integrin) the results seen for alpha 4beta 1 in K562-alpha 4 cells (Figs. 2 and 5B) where it is expressed together with alpha 5beta 1. Shown in Fig. 6B is the cumulative 9EG7 expression for both alpha 5beta 1 and alpha 4beta 1 in K562-alpha 4 cells. Notably, no net effect of EGTA is seen, presumably because positive effects on alpha 5beta 1 are balanced by negative effects on alpha 4beta 1. However, subtraction of the alpha 5beta 1 contribution (Fig. 6D), based on results seen with the K562 reference cells (Fig. 6A), reveals the specific contribution of alpha 4beta 1 (Fig. 6E). Notably, the results for alpha 4beta l in Fig. 6E (after calculation) are very similar to the results in Fig. 6C (where no calculations are necessary). Together, these results not only confirm the unique sensitivity of alpha 4beta 1 to EGTA but help to validate the method utilized for subtraction of background contributions due to alpha 5beta 1 in the various K562 transfectants.


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Fig. 6.   Regulation of the 9EG7 epitope on K562, K562-alpha 4, and Molt-4 cells. 9EG7 expression is indicated as percent of total beta 1 on K562 (A), K562-alpha 4 (B), and Molt-4 cells (C). In the right panel, total 9EG7 expression on K562-alpha 4 cells (B) has been separated into percent of alpha 5beta 1 (D) and percent of alpha 4beta 1 (E). Each experiment represents the mean ± S.D, for n = 3.

To highlight further the unique properties of alpha 4beta 1, we compared the effects of Ca2+ on ligand-induced 9EG7 expression for the various integrins. Calcium markedly inhibited the expression of 9EG7 induced on alpha 5beta 1 by the GRGDSP ligand mimetic peptide (Fig. 7A). Similarly, calcium inhibited collagen-dependent induction of 9EG7 epitope on alpha 2beta 1 in K562-alpha 2 cells (not shown). In contrast, Ca2+ stimulated 9EG7 expression induced by soluble VCAM-1 (Fig. 7B). These results are generally consistent with the divergent effects of Ca2+ inferred from Fig. 5B and further demonstrate that alpha 4beta 1 conformations are regulated in a specialized manner, distinct from other beta 1 integrins.


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Fig. 7.   Effects of calcium on ligand-induced 9EG7 expression. After preincubation in the presence or absence of 1.5 mM CaCl2, K562 cells were then incubated (20 min, 37 °C) with GRGDSP peptide (A), and K562-alpha 4 cells were incubated with soluble VCAM-1 (B).

Effects of alpha 4 Extracellular Domain Mutations on 9EG7 Expression-- The alpha 4 mutations N283E, D346E, and D408E, each within putative divalent cation sites, each have similar negative effects on alpha 4-dependent cell adhesion (43, 48). However, neither the high constitutive expression of 9EG7 on alpha 4beta 1 in K562 cells nor the high level of Mn2+ induction were appreciably altered by the D408E mutation (Fig. 8A) or the N283E and D346E mutations (not shown).


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Fig. 8.   Effects of alpha 4 mutations on 9EG7 expression. Constitutive and Mn2+-induced 9EG7 expressions were analyzed in K562-alpha 4 cells for wild type alpha 4 compared with the alpha 4 D408E mutant (A), or compared with the D489E, D698E, and D811E single mutants, and the LEV triple mutant (D489E/D698E/D811E) (B).

Unlike other integrin alpha  subunits, the alpha 4 sequence contains three extracellular "LDV" motifs (40). Within these motifs, mutations D489E and D698E each caused severe impairment of alpha 4-dependent cell adhesion, whereas D811E had no negative effect (44). Upon analysis of 9EG7 expression (Fig. 8B), we found that alpha 4 D489E and D811E mutations caused little or no loss of the 9EG7 epitope, either constitutively present or induced by Mn2+. In comparison, the D698E mutation caused a marked loss in the constitutive level of 9EG7 on alpha 4beta 1. Furthermore, when all three mutations were present at once (LEV mutant), the loss of constitutive 9EG7 expression was even more obvious. However, these deficits in constitutive expression were substantially overcome upon the addition of 1 mM Mn2+.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

mAb 9EG7 and 15/7 Are Not Universal Reporters of Either Ligand Occupancy or Mn2+ Activation of beta 1 Integrins-- Epitopes defined by anti-beta 1 mAb 9EG7 and 15/7 were initially suggested to correlate with integrin activation, but several striking counter-examples emerged in subsequent studies (6, 7, 16, 23).2 More recently, such antibodies have been shown to recognize ligand-induced binding sites on alpha 4beta 1 and alpha 5beta 1 (6, 7), thus causing them to be assigned to the LIBS or CLIBS category (25). Such results have produced the expectation that CLIBS antibodies would be universal reporters of ligand occupancy for beta 1 integrins. Here we show that expression of the 9EG7 epitope is not indicative of the extent of ligand occupancy, especially for the alpha 2beta 1, alpha 6beta 1, and alpha 3beta 1 integrins. At near-saturating ligand binding conditions, the 9EG7 epitope was indeed induced on the majority of alpha 4beta 1 integrins and on a significant fraction of alpha 5beta 1 integrins. However in contrast, the epitope was induced minimally on alpha 2beta 1 and alpha 6beta 1 and not at all on alpha 3beta 1.

At the high levels of soluble ligand utilized, in the presence of 9EG7 (Fig. 3), we should be near saturation for all of the ligand-integrin combinations tested. In this regard, we are aided by the fact that the 9EG7 antibody not only detects ligand occupancy, but itself stimulates ligand binding (6),3 and thus resembles other integrin-activating antibodies that increase affinity for soluble ligands by 5-50-fold (49, 50). The 9EG7 mAb decreases the alpha 3beta 1/laminin-5 binding Kd from >600 down to ~88 nM,3 which is well below the 300 nM dose of laminin-5 used in Fig. 3. Likewise, mAb 9EG7 markedly stimulated alpha 4beta 1 binding to soluble VCAM-1. Although VCAM binding in the absence of 9EG7 (Mg2+ conditions) was so weak that it was not measurable, binding in the presence of 9EG7 yielded a Kd of ~50 nM (not shown), which is comparable to VCAM-1 binding affinities of 10-100 nM reported elsewhere (48, 51). Thus, the 600 nM soluble VCAM-1 used in Fig. 3 is again well above saturation. In the presence of activating antibodies, Kd values of 9 and 18 nM have been observed for fibronectin binding to alpha 5beta 1 (49) and laminin binding to alpha 2beta 1 (50). Thus we can assume that ligand doses of 1200 nM should be well above saturation for these integrins also. Although estimated Kd values are not yet available for soluble laminin-1 binding to activated alpha 6beta 1, this is a strong interaction (52) that is highly stimulated by mAb 9EG7 (53), and again the Kd values should be well below the 600 nM dose of laminin-1 used in Fig. 3.

It is even more likely that saturation was achieved when soluble ligands were added in the presence of 9EG7 plus Mn2+ (as in Fig. 4). Notably, in those conditions we continued to see a similar hierarchy of 9EG7 induction (alpha 4beta 1 > alpha 5beta 1, alpha 2beta 1, alpha 6beta 1, > alpha 3beta 1). For alpha 3beta 1 binding to laminin-5, 9EG7 alone reduces the Kd by >7-fold (down to ~88 nM), and 1 mM Mn2+ alone reduces the Kd by >22-fold (down to ~27 nM).3 Thus we suspect that both 9EG7 and Mn2+ together would yield a Kd of <5 nM, but nonetheless 300 nM laminin-5 added under those conditions (Fig. 4) still showed no induction of the 9EG7 epitope.

A similar hierarchy of CLIBS epitope induction (alpha 4beta 1 > alpha 5beta 1 > alpha 2beta 1 > alpha 6beta 1 > alpha 3beta 1) was seen in response to ligand plus Mg2+, ligand plus suboptimal Mn2+ levels, Mn2+, or stimulation by Mn2+ alone. Wide differences in integrin responses to Mn2+ seen for K562 cells were confirmed using alpha  subunit-transfected CHO cells, and results obtained by immunoprecipitation of solubilized integrins matched those obtained by flow cytometric analysis at the cell surface. Also, Mn2+ stimulation results obtained with the 9EG7 antibody were confirmed using mAb 15/7.

Results shown here clarify our understanding of the stimulatory effects of Mn2+ on integrin function. From previous results (e.g. Refs. 6 and 7), it appeared that induction of CLIBS epitopes by Mn2+ may be an integral feature of integrin stimulation. Now for the first time, we show definitively that Mn2+-induced function does not necessarily correlate with the induction of CLIBS-type epitopes. As shown elsewhere, 1 mM Mn2+ strongly stimulated the affinity of purified recombinant soluble alpha 3beta 1 integrin for laminin-5,3 whereas we show here that even 10 mM Mn2+ failed to induce any expression of the 9EG7 epitope on cellular alpha 3beta 1 (e.g. Fig. 2A), and 1.0 mM Mn2+ did not induce 9EG7 epitope expression on solubilized alpha 3beta 1 (not shown). Likewise, 0.1-1 mM Mn2+ greatly activated ligand binding by solubilized alpha 2beta 1 (54) and alpha 6beta 1 (55), but 0.1-10 mM Mn2+ had negligible effect on 9EG7 epitope expression by cell surface alpha 2beta 1 or alpha 6beta 1. Thus, 9EG7 (and 15/7) epitope expression is clearly dissociated from integrin activation by Mn2+.

A practical implication of our results is that beta 1 CLIBS epitopes are reporters of ligand occupancy and/or Mn2+ stimulation only for a few beta 1 integrins (e.g. alpha 4beta 1 and alpha 5beta 1). In this regard, it is not surprising that most reports describing expression of the 9EG7 or 15/7 epitopes have involved leukocytes expressing alpha 4beta 1 (7, 27, 56, 57). Conversely, analysis of the other beta 1 integrins with the available "anti-CLIBS" reagents may lead to a substantial underestimation of ligand binding events and underestimation of integrin activation by Mn2+.

What Is the Meaning of 9EG7 Epitope Expression?-- If CLIBS epitopes do not accurately report either ligand occupancy or integrin activation, does that mean that they are irrelevant? Our analysis of CLIBS epitopes provides important information regarding integrin conformational unfolding that transcends considerations of only ligand binding affinity and occupancy. The constellation of events that leads to CLIBS epitope expression on beta 1 (e.g. denaturation, absence of an alpha  chain, ligand binding, etc.) suggests a general correlation with alpha -beta chain unfolding (25). The similar hierarchy of epitope exposure ([alpha 4beta 1] > alpha 5beta 1 > alpha 2beta 1 > alpha 6beta 1 > alpha 3beta 1) induced by three different agents (Mn2+, ligand, and EGTA) emphasizes that there may be fundamental differences in conformational flexibility among these integrins. Consistent with alpha 4 being most flexible, and most readily able to "uncover" beta 1 CLIBS epitopes, the alpha 4 subunit is also most easily dissociated from beta 1. Dissociation of alpha 4beta 1 readily occurs at elevated pH (8.5-9.0) and often occurs even at neutral pH (31, 58). In contrast, the alpha 2beta 1 subunits remain substantially associated, and alpha 3beta 1 is completely associated even after elution from mAb beads at pH 11.5 (59).

As shown schematically (Fig. 9) we indicate that each integrin can readily bind ligand and bind Mn2+. However, there is wide variation in the extent to which these events are accompanied by conformational unfolding to expose CLIBS epitopes. At one extreme, alpha 4beta 1 undergoes dramatic changes in response to either ligand or Mn2+ binding. At the other extreme, the conformation of alpha 3beta 1 is minimally altered, even when both ligand and Mn2+ are added (and bound) simultaneously. It is not integrin activation or ligand binding that differs, but rather the conformational changes that accompany these events.


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Fig. 9.   Schematic diagram of integrin conformational changes. Each beta 1 integrin can bind ligand or be activated by Mn2+. However, there is wide variation in the extent to which these events are accompanied by conformational changes. For example, alpha 4beta 1 shows the most conformational change (and the most induction of 9EG7 epitope), whereas alpha 3beta 1 shows negligible conformational change (and minimal induction of 9EG7 epitope). Also, it should be noted that CLIBS epitopes can sometimes be constitutively expressed, even in the absence of ligand occupancy or Mn2+ (e.g. 9EG7 on alpha 4beta 1).

The alpha 2, alpha 3, alpha 4, alpha 5, and alpha 6 tails have diverse sequences and differentially regulate functions such as cell migration, focal adhesion formation, and collagen gel contraction (60, 61). Also, exchange of cytoplasmic tails may alter adhesive function, through a change in extracellular ligand binding affinity (46). However, our tail deletion and exchange results show that specific alpha  tails do not control the extracellular domain conformational changes that we have measured. Hence, the differing degrees of conformational flexibility seen among the alpha 4beta 1, alpha 5beta 1, alpha 2beta 1, alpha 6beta 1, and alpha 3beta 1 integrins may be intrinsic to the extracellular domains.

Within the beta 1 molecule, 9EG7 binds to a site within residues 495-602 (6), whereas 15/7 mapped within residues 354-425 (16). Both of these regions may lie within the membrane-proximal integrin stalk and are presumed to be distant from ligand and divalent cation binding sites present within the integrin globular head (62, 63). According to a current paradigm, long range propagation of conformational changes within extracellular domains may control integrin cytoplasmic domain "outside-in" signaling events (19). Thus, we hypothesize that integrins showing extensive extracellular conformational changes (e.g. alpha 4beta 1 and alpha 5beta 1) might transmit a larger magnitude of conformational change across the plasma membrane. This would potentially then lead to greater beta -cytoplasmic tail exposure and enhanced formation of signaling complexes of the type stimulated by ligand occupancy (64).

Specialized Regulation of alpha 4beta 1 Conformation-- Integrin exposure to EGTA or EDTA yielded 9EG7 hierarchy (alpha 5beta 1 > alpha 2beta 1 > alpha 6beta 1 > alpha 3beta 1) comparable to that seen with ligand or Mn2+, except that alpha 4beta 1 gave an atypical response. Among the beta 1 integrins tested, a positive effect of calcium on 9EG7 epitope expression was seen only for alpha 4beta 1. This positive effect was manifested as follows: (i) an atypically high level of constitutive 9EG7 on alpha 4beta 1 that was inhibitable by EGTA, and (ii) calcium enhancement of 9EG7 epitope expression induced by soluble VCAM-1. These results are consistent with the ability of calcium to support alpha 4beta 1-mediated adhesion to VCAM-1 (65, 66). In sharp contrast, 9EG7 epitope expression on the alpha 2beta 1 and alpha 5beta 1 integrins was promoted by EGTA treatment and thus was inhibited by calcium. Thus, calcium may stabilize inactive conformations of those integrins. This is consistent with the inhibitory effects of calcium on the adhesive functions of alpha 2beta 1, alpha 5beta 1, and alpha 6beta 1 (67-69). Also, 9EG7 expression on alpha 3beta 1 was not promoted by calcium, in agreement with the general failure of calcium to support alpha 3beta 1-mediated cell adhesion (30, 70).

The D698E mutation in alpha 4 caused a loss of constitutive 9EG7 epitope expression (Fig. 8B) together with a loss of VCAM-1 binding (44). Treatment with EGTA also diminishes both 9EG7 expression and VCAM-1 binding. From these results, we propose that calcium may help to stabilize an active conformation of alpha 4beta 1. This specialized utilization of Ca2+ may allow alpha 4beta 1 on leukocytes to maintain a constitutive level of activation in the presence of high plasma calcium.

Because our D698E mutation and EGTA have similar negative effects on constitutive 9EG7 expression and VCAM binding, we hypothesize that Asp-698 could play a role in the specialized interaction of alpha 4beta 1 with calcium. Notably, Asp-698 in alpha 4 is part of a DXXXXXDXSSXS sequence that partly resembles a consensus DXXXXXDXSXS region that is present in the I domains of six different integrin alpha  chains, and within all eight integrin beta  chains (Fig. 10). Within beta  chains or alpha  chain I domains, Asp, Ser, and Ser residues (at positions 7, 9, and 11 in Fig. 10) play critical roles in divalent cation and ligand binding. Among alpha  chains 1-9, only alpha 2 and alpha 9 have Asp residues comparable to Asp-698 in alpha 4, and only alpha 9 has a sequence in that region (DXXXXXDXSXXS) partly resembling the DXXXXXDXSSXS sequence in alpha 4. Within alpha 4, further mutations (e.g. at positions 1, 9, 10, and 12) will be required to verify the importance of the alpha 4 DXXXXXDXSSXS sequence. Also it remains to be determined whether this region directly binds to Ca2+.


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Fig. 10.   Putative novel divalent cation binding site within the alpha 4 subunit. Consensus I domain and beta  chain sequences that contact divalent cation are derived from alignments of six different I domains and eight different integrin beta  sequences (44, 71). The nine different alpha  sequences (24, 45, 72, 73) have been described and aligned previously. Consensus residues are enclosed within double lines. Conservative substitutions are enclosed within single lines. Serines offset by one position are shaded.

In conclusion, we have demonstrated that monoclonal antibodies such as 9EG7 and 15/7 are not universal indicators of ligand occupancy or Mn2+ activation but nonetheless provide important insights into integrin function. First, we have provided evidence for major differences among beta 1 integrins in the extent to which conformational changes may accompany Mn2+, Ca2+, and ligand binding. Second, our results support the hypothesis that differences in extracellular conformational changes may be highly relevant toward understanding fundamental differences in outside-in signaling. Third, only through analysis of the 9EG7 epitope have we discovered that atypical positive effects of calcium on alpha 4beta 1 functions are paralleled by the presence of a conformation that requires ASP-698 in alpha 4.

    ACKNOWLEDGEMENTS

We thank Bob Burgeson (MGH East, Charlestown, MA) for purified laminin-5; Roy Lobb (Biogen) for VCAM-1; Dietmar Vestweber (Institute of Cell Biology, Münster, Germany) for mAb 9EG7; Ted Yednock (Athena Neurosciences) for mAb 15/7; Amy Skubitz (University of Minnesota) for GD6 peptide; and Isao Tachibana for assistance with control experiments.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant CA42368 and an American-Italian Cancer Foundation postdoctoral fellowship award (to G. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dana-Farber Cancer Institute, Rm. M-413, 44 Binney St., Boston, MA 02115. Tel.: 617-632-3410; Fax: 617-632-2662; E-mail: Martin_Hemler{at}DFCI.HARVARD.EDU.

1 The abbreviations used are: LIBS, ligand-induced binding site; CHO, Chinese hamster ovary cell; CLIBS, cation or ligand-influenced binding site; mAb, monoclonal antibody; VCAM-1, vascular cell adhesion molecule-1; PCR, polymerase chain reaction.

3 J. A. Eble, K. W. Wucherpfennig, L. Gauthier, P. Dersch, E. Krukonis, R. R. Isberg, and M. E. Hemler, submitted for publication.

2 D. Crommie and M. E. Hemler, submitted for publication.

    REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
References

  1. Hynes, R. O. (1992) Cell 69, 11-25[Medline] [Order article via Infotrieve]
  2. Zhu, X. Y., Ohtsubo, M., Bohmer, R. M., Roberts, J. M., Assoian, R. K. (1996) J. Cell Biol. 133, 391-403[Abstract]
  3. Zhang, Z. H., Vuori, K., Reed, J. C., Ruoslahti, E. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 6161-6165[Abstract/Free Full Text]
  4. Yurochko, A. D., Liu, D. Y., Eierman, D., and Haskill, S. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 9034-9038[Abstract]
  5. Ginsberg, M. H., Du, X., and Plow, E. F. (1992) Curr. Opin. Cell Biol. 4, 766-771[Medline] [Order article via Infotrieve]
  6. Bazzoni, G., Shih, D.-T., Buck, C. A., Hemler, M. E. (1995) J. Biol. Chem. 270, 25570-25577[Abstract/Free Full Text]
  7. Yednock, T. A., Cannon, C., Vandevert, C., Goldbach, E. G., Shaw, G., Ellis, D. K., Liaw, C., Fritz, L. C., Tanner, I. L. (1995) J. Biol. Chem. 270, 28740-28750[Abstract/Free Full Text]
  8. Mould, A. P., Garratt, A. N., Askari, J. A., Akiyama, S. K., Humphries, M. J. (1995) FEBS Lett. 363, 118-122[CrossRef][Medline] [Order article via Infotrieve]
  9. Cabañas, C., and Hogg, N. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 5838-5842[Abstract]
  10. Frelinger, A. L., III, Cohen, I., Plow, E., Smith, M. A., Roberts, J., Lam, S. C.-T., Ginsberg, M. H. (1990) J. Biol. Chem. 265, 6346-6352[Abstract/Free Full Text]
  11. Frelinger, A. L., III, Du, X. P., Plow, E. F., Ginsberg, M. H. (1991) J. Biol. Chem 266, 17106-17111[Abstract/Free Full Text]
  12. Frelinger, A. L., III, Lam, S. C.-T., Plow, E. F., Smith, M. A., Loftus, J. C., Ginsberg, M. H. (1988) J. Biol. Chem. 263, 12397-12402[Abstract/Free Full Text]
  13. Kouns, W. C., Wall, C. D., White, M. M., Fox, C. F., Jennings, L. K. (1990) J. Biol. Chem. 265, 20594-20601[Abstract/Free Full Text]
  14. Faull, R. J., Kovach, N. L., Harlan, J. M., Ginsberg, M. H. (1993) J. Cell Biol 121, 155-162[Abstract]
  15. Takada, Y., and Puzon, W. (1993) J. Biol. Chem. 268, 17597-17601[Abstract/Free Full Text]
  16. Puzon-Mclaughlin, W., Yednock, T. A., Takada, Y. (1996) J. Biol. Chem. 271, 16580-16585[Abstract/Free Full Text]
  17. Luque, A., Gómez, M., Puzon, W., Takada, Y., Sánchez-Madrid, F., and Cabañas, C. (1996) J. Biol. Chem. 271, 11067-11075[Abstract/Free Full Text]
  18. Honda, S., Tomiyama, Y., Pelletier, A. J., Annis, D., Honda, Y., Orchekowski, R., Ruggeri, Z., Kunicki, T. J. (1995) J. Biol. Chem. 270, 11947-11954[Abstract/Free Full Text]
  19. Du, X., Gu, M., Weisel, J. W., Nagaswami, C., Bennett, J. S., Bowditch, R., Ginsberg, M. H. (1993) J. Biol. Chem. 268, 23087-23092[Abstract/Free Full Text]
  20. Dransfield, I., and Hogg, N. (1989) EMBO J. 8, 3759-3765[Abstract]
  21. Ginsberg, M. H., Lightsey, A., Kunicki, T. J., Kaufmann, A., Marguerie, G., Plow, E. F. (1986) J. Clin. Invest. 78, 1103-1111[Medline] [Order article via Infotrieve]
  22. D'Souza, S. E., Haas, T. A., Piotrowicz, R. S., Byers-Ward, V., McGrath, D. E., Soule, H. R., Cierniewski, C., Plow, E. F., Smith, J. W. (1994) Cell 79, 659-667[Medline] [Order article via Infotrieve]
  23. Lenter, M., Uhlig, H., Hamann, A., Jeno, P., Imhof, B., and Vestweber, D. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 9051-9055[Abstract]
  24. Bossy, B., Bossy-Wetzel, E., and Reichardt, L. F. (1991) EMBO J. 10, 2375-2385[Abstract]
  25. Bazzoni, G., and Hemler, M. E. (1997) Trends Biochem. Sci. 23, 30-34[CrossRef]
  26. Akiyama, S. K., Yamada, S. S., Chen, W.-T., and Yamada, K. M. (1989) J. Cell Biol. 109, 863-875[Abstract]
  27. Picker, L. J., Treer, J. R., Nguyen, M., Terstappen, L. W. M. M., Hogg, N., Yednock, T. (1993) Eur. J. Immunol. 23, 2751-2757[Medline] [Order article via Infotrieve]
  28. Hemler, M. E., Ware, C. F., and Strominger, J. L. (1983) J. Immunol. 131, 334-340[Abstract/Free Full Text]
  29. Bergelson, J. M., St. John, N., Kawaguchi, S., Pasqualini, R., Berdichevsky, F., Hemler, M. E., Finberg, R. W. (1994) Cell Adhes. Commun. 2, 455-464[Medline] [Order article via Infotrieve]
  30. Weitzman, J. B., Pasqualini, R., Takada, Y., and Hemler, M. E. (1993) J. Biol. Chem. 268, 8651-8657[Abstract/Free Full Text]
  31. Hemler, M. E., Huang, C., Takada, Y., Schwarz, L., Strominger, J. L., Clabby, M. L. (1987) J. Biol. Chem. 262, 11478-11485[Abstract/Free Full Text]
  32. Pujades, C., Teixidó, J., Bazzoni, G., and Hemler, M. E. (1996) Biochem. J. 313, 899-908[Medline] [Order article via Infotrieve]
  33. Lee, R. T., Berditchevski, F., Cheng, G. C., Hemler, M. E. (1995) Circ. Res 76, 209-214[Abstract/Free Full Text]
  34. Lemke, H., Hammerling, G. J., Hohmann, C., and Rajewsky, K. (1978) Nature 271, 249-251[Medline] [Order article via Infotrieve]
  35. Lobb, R. R., Chi-Rosso, G., Leone, D. R., Rosa, M. D., Newman, B. M., Luhowskyj, S., Osborn, L., Schiffer, S. G., Benjamin, C. D., Dougas, I. G., Hession, C., Chow, E. P. (1991) Biochem. Biophys. Res. Commun. 178, 1498-1504[Medline] [Order article via Infotrieve]
  36. Mannion, B. A., Berditchevski, F., Kraeft, S.-K., Chen, L. B., Hemler, M. E. (1996) J. Immunol. 157, 2039-2047[Abstract]
  37. Berditchevski, F., Bazzoni, G., and Hemler, M. E. (1995) J. Biol. Chem. 270, 17784-17790[Abstract/Free Full Text]
  38. Takada, Y., and Hemler, M. E. (1989) J. Cell Biol. 109, 397-407[Abstract]
  39. Takada, Y., Murphy, E., Pil, P., Chen, C., Ginsberg, M. H., Hemler, M. E. (1991) J. Cell Biol. 115, 257-266[Abstract]
  40. Takada, Y., Elices, M. J., Crouse, C., and Hemler, M. E. (1989) EMBO J. 8, 1361-1368[Abstract]
  41. Higuchi, R., Krummel, B., and Saiki, R. K. (1988) Nucleic Acids Res. 16, 7351-7367[Abstract]
  42. Hahn, W. C., Menzin, E., Saito, T., Germain, R. N., Bierer, B. E. (1993) Gene (Amst.) 127, 267-268[CrossRef][Medline] [Order article via Infotrieve]
  43. Masumoto, A., and Hemler, M. E. (1993) J. Cell Biol. 123, 245-253[Abstract]
  44. Ma, L., Conrad, P. J., Webb, D. L., Blue, M.-L. (1995) J. Biol. Chem. 270, 18401-18407[Abstract/Free Full Text]
  45. Palmer, E. L., Rüegg, C., Ferrando, R., Pytela, R., and Sheppard, D. (1993) J. Cell Biol. 123, 1289-1297[Abstract]
  46. O'Toole, T. E., Katagiri, Y., Faull, R. J., Peter, K., Tamura, R. N., Quaranta, V., Loftus, J. C., Shattil, S. J., Ginsberg, M. H. (1994) J. Cell Biol. 124, 1047-1059[Abstract]
  47. Gehlsen, K. R., Sriramarao, P., Furcht, L. T., Skubitz, A. P. N. (1992) J. Cell Biol. 117, 449-459[Abstract]
  48. Pujades, C., Alon, R., Yauch, R. L., Masumoto, A., Burkly, L. C., Chen, C., Springer, T. A., Lobb, R. R., Hemler, M. E. (1997) Mol. Biol. Cell 8, 2635-2645
  49. Faull, R. J., Kovach, N. L., Harlan, J. M., Ginsberg, M. H. (1994) J. Exp. Med. 179, 1307-1316[Abstract]
  50. Pfaff, M., Göring, W., Brown, J. C., Timpl, R. (1994) Eur. J. Biochem. 225, 975-984[Abstract]
  51. Jakubowski, A., Rosa, M. D., Bixler, S., Lobb, R., and Burkly, L. C. (1995) Cell Adhes. Commun. 3, 131-142[Medline] [Order article via Infotrieve]
  52. Delwel, G. O., de Melker, A. A., Hogervorst, F., Jaspars, L. H., Fles, D. L. A., Kuikman, I., Lindblom, A., Paulsson, M., Timpl, R., Sonnenberg, A. (1994) Mol. Biol. Cell 5, 203-215[Abstract]
  53. Driessens, M. H. E., Van Rijthoven, E. A. M., Kemperman, H., Roos, E. (1995) Cell Adhes. Commun. 3, 327-336[Medline] [Order article via Infotrieve]
  54. Elices, M. J., and Hemler, M. E. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 9906-9910[Abstract]
  55. Sonnenberg, A., Gehlsen, K. R., Aumailley, M., and Timpl, R. (1991) Exp. Cell Res. 197, 234-244[Medline] [Order article via Infotrieve]
  56. Arroyo, A. G., García-Vincuña, R., Marazuela, M., Yednock, T. A., González-Amaro, R., Sánchez-Madrid, F. (1995) Eur. J. Immunol. 25, 1720-1728[Medline] [Order article via Infotrieve]
  57. Kovach, N. L., Lin, N., Yednock, T., Harlan, J. M., Broudy, V. C. (1995) Blood 85, 159-167[Abstract/Free Full Text]
  58. Hemler, M. E., Huang, C., and Schwarz, L. (1987) J. Biol. Chem. 262, 3300-3309[Abstract/Free Full Text]
  59. Takada, Y., Strominger, J. L., and Hemler, M. E. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 3239-3243[Abstract]
  60. Chan, B. M. C., Kassner, P. D., Schiro, J. A., Byers, H. R., Kupper, T. S., Hemler, M. E. (1992) Cell 68, 1051-1060[Medline] [Order article via Infotrieve]
  61. Kassner, P. D., Alon, R., Springer, T. A., Hemler, M. E. (1995) Mol. Biol. Cell 6, 661-674[Abstract]
  62. Nermut, M. V., Green, N. M., Eason, P., Yamada, S., and Yamada, K. M. (1988) EMBO J. 7, 4093-4099[Abstract]
  63. Loftus, J. C., Smith, J. W., and Ginsberg, M. H. (1994) J. Biol. Chem. 269, 25235-25238[Free Full Text]
  64. Miyamoto, S., Teramoto, H., Coso, O. A., Gutkind, J. S., Burbelo, P. D., Akiyama, S. K., Yamada, K. M. (1995) J. Cell Biol. 131, 791-805[Abstract]
  65. Masumoto, A., and Hemler, M. E. (1993) J. Biol. Chem. 268, 228-234[Abstract/Free Full Text]
  66. Shimizu, Y., and Mobley, J. L. (1993) J. Immunol. 151, 4106-4115[Abstract/Free Full Text]
  67. Staatz, W., Rajpara, S. M., Wayner, E. A., Carter, W. G., Santoro, S. A. (1989) J. Cell Biol. 108, 1917-1924[Abstract]
  68. Mould, A. P., Akiyama, S. K., and Humphries, M. J. (1995) J. Biol. Chem. 270, 26270-26277[Abstract/Free Full Text]
  69. Sonnenberg, A., Modderman, P. W., and Hogervorst, F. (1988) Nature 360, 487-489
  70. Elices, M. J., Urry, L. A., and Hemler, M. E. (1991) J. Cell Biol. 112, 169-181[Abstract]
  71. Loftus, J. C., O'Toole, T. E., Plow, E. F., Glass, A., Frelinger, A. L., Ginsberg, M. H. (1990) Science 249, 915-918[Medline] [Order article via Infotrieve]
  72. Briesewitz, R., Epstein, M. R., and Marcantonio, E. E. (1993) J. Biol. Chem. 268, 2989-2996[Abstract/Free Full Text]
  73. Song, W. K., Wang, W., Forster, R. F., Bielser, D. A., Kaufman, S. J. (1992) J. Cell Biol. 117, 643-657[Abstract]


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