©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
(CD18) Mutations Abolish Ligand Recognition by I Domain Integrins LFA-1 (, CD11a/CD18) and MAC-1 (, CD11b/CD18) (*)

(Received for publication, July 18, 1994; and in revised form, October 11, 1994)

Mary Lynn Bajt (§) Tom Goodman Sarah Lea McGuire

From the From Cell Biology and Inflammation Research, Upjohn Laboratories, Kalamazoo, Michigan 49001

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The ``I'' domains of the beta(2) (CD18) leukocyte integrins are implicated in ligand binding function. Moreover, rather than recognizing linear peptide sequences, this class of integrins generally recognizes multiple discontinuous sites on immunoglobulin superfamily adhesion receptors. A conserved cluster of oxygenated residues is involved in ligand recognition by beta(1) and beta(3) integrins. In the present study, we evaluated the role of this region in the I domain-containing beta(2) integrins. Recombinant alpha(L)beta(2) (LFA-1, CD11a/CD18) and alpha(M)beta(2) (MAC-1, CD11b/CD18) were expressed on COS cells, and function was assessed by adhesion to ICAM-1 or iC3b, respectively. Alanine substitution at position Asp or Ser in beta(2) produced a complete loss in the capacity of both alpha(L)beta(2) and alpha(M)beta(2) to support cell adhesion. In contrast, substitution at Asp or Ser resulted in loss of beta(2) surface expression when co-transfected with alpha(L) (CD11a) or alpha(M) (CD11b). These data provide the first evidence for involvement of the beta(2) subunit in ligand binding to I domain integrins.


INTRODUCTION

The beta(2) integrin subfamily mediates leukocyte interactions during normal immune and inflammatory responses(1, 2, 3) . The leukocyte integrins, alpha(L)beta(2) (LFA-1, CD11a/CD18), alpha(M)beta(2) (MAC-1, CD11b/CD18), and alpha(X)beta(2) (p150, 95, CD11c/CD18), share a common beta subunit (CD18) that associates with three unique but homologous alpha subunits(4) . Unique to the three alpha subunits of the beta(2) integrins and alpha(1) and alpha(2) subunits of the beta(1) integrins is a 200-amino acid inserted or ``I'' domain(5, 6, 7, 8, 9, 10) .

Each of the beta(2) integrins contributes to leukocyte adhesive interaction by recognition of a multiplicity of protein ligands. alpha(L)beta(2) recognizes three members of the immunoglobulin superfamily: ICAM-1(11) , ICAM-2(12) , and ICAM-3 (13) . alpha(M)beta(2) binds the complement protein iC3b(14, 15) , ICAM-1(16) , fibrinogen(17) , and factor X(18) . Little information about the structural features involved in the binding of the various adhesive proteins to the beta(2) integrins is known. Perhaps the best characterized domain implicated in ligand recognition is a discrete region of the beta(3) (GPIIIa) subunit of the platelet integrin alphabeta(3) (GPIIb/IIIa)(19) . Based on mutational analysis, a highly conserved cluster of oxygenated residues within this region of beta(3) has been implicated in the ligand recognition function of alphabeta(3)(20, 21) . Further, substitution of the corresponding residue in beta(1) integrin abolished ligand recognition of alpha(5)beta(1)(22) . The remarkable conservation of this region among the known integrin beta subunits suggests that this region may also be involved in ligand recognition in all other integrins (Fig. 1). However, unlike the beta(1) and beta(3) integrins, the beta(2) leukocyte integrins do not appear to utilize small linear peptide sequences to bind but apparently recognize multiple discontinuous sites on their adhesive proteins(32, 33, 34) . In addition, epitope mapping of functional blocking monoclonal antibodies implicate the I domains as a ligand binding site in alpha(L)beta(2), alpha(M)beta alpha(X)beta(2), and alpha(2)beta(1)(35, 36, 37, 38, 39) . Thus, I domain integrins may utilize a distinct ligand recognition mechanism from other integrins.


Figure 1: Alignment of the conserved cluster of oxygenated residues of the human beta integrin subunits. The amino acid sequences of human beta(1)(23) , beta(2)(24) , beta(3)(25) , beta(4)(26, 27) , beta(5)(28) , beta(6)(29) , beta(7)(30) , and beta(8)(31) are shown using the single letter code. Squaredresidues were mutated to alanine. Asterisks indicate previously identified residues in beta(1) and beta(3) critical for ligand recognition (20, 21, 22) .



To test this idea, we evaluated the role of the integrin beta(2) subunit in ligand recognition by the I domain integrins alpha(L)beta(2) and alpha(M)beta(2). Recombinant alpha(L)beta(2) and alpha(M)beta(2) were expressed on COS cells, and function was assessed by the adhesion to ICAM-1 and iC3b, respectively. Alanine substitution at the conserved Asp or Ser of beta(2) resulted in a complete loss of the capacity of both alpha(L)beta(2) and alpha(M)beta(2) to support cell adhesion. These data implicate the beta subunits of I domain integrins in ligand binding and implicate Asp and Ser as part of a ligand binding domain in alpha(L)beta(2) and alpha(M)beta(2).


MATERIALS AND METHODS

Monoclonal Antibodies

Purified murine monoclonal anti-alpha(L) (mAb (^1)38) (40) was kindly provided by Nancy Hogg (Imperial Cancer Research Fund, London). Murine monoclonals anti-alpha(L) (mAb TS1/22)(41) , anti-alpha(M) (mAb LM2/1)(42) , and anti-beta(2) (TS1/18) (41) were prepared as IgG fraction from ascites using hybridoma cells obtained from the American Type Culture Collection. Murine monoclonal anti-ICAM-1 (mAb 8.4A6) (43) were purified by protein A chromatography. The following murine mAbs against human antigens were used as culture supernatants: 904 (anti-alpha(M))(36) , M1/70 (anti-alpha(M))(14) , TS2/4 (anti-alpha(L))(41) , and R15.7 (anti-beta(2))(44) . Murine anti-human alpha(M) (mAb 3H5) and anti-human beta(2) (mAb 8H1) were prepared in our laboratory by immunizing BALB/c mice with affinity-purified alpha(M)beta(2) from polymorphonuclear leukocytes obtained from healthy donors(16) . These antibodies have been evaluated for subunit specificity and their ability to inhibit neutrophil adhesion to interleukin-1-stimulated HUVEC monolayers, keyhole limpet hemocyanin, fibrinogen, and affinity-purified human ICAM-1. The purified murine monoclonal anti-iC3b antibody was purchased from Quidel (San Diego, CA).

Mutagenesis and Transfection

The full-length wild-type beta(2) cDNA (24) cloned into Bluescript (Stratagene) was used to introduce nucleotide base substitutions by site-directed mutagenesis(45) . Mutant clones were confirmed by nucleotide sequencing of the mutated region. A 2.8-kilobase insert containing portions of the 3`- and 5`-untranslated sequences was isolated by digestion with HindIII and NotI and subcloned into the HindIII and NotI sites of the expression vector pcDNA1/neo or pcDNA3 (Invitrogen Corp.). Mutant clones were sequenced to verify the absence of any other substitutions.

COS-7 cells were transfected by electroporation with the wild-type beta(2) or the mutant beta(2) constructs together with the wild-type alpha(L) subunit (9) or alpha(M) subunit (47) cDNAs subcloned into the expression vector pCDM8 (Invitrogen Corp.). Cells were evaluated for surface expression and adhesion 48 h after electroporation. Flow cytometric and immunoprecipitation analyses were carried out as previously described (48) .

Adhesion Assays

Microtiter plates were coated with 100 µl of purified ICAM-1 diluted 1:10 in 0.1 M sodium bicarbonate, pH 8.0, overnight at 4 °C. The amount of ICAM-1 used to coat the microtiter wells (150 ng/well) was found in preliminary studies to be sufficient to support maximal JY cell adhesion. The remaining binding sites on the plastic were blocked with 1% bovine serum albumin. Microtiter plates were coated with iC3b as previously described(49) . Transfected cell lines were fluorescently labeled with 2`7`-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (Molecular Probes, Inc., Eugene, OR)(13) . A 50-µl aliquot of 8 times 10^4 cells was plated in triplicate to the microtiter wells. In addition, some of the wells were coated with 15 µg/ml mAb TS1/18 (anti-CD18). To inhibit adhesion to iC3b-coated wells, 50 µl of mAb 3H5 (anti-CD11b) or TS1/18 (anti-CD18) was added to some of the wells at a final concentration of 20 µg/ml before the addition of the cells. To inhibit adhesion to ICAM-1-coated wells, 50 µl of anti-ICAM mAb 8.4 (25 µg/ml) was added to the wells or anti-CD18 mAb TS1/18 (25 µg/ml) was added to the cells for 10 min before addition to the wells. Following incubation for 30 min at 37 °C, the plates were washed three times with phosphate-buffered saline. Fluorescence was quantitated in the wells using a Pandex fluorescence concentration analyzer (Baxter Healthcare Corp., Mundelein, IL).

Purification of Human ICAM-1

ICAM-1 was purified from human placental lysate by affinity chromatography using the anti-ICAM-1 monoclonal antibody 8.4A6 as previously described with modification(50) . Preparation of placental tissue and all subsequent work was carried out at 10 °C. 70 g of human placenta was frozen at -80 °C and then thinly sliced with a scalpel (1-2 mm in thickness). Placental tissue was then added to 250 ml of lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 0.01% NaN(3), 5 mM EDTA, 10 µg/ml aprotinin, 5 µg/ml leupeptin, 0.5 mM iodoacetamide) and homogenized in a blender for 30 s. 250 ml of lysis buffer containing 2% Triton X-100 was added for a final volume of 500 ml and gently stirred for 1 h in the cold. The lysate was then put through a low speed centrifugation (2000 rpm for 15 min) followed by a high speed centrifugation (50,000 times g for 60 min). The supernatant was then passed over the antibody affinity column (bed volume of 4 ml) twice at a rate of 0.5 ml/min. The column was washed with 40 ml of TSA buffer, pH 8.0 (20 mM Tris, pH 7.4, 150 mM NaCl, 0.01% NaN(3)) containing 0.1% Triton X-100 followed by 40 ml of TSA buffer, pH 8.0, containing 1% n-octyl beta-D-glucopyranoside. ICAM-1 was eluted with TSA buffer, pH 11.0, containing 1% n-octyl beta-D-glucopyranoside at a rate of 0.5 ml/min and immediately neutralized with 0.1 volume of 1 M Tris, pH 6.8. 1-ml fractions were collected, and the eluted protein was analyzed by SDS-gel electrophoresis and visualized by silver staining.


RESULTS

Expression of Recombinant Mutant alpha(L)beta(2)and alpha(M)beta(2)Receptors

To investigate the role that the highly conserved residues (Asp, Asp, Ser, and Ser) in beta(2) play in ligand recognition, each of these residues was individually substituted with alanine (Fig. 1). After co-transfection into COS cells with alpha(L) or alpha(M), cell surface expression and heterodimer association were evaluated by immunoprecipitation of detergent-lysed surface-labeled cells. Alanine substitution at Asp or Ser did not have any detectable effect on alpha(L)beta(2) or alpha(M)beta(2) heterodimer formation or surface expression (Fig. 2). alpha(L)- or beta(2)-specific antibodies immunoprecipitated both alpha and beta subunits on COS cells transfected with alpha(L)beta(2)(D134A), alpha(L)beta(2)(S136A), or the wild type alpha(L)beta(2), indicating that both subunits were associated on the cell surface. In addition, alpha(M)- or beta(2)-specific antibodies immunoprecipitated both alpha and beta subunits on COS cells transfected with alpha(M)beta(2)(D134A), alpha(M)beta(2)(S136A), or the wild-type alpha(M)beta(2). In contrast, substitution of beta(2) at Asp or Ser resulted in loss of surface expression when co-transfected with alpha(L) or alpha(M) (Fig. 2). alpha(M)-, alpha(L)-, or beta(2)-specific antibodies did not precipitate alphabeta heterodimers. In most experiments, no material was immunoprecipitated; however, occasionally the anti-alpha(L) or anti-alpha(M) precipitated the transfected alpha subunit only. Surface expression of beta(2)(D134A) or beta(2)(S136A) co-transfected with alpha(L) or alpha(M) was also evaluated by flow cytometry utilizing anti-alpha and -beta(2) antibodies (Fig. 3). FACS profiles of the recombinant wild-type alpha(L)beta(2), alpha(M)beta(2), or mutant receptor heterodimers were very similar and were expressed on the cell surface of 30-40% of the cells. Both beta(2)(D134A) and beta(2)(S136A) co-transfected with alpha(L) or alpha(M) were recognized by three anti-alpha(L) and four anti-alpha(M), respectively, and three anti-beta(2) antibodies (Table 1). In contrast, surface expression of beta(2)(D128A) or beta(2)(S138A) co-transfected with alpha(L) or alpha(M) was not detected on COS cells as determined by flow cytometry, in agreement with the immunoprecipitation analysis (data not shown).


Figure 2: SDS-polyacrylamide gel electrophoresis analysis of wild-type or mutant alpha(L)beta(2) and alpha(M)beta(2) receptors transiently expressed on COS cells. A, COS cells co-transfected with alpha(L)beta(2) or alpha(L)beta(2) mutants were surface-iodinated, lysed, and immunoprecipitated with mAb 38 (anti-alpha(L), lanesa) or mAb TS1/18 (anti-beta(2), lanesb). B, COS cells co-transfected with alpha(M)beta(2) or alpha(M)beta(2) mutants were surface-iodinated, lysed, and immunoprecipitated with mAb 3H5 (anti-alpha(M), lanesa) or mAb TS1/18 (anti-beta(2), lanesb). Immune complexes were isolated, and reduced samples were resolved by SDS-polyacrylamide gel electrophoresis on 7.5% acrylamide gels and detected by autoradiography. Molecular mass markers are indicated on the left in kDa.




Figure 3: FACS histograms of wild-type or mutant alpha(L)beta(2) and alpha(M)beta(2) receptors transiently expressed on COS cells. A, COS cells co-transfected with alpha(L)beta(2) or alpha(L)beta(2) mutants were incubated in the presence of mAb 38 (anti-alpha(L)) (a) or mAb TS1/18 (anti-beta(2)) (b). B, COS cells co-transfected with alpha(M)beta(2) or alpha(M)beta(2) mutants were incubated in the presence of mAb 3H5 (anti-alpha(M)) (a) or mAb TS1/18 (anti-beta(2)) (b). After 30 min, cells were washed, further incubated with fluorescein isothiocyanate-conjugated goat anti-mouse F(ab`)(2) fragments for 30 min, and analyzed on a FACScan. Abscissa, log of the fluorescence; ordinate, cell number.





Functional Integrity of Expressed Mutant alpha(L)beta(2)and alpha(M)beta(2)Receptors

To determine whether these residues of CD18 were essential for alpha(L)beta(2) ligand recognition, the capacity of the transfected COS cells to adhere to microtiter wells coated with immunopurified ICAM-1 was examined. COS cells bearing the recombinant wild-type alpha(L)beta(2) attached to affinity-purified ICAM-1-coated microtiter wells (Fig. 4A). Attachment was specific since it was inhibitable by anti-beta(2)- (TS1/18) or anti-ICAM (mAb 8.4)-specific antibody. In contrast, COS cells expressing alpha(L)beta(2)(D134A) or alpha(L)beta(2)(S136A) failed to attach to ICAM-1-coated microtiter wells.


Figure 4: Adhesion of cells expressing wild-type or mutant alpha(L)beta(2) and alpha(M)beta(2) receptors to purified ICAM-1 and iC3b, respectively. Fluorescently labeled cells were allowed to attach to microtiter wells coated with immunoaffinity-purified ICAM-1 (A) or iC3b (B) in the absence or presence of the indicated monoclonal antibodies for 30 min at 37 °C. Unbound cells were removed, and adherent cells were quantitated by fluorescence using a Pandex fluorescence concentration analyzer. The data are expressed as the percentage bound where 100% equals the total number of cells adhered to the mAb TS1/18 (anti-beta(2))-coated wells to correct for the levels of integrin expressed by the different transfectants. Results are representative of three separate experiments. Bars represent the mean ± S.E. of three determinations.



To determine whether these residues of beta(2) also affected alpha(M)beta(2) ligand recognition, the ability of the transfected cells expressing this integrin to adhere to microtiter wells coated with iC3b was examined (Fig. 4B). Cells bearing the recombinant alpha(M)beta(2) attached to iC3b and were blocked by anti-alpha(M) (mAb 3H5)-specific antibody. In contrast, cells bearing alpha(M)beta(2)(D134A) or alpha(M)beta(2)(S136A) failed to adhere to iC3b-coated microtiter wells. Consequently, Asp along with Ser play an integral role in the ligand binding function of both alpha(L)beta(2) and alpha(M)beta(2).


DISCUSSION

The major findings in this work are as follows. 1) Alanine substitution of two of the conserved oxygenated residues in beta(2), Asp, or Ser did not affect heterodimer formation or surface expression when co-transfected with alpha(L) or alpha(M) in COS cells. 2) Substitution of these residues to alanine resulted in deficits in both alpha(L)beta(2) and alpha(M)beta(2) ligand binding function. 3) Alanine substitution of Asp or Ser resulted in loss of beta(2) surface expression when co-transfected with alpha(L) or alpha(M). These data demonstrate the role of the beta subunit in ligand recognition by the I domain-containing leukocyte integrins.

Two of the individual alanine substitutions, Asp and Ser, impede heterodimer formation of beta(2) with the co-transfected alpha(L) or alpha(M). The lack of surface expression may be due to faulty folding and rapid intracellular degradation. However, previous studies identifying mutations from patients with genetic deficiencies of beta(2) (leukocyte adhesion deficiency) suggest that mutations within this critical region of beta(2) (residues 128-361) represent critical contact sites between the alpha and beta chain precursors. These contact sites are presumably required for alphabeta precursor association and biosynthesis (reviewed in (51) ). In addition, extensive investigation of bovine leukocyte adhesion deficiency, prevalent among Holstein cattle, led to the identification of two beta(2) mutations, one of which results in the substitution of Asp for Gly at position 128 (D128G)(52) . Therefore, the present results further implicate that this region is critical in alphabeta heterodimer surface expression in the leukocyte integrins.

In contrast, alanine substitution at residue Asp or Ser did not affect the capacity of the mutant beta(2) to form heterodimers with co-transfected alpha(L) or alpha(M). This is based on the finding that anti-alpha or anti-beta(2) coprecipitated beta(2) or alpha, respectively. Furthermore, surface expression was confirmed by reactivity with a panel of anti-alpha or beta(2) antibodies, suggesting that the amino acid substitutions did not affect receptor structure. The present work suggests that these residues of beta(2) appear to be critical for ligand binding function of the leukocyte integrins. This conclusion is based on the failure of cells expressing beta(2)(D134A) or beta(2)(S136A) co-transfected with alpha(L) or alpha(M) to adhere to immobilized ICAM-1 or iC3b, respectively. Considered together, these data suggest that this region of beta(2) is critical to the common ligand binding structural characteristics of beta(2) integrins alpha(L)beta(2) and alpha(M)beta(2).

These beta(2) residues (Asp and Ser) are highly conserved among the beta subunits and play a role in ligand recognition in beta(3) (corresponding residues Asp and Ser)(20, 21) . Substitution of Asp to tyrosine in beta(3) results in a complete loss of RGD-dependent ligand binding function of alphabeta(3). In addition, amino acid substitution of the corresponding residue in beta(1) (Asp) (22) abrogates the RGD-dependent binding of alpha(5)beta(1) to the 110-kDa cell binding fragment of fibronectin. However, the beta(2) integrins, unlike certain beta(1) and beta(3) integrins, do not utilize RGD-like sequences to interact with their ligands. The present data suggest that the beta(1), beta(2), and beta(3) integrins utilize these same conserved residues to interact with their respective ligands despite the very dissimilar nature of their ligands. This suggests the possibility of a common mechanism of ligand recognition by integrins.

The linear spacing of the conserved cluster of oxygenated residues approximates that of the calcium binding residues in EF-hand proteins (53) . Given the absolute requirement of divalent cations for the function of all integrins and the high degree of conservation of this cluster of oxygenated residues, it has been hypothesized that a common feature of ligand binding to integrins is the interaction of ligand with divalent cations occupying a divalent cation binding site in the integrins(20, 54) . In this working hypothesis, oxygenated residues in the ligand, such as the Asp in RGD, would contribute to the coordination of cation binding. This is further supported by the finding that substitution of Asp to tyrosine in beta(3) also resulted in disruption of divalent cation binding of alphabeta(3) as determined by the binding of a divalent cation-dependent antibody, PMI-1(20) .

Since the I domain beta(2) integrins do not utilize RGD-like sequences in ligand recognition, we hypothesize that an unidentified oxygenated residue(s) in their ligands contributes to the coordination of cation binding similarly to the Asp in RGD-containing ligands for beta(1) and beta(3) integrins. This is further supported by the finding that the substitution of Asp to alanine in beta(1) also results in a loss of ligand binding of alpha(5)beta(1) to the non-RGD-containing ligand, invasin(22) . In addition, other small peptide ligands that interact with beta(1) and beta(3) integrins, such as LGGAKQAGDV (55) and EILDVPST(46) , contain oxygenated residues that may be capable of coordinating divalent cations.

In addition, results of previous studies implicate the I domain of the beta(2) integrins as an integral part of the ligand binding function of alpha(L)beta(2), alpha(M)beta(2), and alpha(X)beta(2)(35, 37, 39) . Inhibitory anti-alpha antibodies have been mapped to the I domains of the alpha(L), alpha(M), and alpha(X). Moreover, alanine substitution of certain conserved oxygenated residues in the alpha(M) I domain (D140GS/AGA) abolished divalent cation-dependent binding of alpha(M)beta(2) to iC3b(36) . Alignment of these critical residues of the I domains with the presently described region of the beta subunits demonstrates a remarkable conservation of these oxygenated residues between the I domain and the beta subunits, suggesting that these sequences may comprise a ligand binding motif essential for all integrin receptor function(21) . This is further supported by the finding that alanine mutagenesis of one of these conserved residues in the I domain of alpha(2) (Asp) blocked the binding of alpha(2)beta(1) to collagen(38) . However, further mutational analysis is in progress to determine whether other conserved and nonconserved beta(2) residues are essential for either ligand binding or surface expression.

In summary, we have verified a ligand-interactive region in the beta(2) subunit of the leukocyte integrins alpha(L)beta(2) and alpha(M)beta(2). This is the first evidence for a ligand binding site in the beta(2) subunit of the ``I'' domain integrins. Moreover, that Asp along with Ser play a role in the ligand recognition of both alpha(L)beta(2) and alpha(M)beta(2) further supports the proposal of a common ligand binding mechanism(20, 21) , which is essential for integrin receptor function irrespective of the presence of the RGD sequence in the ligand. Since the I domain as well as domains V and VI of alpha(L)(56) have been implicated in ligand binding function, it is likely that multiple sites in integrins cooperate in recognition of ligands.


FOOTNOTES

*
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.

§
To whom all correspondence should be addressed: Upjohn Laboratories, Cell Biology and Inflammation Research, 7239-267-302, Kalamazoo, MI 49001.

(^1)
The abbreviations used are: mAb, monoclonal antibodies; FACS, fluorescence-activated cell sorter.


ACKNOWLEDGEMENTS

We thank Dr. Mark H. Ginsberg for critical review of the manuscript.


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