©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Critical Threonine and Aspartic Acid Residues within the I Domains of 2 Integrins for Interactions with Intercellular Adhesion Molecule 1 (ICAM-1) and C3bi (*)

Tetsuji Kamata (§) , Richard Wright (1), Yoshikazu Takada (¶)

From the (1) Department of Vascular Biology, The Scripps Research Institute, La Jolla, California 92037 and Oncology Research Program, Preclinical Research, Sandoz Research Institute, Sandoz Pharmaceutical Corporation, East Hanover, New Jersey 07936

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Integrins mediate signal transduction through interactions with multiple cellular or extracellular matrix ligands. Evidence is accumulating that the I (or A) domain, a 200-residue inserted sequence in some integrin subunits, mediates ligand binding. We have previously shown that Thr-221 of the putative ligand binding sites within 2 I domain of 21 is critical for binding to collagen (Kamata, T., and Takada, Y. (1994) J. Biol. Chem. 269, 26006-26010). Here we report that the mutation of Thr-206 of L blocks intercellular adhesion molecule 1 (ICAM-1) binding to L2 and mutation of Thr-209 of M blocks ICAM-1 and C3bi binding to M2. The data indicate the Thr residues of M and L corresponding to Thr-221 of 2 are critically involved in the ligand interaction with 2 integrins. The mutations of the Asp-137 and Asp-239 of L also block ICAM-1 binding to L2, as do the corresponding Asp residues of 2 or M in collagen/21 or C3bi/M2 interactions, respectively. These data suggest that these Thr and Asp residues, conserved among I domains, are critical for interaction with structurally distinct ligands (e.g. ICAMs, C3bi, and collagen).


INTRODUCTION

Integrins are a supergene family of cell adhesion receptor heterodimers, which recognize multiple ligands and mediate cell-cell and cell-extracellular matrix interactions (1, 2, 3, 4, 5, 6) . Integrins L2 and M2, which share a common 2 (CD18) subunit (M 95,000), are broadly distributed on all leukocytes of lymphoid and myelomonocytic lineage and mediate crucial adhesive recognition through their ability to bind multiple ligands (intercellular adhesion molecules (ICAMs),() fibrinogen, Factor X) (2, 7) .

Some integrin subunits have an I (or A) domain that consists of about 200 amino acids and is located close to the three metal binding sites (8, 9, 10, 11, 12, 13, 14, 15) . The I domain is present in the integrin L (8), M (9, 10, 11) , and X subunits (12) of the 2 subfamily, in the 1 (13, 14) and 2 (15) subunits of the 1 subfamily, and in the E subunit (16) of the 7 subfamily. The I domains are also present in other proteins, including von Willebrand factor (17) , cartilage matrix protein (18) , complement factors C2 and B (19, 20) , and collagen type VI (21, 22) . There is growing evidence that the I domains are important in ligand binding and receptor activation. A number of function-blocking or activation-dependent antibodies map to the I domains of L2 (23, 24) , M2 (25, 26) , 21 (27) , and 11 (28) (collagen/laminin receptors). The recombinant M I domain fusion protein binds to fibrinogen, ICAM-1 (26) , C3bi (29) , or hookworm neutrophil inhibitory factor (30, 31) . Also, an L I domain fusion protein binds to ICAM-1 (24) and an 2 I domain fusion protein to collagen (32) . Asp-140 and Asp-242 within the M I domain are critical for the binding of divalent cations to the I domain, and for binding of M2 to C3bi (33) (Fig. 1). Asp-151 and Asp-254 in the 2 I domain (corresponding to Asp-140 and Asp-242 of the M I domain) were shown to be critical for collagen/21 interaction as well (27) . The Asp-253 of the 1 I domain (corresponding to Asp-242 of M) has been shown to be critical in collagen IV/11 interactions (28) . Since Asp-151 or Asp-254 of 2 are not required for the recombinant 2 I domain/collagen interaction (27) , it is likely that the Asp residues are indirectly involved in the collagen binding to 21 probably through divalent cation binding to the I domain (Fig. 1).


Figure 1: Conserved Asp and Thr residues among I domains potentially important in ligand binding. The putative ligand binding sites of the 2 I domain were recognized by function-blocking anti-2 mAbs (27). Thr-221 of 2 has been identified as critical in collagen binding by alanine scanning mutagenesis of the 2 I domain (32). Asp-140 and Asp-242 were critical in divalent-cation binding to the M I domain and in binding to C3bi (33). Asp-151 and Asp-254 of 2 were critical in binding to collagen (27). Asp-137, Asp-239, and Thr-206 of L, and Thr-209 of M were tested for ligand interactions in this study.



Recently we have shown that function-blocking anti-2 mAbs recognize a small region (residues 173-259) of the 2 I domain, suggesting that the region may be involved in ligand binding to 21 (putative ligand binding sites) (27) . Notably, we identified Thr-221 of the putative ligand binding site as critical residue for binding of collagens to both the recombinant 2 I domain and intact 21 (32), suggesting that Thr-221 is directly involved in collagen binding. In the present study we introduced mutations of the conserved Thr and Asp residues to study their role in 2 integrin/ICAM-1 interactions. The data suggest that the conserved Thr and Asp residues are equally critical in 2 integrin/ICAM-1 or C3bi interactions, and that there may be similar ligand recognition mechanisms among different I domains that recognize structurally distinct ligands (e.g. C3bi, collagens, ICAM-1).


EXPERIMENTAL PROCEDURES

Materials

Monoclonal antibodies TS1/22 (L) (34) and TS1/18 (2) (34) were obtained from ATCC. mAbs OKM1 (M) (35) and 60.3 (2) (36) are gifts of D. Altieri (Yale University, New Haven). M and 2 (37) cDNAs were obtained from D. D. Hickstein (University of Washington, Seattle). L cDNA was obtained by amplifying partial cDNAs from a human peripheral leukocyte cDNA library by a polymerase chain reaction using synthetic oligonucleotides from the published sequence (8) and ligating the cDNA fragments. ICAM-1/mouse C fusion protein was obtained from D. Dottavio (Sandoz Research Institute, East Hanover, NJ). C3bi was purified as described elsewhere (38) .

Adhesion Assay

Wells of 96-well Immulon-2 plates were coated with goat anti-mouse C chain polyclonal antibody (Caltag, South San Francisco, CA) (0.2 µg/well in 100 µl of PBS) overnight at 4 °C. After washing the wells with PBS, wells were incubated with ICAM-1/mouse C fusion protein (0.4 µg/well) at room temperature for 1 h. Unoccupied protein binding sites were blocked by incubating the wells with 1% heat-denatured bovine serum albumin (BSA) for 1 h at room temperature. After washing with PBS, cells (10/well) were added and incubated for 1 h at 37 °C in 100 µl of Tyrode, 5 mM Hepes buffer, pH 7.4, in the presence of 0.1% BSA and 0.1 mM MnCl. After rinsing the wells to remove unbound cells, bound cells were quantified by assaying endogenous phosphatase activity (39) . Adhesion of cells to C3bi was measured as described above except that purified C3bi (1 µg/well in 100 µl of PBS) was directly coated onto wells.

Other Methods

Site-directed mutagenesis was carried out using unique restriction site elimination (40) . The presence of mutations was confirmed by DNA sequencing. M, L, and 2 cDNAs were subcloned into pBJ-1 (41) . Transfection into CHO cells by electroporation, selection of the transfected cells with G418, immunoprecipitation, and flow cytometric analysis were carried out as previously described (42) .


RESULTS AND DISCUSSION

Expression of Wild-type (wt) and Mutant 2 Integrins on CHO Cells

We introduced T206A (Thr-206 to Ala), T208A, D137A, or D239A mutations into the I domain of the L subunit. wt and mutant L cDNA in pBJ-1 expression vector were used to transfect CHO cells together with wt 2 cDNA. Forty-eight h after transfection, CHO cells transiently expressing wt and mutant L2 were surface I-labeled and analyzed by immunoprecipitation with anti-L and anti-2 antibodies. Fig. 2a demonstrates that wt and mutant L (M 175,000) and 2 (M 95,000) were co-precipitated in each case. The data indicate that wt and mutant L are expressed on the surface of CHO cells in association with wt 2, suggesting that the mutations do not affect the subunit association of L2. We cloned CHO cell lines stably expressing wt or mutant L2 using cell sorting to obtain cells expressing high levels of wt or mutant L2 (Fig. 2b). While the transient expression of T208A mutant was low, we obtained clonal CHO cells stably expressing the mutant.


Figure 2: Surface expression of wt and mutant L2 on CHO cells. a, immunoprecipitation of L and 2 subunits from CHO cells transiently expressing wt or mutant L2. Cells were surface I-labeled and lysates were immunoprecipitated with anti-L (TS1/22) or anti-2 (60.3) mAbs. Immunoprecipitated samples were analyzed by SDS-polyacrylamide gel electrophoresis (7% gel) under nonreducing conditions. Dried gels were exposed for 3 days. Expression level of T208A L mutant was low as compared to other mutants. b, CHO cells stably expressing wt or mutant L2 were cloned using cell sorting. Clones were stained with anti-L (TS1/22) or anti-2 (TS1/18) mAbs and analyzed in a cell sorter. Levels of L2 expression were similar in each case, except for CHO cells expressing T208A L mutant/2, which show a significantly lower expression level.



We also introduced the T209A and T211A mutations into M cDNA to examine the effects of the mutations on M2/ICAM-1 interaction. CHO cell lines expressing wt and mutant M in association with wt 2 were developed using the same strategy described above. Upon immunoprecipitation, wt and mutant M were shown to be expressed on CHO cells in association with wt 2 (Fig. 3). The cloned CHO cells homogeneouslyexpressed high level M2 assayed by flow cytometry (data not shown).


Figure 3: Surface expression of wt and mutant M2 on CHO cells. CHO cells stably expressing wt or mutant M2 were cloned using cell sorter. Cells were surface I-labeled and lysates were immunoprecipitated with anti-M (OKM1) or anti-2 (60.3) mAbs. Immunoprecipitated samples were analyzed by SDS-polyacrylamide gel electrophoresis (7% gel) under nonreducing conditions. Dried gels were exposed for 3 days.



The Effects of Mutations within the I Domain of L or M on the Interaction with ICAM-1

We examined the adhesion of CHO cells expressing wt or mutant L or M (with wt 2). All the CHO cell lines examined adhered very well to fibronectin via endogenous 51 (43), but not to heat-denatured BSA. The ICAM-1/C fusion protein concentration at approximately 0.2 µg/well gives adhesion of 50% of added CHO cells expressing wt L (Fig. 4a) and the concentration at 0.4 µg/well was used throughout the experiments. Mn was required to induce the adhesion of wild-type L transfectants. CHO cells expressing wt L/wt 2 or T208A mutant L/wt 2 bound to the ICAM-1, but the CHO cells expressing T206A mutant L/wt 2 did not (Fig. 4b). T206A mutant L did not respond to the increase in coating concentration of ICAM-1 up to 1.6 µg/well (Fig. 4a). Also, CHO cells expressing T211A mutant M/wt 2 adhered to ICAM-1, but those expressing T209A mutant M/wt 2 did not (Fig. 5, a and b). Cells expressing the T209A mutant M did not respond to the increase in ICAM-1 coating concentration up to 1.6 µg/well (Fig. 5a). The T209A mutant M did not show binding to C3bi, but T211A had no effect on the binding (Fig. 5c). These data clearly show that Thr-206 of L or Thr-209 of M that correspond to Thr-221 of 2, are critical for adhesion to ligands. By contrast, mutation of nearby residues Thr-208 of L or Thr-211 of M have no effect.


Figure 4: Effects of mutations within L I domain on ICAM-1/L2 interactions. a, cells (10/well) were added to wells of 96-well Immulon-2 plates coated with ICAM-1/mouse C fusion protein (0-2.0 µg/well in 100 ml of PBS) and incubated for 1 h at 37 °C in 100 µl of Tyrode, 5 mM Hepes buffer, pH 7.4, in the presence of 0.1% BSA and 0.1 mM MnCl. After rinsing the wells to remove unbound cells, bound cells were quantified as described under ``Experimental Procedures.'' b, cell adhesion assay was performed with saturating concentrations of ICAM-1/mouse C fusion protein (0. 4 µg/well) as described above. Most (91.8-98.9%) of added cells of all cell lines bound to fibronectin (coating concentration at 1 µg/well in 100 µl of PBS). Expression (%) and mean fluorescent intensity of L on CHO cell lines with mAb TS1/22: parent CHO cells (1.8%, 2.8), wt L (94.3%, 458), T206A L (94.6%, 428), T208A L (99.0%, 135), D137AL (99.8%, 532), and D239A L (98.3%, 437). The data indicate that mutation of Thr-206, Asp-137, and Asp-239 blocked the ICAM-1/L2 only or C3bi interaction but that mutation of Thr-208 did not.




Figure 5: Effects of Thr-209 and Thr-211 mutations of M on the interaction of M2 with ICAM-1 or C3bi. Cell adhesion to ICAM-1 (a, b) or to C3bi (c) were determined as described above except that C3bi (1 µg/well) was directly coated onto wells. Most (93.4-99.3%) added cells of all cell lines bound to fibronectin. Expressions (%) and mean fluorescent intensity of M on CHO cell lines with mAb OKM1: parent CHO cells (1.4%, 2.1), wt M (97.4%, 133), T209A M (93.4%, 152), T211A M (99.3%, 334). The data indicate that mutation of Thr-209 in M blocked the ICAM-1 or C3bi/M2 interactions but that mutation of Thr-211 did not.



CHO cells expressing D137A or Asp-239 mutant L/wt 2 did not show significant binding to ICAM-1 (Fig. 4b). These data suggest the conserved Asp-137 and Asp-239 of L are critical for ICAM-1/L2 interaction, as Asp-151 and Asp-254 of 2 are for collagen/21 (27) or Asp-140 and Asp-242 of M are required for C3bi/M2 (33) interactions.

Potential Roles of the Conserved Asp Residues in Ligand/I Domain Interactions

While mutation of Asp-151 or Asp-254 of 2 completely block the binding of 21 to collagen, Asp-151 (and probably Asp-254 as well) are not critical in collagen binding to the recombinant 2 I domain fusion protein (27, 32) . We demonstrated that recombinant 2 I domain/collagen interaction is not divalent cation dependent, while 21/collagen interaction requires cations (32) . This suggests that Asp-151 and Asp-254 are involved in the regulation of 21 conformation rather than in the actual binding to collagen. Such regulation could occur through effects on accessibility of ligands to the binding sites or maintenance of active conformation of the ligand binding site. These effects are likely to be mediated through binding of divalent cations to the I domain. In this study we established that the Asp residues at position 137 and 239 of L I domain are also critical for binding to ICAM-1. These data indicate the possibility that there is a regulatory mechanism of ligand binding through divalent cation binding common to I domain-containing integrins.

Critical Roles of the Conserved Thr Residues in Ligand/I Domain Interaction

We have reported that the T221A mutation of 2 almost completely blocks binding of collagen to both intact 21 and recombinant 2 I domain (32) . By contrast, D151A or D254A mutations block binding of 21 but not recombinant I domain (27) . The present study establishes that the corresponding Thr residues in L and M at positions 206 and 209, respectively, are critical for the interaction with ICAM-1. The present data suggest that there may be a common mechanism of interaction (requiring the conserved Thr residues) between I domains and ligands as structurally distinct as the IgG superfamily protein ICAM-1, C3bi, and collagen type I. The Thr-206 of L are well conserved among integrin I domains (Fig. 6), whereas sequences around these Thr residues are not. It will be interesting to examine if the conserved Thr residues are equally involved in ligand interactions of other I domain-containing integrins, 11, X2, and E7, or in the interactions between 2 integrins and the other ligands (e.g. ICAM-2, fibrinogen, neutrophil inhibitory factor).


Figure 6: The critical Thr residues are conserved among I domains. The Thr residues (marked by *) critical in ligand binding in 2, L, and M are conserved among I domains, but amino acid sequences around the Thr residues are not. L (8), M (9-11), X (12), 1 (13, 14), 2 (15), E (16), von Willebrand factor (vWf) (17), cartilage matrix protein (CMP) (18), complement factors C2 and B (19, 20), and collagen type VI (21, 22).



L2 and M2 integrins play important roles in the inflammatory response such as arthritis, glomerulonephritis, and tissue injury induced by ischemia-reperfusion and therefore the efforts have been made toward modulating their in vivo functions using mAbs to 2 and ligands (1, 2, 7) . The present data may be useful to focus the study for designing specific inhibitors of ICAM-1/2 integrin interactions on a region of the I domain.

Ueda et al.(29) recently reported that C3bi directly bound to recombinant M I domain and a linear peptide A7 (residue 232-245) of the I domain blocked the binding. The peptide A7 includes Asp-242, but not Thr-209 (29) . The peptide A5 that contains Thr-209 had no effect on C3bi/M I domain interaction (29) . These findings are not necessarily contradictory to the data in the present study if more than one potential binding site is involved in ligand/I domain interactions. Also, linear peptides are likely to have random conformation and might not be inhibitory if conformation of the corresponding protein sequence is critical for ligand recognition. More structural and mutagenesis studies will be required for detailed understanding of the ligand/I domain interactions, especially the roles of the critical Thr and Asp residues.

After this study was submitted, a report describing the crystal structure of the I domain of M appeared (44) . The domain adopts a classic / ``Rossmann'' fold and contains an unusual Mg coordination site at its surface. Interestingly, Asp-140, Asp-242, and Thr-209 are all involved in the coordination of Mg on the surface, underscoring the importance of these residues in ligand binding.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants GM47157 and GM49899 and funding from Sandoz Pharmaceuticals Corporation. This is Publication 9112-VB from The Scripps Research Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a research Fellowship from the American Heart Association, California Affiliate.

To whom correspondence should be addressed: Dept. of Vascular Biology, The Scripps Research Institute, VB-4, 10666 North Torrey Pines Rd., La Jolla, CA 92037. Tel.: 619-554-7122; Fax: 619-554-6408.

The abbreviations used are: ICAM-1, intercellular adhesion molecule 1; mAb, monoclonal antibody; PBS, phosphate-buffered saline; BSA, bovine serum albumin; CHO, Chinese hamster ovary; wt, wild type.


ACKNOWLEDGEMENTS

We thank to D. Altieri for antibodies; D. Dottavio for recombinant ICAM-1; D. Hickstein for cDNAs; and Drs. M. Schwartz, S. Shattil, and J. Smith for critical reading of the manuscript.


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