The Structure of the beta -Propeller Domain and C-terminal Region of the Integrin alpha M Subunit
DEPENDENCE ON beta  SUBUNIT ASSOCIATION AND PREDICTION OF DOMAINS*

Chafen Lu, Claus Oxvig, and Timothy A. SpringerDagger

From the Center for Blood Research and Harvard Medical School, Department of Pathology, Boston, Massachusetts 02115

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The alpha M subunit of integrin Mac-1 contains several distinct regions in its extracellular segment. The N-terminal region has been predicted to fold into a beta -propeller domain composed of seven beta -sheets each about 60 amino acid residues long, with the I-domain inserted between beta -sheets 2 and 3. The structure of the C-terminal region is unknown. We have used monoclonal antibodies (mAbs) as probes to study the dependence of the structure of different regions of the alpha M subunit on association with the beta 2 subunit in the alpha M/beta 2 heterodimer. All of the mAbs to the I-domain immunoprecipitated the unassociated alpha M precursor and reacted with the alpha M subunit expressed alone on the surface of COS cells. By contrast, four mAbs to the beta -propeller domain did not react with the unassociated alpha M precursor nor with the uncomplexed alpha M subunit expressed on COS cell surface. The four mAbs were mapped to three subregions in three different beta -sheets, making it unlikely that each recognized an interface between the alpha  and beta  subunits. These results suggest that folding of different beta -propeller subregions is coordinate and is dependent on association with the beta 2 subunit. The segment C-terminal to the beta -propeller domain, residues 599-1092, was studied with nine mAbs. A subset of four mAbs that reacted with the alpha M/beta 2 complex but not with the unassociated alpha M subunit were mapped to one subregion, residues 718-759, and five other mAbs that recognized both the unassociated and the complexed alpha M subunit were localized to three other subregions, residues 599-679, 820-882, and 943-1047. This suggests that much of the region C-terminal to the beta -propeller domain folds independently of association with the beta 2 subunit. Our data provide new insights into how different domains in the integrin alpha  and beta  subunits may interact.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The integrin family of adhesion molecules participate in important cell-cell and cell-extracellular matrix interactions in a diverse range of biological processes (1). Integrins are noncovalently associated alpha /beta heterodimers, with each subunit consisting of a large extracellular domain (>100 kDa for alpha  subunits and >75 kDa for beta  subunits), a single transmembrane region, and a short cytoplasmic tail (50 amino acids or less, except for the beta 4 subunit) (1). The adhesiveness of integrins is dynamically regulated in response to cytoplasmic signals, termed "inside-out" signaling (2-4). The leukocyte integrin subfamily consists of four members that share the common beta 2 subunit (CD18) but have distinct alpha  subunits, alpha L (CD11a), alpha M (CD11b), alpha X (CD11c), and alpha d for LFA-1, Mac-1, p150, 95, and alpha d/beta 2, respectively (5-7). The leukocyte integrins mediate a range of adhesive interactions that are essential for normal immune and inflammatory responses (5).

Although the overall structure of integrins is unknown, several structurally distinct domains in the extracellular portions of both alpha  and beta  subunits have been predicted or identified. The N-terminal region of the integrin alpha  subunits contains seven repeats of about 60 amino acids each (8) and has recently been predicted to fold into a beta -propeller domain that consists of seven beta -sheets, with each beta -sheet containing four anti-parallel beta -strands (9). The leukocyte integrin alpha  subunits (10), the alpha 1 (11) and alpha 2 (12) subunits of the beta 1 subfamily, and the alpha E subunit (13) of the beta 7 subfamily contain an inserted domain or I-domain of about 200 amino acids that is predicted to be inserted between beta -sheets 2 and 3 of the beta -propeller domain (9). The three-dimensional structure of the I-domain from the Mac-1, LFA-1, and alpha 2beta 1 integrins has been solved and shows that it adopts the dinucleotide-binding fold with a unique divalent cation coordination site designated the metal ion-dependent adhesion site (14-17). The integrin beta  subunits contain a conserved domain of about 250 amino acids in the N-terminal portion. This domain has been predicted to have an "I-domain-like" fold (14, 18, 19). Very little is known about the structure of the C-terminal half of the extracellular portions of both alpha and beta  subunits. Electron microscopic images of integrins reveal that the N-terminal portions of the alpha  and beta  subunits fold into a globular head that is connected to the membrane by two rod-like segments about 16 nm long corresponding to the C-terminal portions of the alpha  and beta  extracellular domains (20-22). This would suggest that the C-terminal portions of both subunits are quite extended.

Previous studies using mAbs1 as probes have shown that the structure of specific domains in LFA-1 requires association of the alpha L and beta 2 subunits. mAbs to the beta 2 subunit conserved domain do not react with the unassociated beta 2 subunit, whereas mAbs to the regions preceding and following this domain do, indicating that the structure of the conserved domain is dependent on association with the alpha L subunit (23). mAbs to the I-domain react with the unassociated alpha L subunit (24). This finding together with the fact that the I-domain can be expressed as an isolated domain (14, 16, 25, 26) show that the I-domain assumes a native structure independently of the beta 2 subunit. By contrast, two mAbs (S6F1 and TS2/4) mapped to the N-terminal region of the beta -propeller domain, and one mAb (G-25.2) that maps to a region of 212 amino acids with 159 amino acids located in the beta -propeller domain and the remainder in the C-terminal region, do not recognize the alpha L subunit in the absence of association with the beta 2 subunit (24). Another mAb (CBRLFA-1/1) that maps to a region overlapping the I-domain and beta -propeller domain reacts weakly with the uncomplexed alpha L subunit. These results indicate that at least one region in the beta -propeller domain is dependent on association with the beta 2 subunit for mAb reactivity, and it has been suggested that the most likely explanation is that folding of the beta -propeller domain is not completed until after association with the beta  subunit (24). Since mAbs specific for the region of the alpha L subunit C-terminal to the beta -propeller domain have not been described, it is not known whether folding of this region is dependent on association with the beta  subunit.

In this study, we have used mAb probes to study the structure of the Mac-1 alpha  subunit in the presence and absence of association with the beta 2 subunit. We have studied the beta -propeller domain, the I-domain, and the extensive region C-terminal to the beta -propeller domain. Compared with the previous studies on LFA-1, our studies on the beta -propeller domain are more definitive, since mAb specificity is defined to individual amino acid substitutions between mouse and human, and mAb to epitopes that are widely separated in the predicted beta -propeller structure all show a dependence on beta  subunit association for reactivity. Furthermore, we employ a panel of mAbs that defines four different subregions within the C-terminal region of the alpha  subunit. The results show that epitopes in three of these regions have a native structure in the absence of beta  subunit association, whereas a fourth epitope is dependent on association with the beta  subunit. Thus, much of the C-terminal region of the alpha M subunit appears to assume a native fold independently of association with the beta 2 subunit.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cell Lines-- U937, a human monoblast-like cell line, was cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 50 µg/ml gentamicin, and 50 µM 2-mercaptoethanol (complete medium). COS cells (SV40-transformed monkey kidney fibroblasts) were maintained in RPMI 1640 supplemented with 10% FBS and 50 µg/ml gentamicin. Human embryonic kidney 293 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% FBS, 2 mM glutamine, and 50 µg/ml gentamicin.

mAbs-- The following murine mAbs against the alpha M subunit of human Mac-1 were previously described: OKM1, OKM9 (27), TGM-65 (28), CBRM1/1, CBRM1/2, CBRM1/29, CBRM1/20, CBRM1/32, CBRM1/10, CBRM1/16, CBRM1/17, CBRM1/18, CBRM1/23, CBRM1/25, CBRM1/26, and CBRM1/30 (29). All these mAbs were used as ascites except for CBRM1/29 that was used as concentrated hybridoma supernatant. CBRN1/6 and CBRN3/4 against the alpha M subunit of Mac-12 were used as hybridoma supernatant. TS1/18 and CBRLFA-1/2 against human leukocyte integrin beta 2 subunit were described previously (30, 31) and used as purified IgG.

DNA Constructs and Mutagenesis-- The human wild-type alpha M subunit cDNA was subcloned in the expression vector pCDNA3.1+ (Invitrogen, Carlsbad, CA) as described.3 For generating human-mouse alpha M chimeras, a SacII site was created immediately after the stop codon (nucleotides 3532-3534). By specifically primed reverse transcription of murine spleen mRNA (CLONTECH, Palo Alto, CA) from approximately 50 nucleotides downstream of the stop codon, the first strand of the mouse alpha M cDNA (33) was generated with Moloney murine leukemia virus reverse transcriptase (Stratagene, La Jolla, CA). By using this as a template for PCR, a 2-kilobase pair mouse alpha M cDNA fragment covering nucleotides from the SfiI site (nucleotide 1688) to the stop codon and having a SacII site immediately after the stop codon was made. This mouse alpha M SfiI-SacII fragment was used to replace the corresponding human alpha M SfiI-SacII fragment to generate the initial chimeric alpha M cDNA encoding the N-terminal 529 residues of human sequence and the remaining C-terminal sequence from mouse. Using this initial chimeric construct as template, eight human-mouse alpha M chimeras with a variable mouse C-terminal portion were generated by overlap extension PCR (34, 35). Briefly, outer primers for overlap PCR were just 5' to the SfiI site and 3' to the SacII site, and the first set of reactions was carried out using the human wild-type alpha M and the initial chimeric construct as templates. After the overlap extension reaction, the chimeric products were digested with SfiI and SacII, and the SfiI-SacII fragments were swapped into the human wild-type alpha M in vector pCDNA3.1+. Human to mouse individual amino acid substitutions in the region from amino acids 718-759 of human alpha M were made by overlap extension PCR (34, 35). Briefly, the overlapping primers contained the desired mutations, and the outer primers were 5' to the SfiI site and 3' to the NdeI site, respectively. The overlap extension PCR products were digested with SfiI and NdeI and swapped into human wild-type alpha M in expression vector pEFpuro (36).

For mapping mAb epitopes in the beta -propeller domain of the human alpha M subunit, 32 different chimeric alpha M subunits were made in which a short segment of mouse sequence comprising a predicted loop or a strand 4 was inserted in the human sequence. Mutagenesis was done by inverse PCR on plasmid pBluescript II containing Mac-1 alpha M cDNA fragments that included the NotI site 5' to the coding region and the BspEI site at amino acid residue 180 or included the BspEI-BbsI fragment from 180 to 672, as described elsewhere.3 The mutated cDNA fragments were excised with NotI and BspEI or BspEI and BbsI and swapped into wild-type alpha M cDNA contained in plasmid pCDNA3.1+. Mutants were named after the sheet (W) and the loop (L) or the strand (S) that was exchanged, e.g. hu(W7L3-4)mo has mouse sequence in the loop between strands 3 and 4 of W7, and hu(W1S4)mo contains mouse sequence in strand 4 of W1. In the following list, the amino acid segment or individual amino acid residue that was of murine origin is indicated for each mutant in the numbering system for the mature human alpha  subunit. These mutants are as follows: hu(W7L3-4)mo, 7-8; hu(W7L4-1)mo, 16; hu(W1L1-2)mo, 26-29; hu(W1L2-3)mo, 38-44; hu(W1L3-4)mo, 55-56; hu(W1S4)mo, 58-61; hu(W1L4-1)mo, 66; hu(W2L1-2)mo, 82-84; hu(W2L2-3)mo a, 96-98; hu(W2L2-3)mo b, 104; hu(W2S3-I-domain)mo a, 115-120; hu(W2S3-I-domain)mo b, 127; hu(I-domain-W3S1)mo, 327; hu(W3L2-3)mo, 356; hu(W3L3-4)mo, 369-371; hu(W3S4)mo, 376; hu(W4L3-4)mo, 421-425; hu(W4S4), 428-432; hu(W4L4-1)mo, 435-439; hu(W5L1-2)mo a, 450-455; hu(W5L1-2)mo b, 457; hu(W5L2-3)mo, 469; hu(W5L3-4)mo, 484; hu(W5L4-1)mo, 495-500; hu(W6L2-3)mo, 531-534; hu(W6L3-4)mo a, 541; hu(W6L3-4)mo b, 543-550; hu(W6L3-4)mo c, 554; hu(W6S4)mo, 557-559; hu(W6L4-1)mo, 460-464; hu(W7L1-2)mo, 576; hu(W7S3-)mo, 599-606.

All mutations were verified by DNA sequencing. At least two independent clones of each mutant were used for transfection, and identical results were obtained.

Transient Transfection-- COS cells were transfected by the DEAE-dextran method (36) with the alpha M cDNA alone or were co-transfected with the wild-type or chimeric alpha M and beta 2 cDNA. The wild-type and chimeric alpha M cDNA were in plasmid pCDNA3.1+, and the beta 2 cDNA was contained in plasmid pEF-BOS (36). Three days after transfection, COS cells were detached with Hanks' balanced salt solution supplemented with 5 mM EDTA for flow cytometric analysis. 293 cells were transfected with the calcium phosphate method (37, 38). Briefly, 7.5 µg of wild-type or mutant alpha M cDNA in plasmid pEFpuro and 7.5 µg of beta 2 cDNA in plasmid pEF-BOS were used to transfect one 6-cm plate of 70-80% confluent cells. Two days after transfection, cells were detached with Hanks' balanced salt solution, 5 mM EDTA for flow cytometric analysis.

Flow Cytometry-- COS cells and 293 cells were washed twice with L15 medium containing 2.5% FBS (L15/FBS) and resuspended to 1-2 × 106 cells/ml in the same medium. 50 µl of the cell suspension was incubated with an equal volume of the primary antibody (20 µg/ml purified mAb, 1:100 dilution of mAb ascites, or 1:2 dilution of hybridoma supernatant in PBS) on ice for 30 min. Cells were then washed three times with L15/FBS and incubated with fluorescein isothiocyanate-conjugated goat anti-mouse IgG (heavy and light chain, Zymed Laboratories, San Francisco, CA) for 30 min on ice. For staining with mAb CBRM1/20 that requires Ca2+,3 the primary and secondary antibodies were diluted in PBS supplemented with 1 mM Ca2+. After washing, cells were resuspended in cold PBS and analyzed on a FACScan (Becton Dickinson, San Jose, CA).

Radiolabeling, Immunoprecipitation, and Gel Electrophoresis-- For metabolic labeling, U937 cells were plated in four 10-cm Petri dishes and induced with PMA for 3 days as described previously (39). Cells in each dish were washed twice with methionine-free RPMI 1640 medium and labeled with 0.625 mCi of [35S]methionine in 5 ml of methionine-free RPMI 1640 containing 15% dialyzed FBS. After incubation at 37 °C for 30 min, cells in two dishes were washed twice with cold PBS and lysed by addition of 3 ml of lysis buffer (1% Triton X-100, 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM MgCl2, 1 mM iodoacetamide, 1 mM phenylmethylsulfonyl fluoride, 0.24 TIU/ml aprotinin, and 10 µg/ml each of pepstatin A, antipain, and leupeptin) and incubation for 30 min at 4 °C with gentle agitation. For chase labeling, 5 ml of complete medium supplemented with 100 µg/ml unlabeled methionine was added to each of the remaining dishes, and incubation at 37 °C was continued for 16 h. The chase-labeled cells were lysed identically to pulse-labeled cells, and lysates were clarified by centrifugation at 12,000 rpm for 10 min at 4 °C.

For surface labeling, COS transfectants (2 × 106 cells) were washed three times with PBS and resuspended in 1 ml of PBS. The cells were surface-labeled with 1 mCi of Na125I using two IODO-BEADS (Pierce) following the manufacturer's instructions. The labeled cells were washed three times with PBS containing 10% FBS and once with PBS and lysed as described above.

For immunoprecipitation, cell lysates were precleared by addition of 1/10 volume of recombinant protein G agarose (50% suspension in PBS) (Life Technologies, Inc.) and incubation at 4 °C for 2-3 h with agitation. The precleared lysates were split into 250-µl aliquots, and to each aliquot, 2.5 µl of mAb ascites or 10 µg of purified mAb or 250 µl of mAb supernatant was added, and the final volume was adjusted to 500 µl with lysis buffer. After incubation overnight at 4 °C, followed by centrifugation at 12,000 rpm for 10 min at 4 °C to remove protein aggregates, the antigen/antibody mixture was incubated with 50 µl of protein G-agarose beads for 1.5-2 h at 4 °C with agitation. Beads were washed three times with lysis buffer and once with lysis buffer without detergent. For immunoprecipitation with mAb CBRM1/20, lysis buffer and wash buffer were supplemented with 1 mM Ca2+. Bound proteins were eluted from beads with 50 µl of Laemmli sample buffer by heating for 5 min at 100 °C, and the immunoprecipitates were analyzed by 7.5% SDS-polyacrylamide gel electrophoresis (40). The gels were processed for fluorography for [35S]methionine-labeled proteins or autoradiography for 125I-labeled proteins.

Secondary Structure Prediction-- The amino acid sequences between the beta -propeller domain and the transmembrane segment of 36 integrin alpha  subunits (9) were aligned with ClustalW, and then the alignment was iteratively refined using default settings with PRRP and the Gonnet amino acid substitution matrix, and an evolutionary tree was prepared with PHYLP (41). The alpha M and alpha IIb subunits fall in different branches of this tree, each of which is well populated. One branch containing 11 subunits most closely related to human alpha M, i.e. murine alpha M, human alpha D, and alpha X, murine and human alpha L, human and rat alpha 1, and bovine, human, and mouse alpha 2, were realigned with one another using PRRP. They are 21-70%, &xmacr; = 34% identical to human alpha M. Another branch containing the 17 subunits most closely related to human alpha IIb, i.e. hamster, human, and mouse alpha 3, human and Xenopus alpha 5, chicken and human alpha 6, mouse alpha 7, chicken and human alpha 8, and chicken, human, mouse, and Pleurodes alpha V, and YMA1 of Caenorhabditis elegans, were realigned in a separate group. They are 20-38%, &xmacr; = 28% identical to human alpha IIb. The alignments in MSF format, with gaps in human alpha M and human alpha IIb removed to increase prediction accuracy, were separately submitted for secondary structure prediction to PHD (42).4 Smaller subgroups containing a higher degree of relationship to alpha M (6 alpha  subunits, with 27-70% identity to alpha M) or to alpha IIb (9 alpha  subunits, with 33-38% identity to alpha IIb) gave very similar predictions but with a slightly lower correlation between the alpha M and alpha IIb predictions.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

mAbs to the beta -Propeller Domain and a Subset of mAbs to the C-terminal Region Do Not React with the Unassociated alpha M Subunit-- To study whether folding of the alpha M subunit is dependent on association with the beta 2 subunit, we examined the expression of mAb epitopes on the unassociated alpha M subunit. Eighteen mAbs that have previously been mapped to different regions in the alpha M subunit were used (29)2 (Fig. 1). Previous studies on leukocyte integrin biosynthesis have shown that the alpha  and beta  subunit precursors are initially unassociated in the endoplasmic reticulum and that transport to the Golgi apparatus and processing from high mannose N-linked carbohydrates to complex carbohydrates are dependent on the formation of alpha  and beta  complex (39, 43, 44). We therefore examined whether mAbs to the I-domain, to the beta -propeller domain, and to the C-terminal region immunoprecipitated the unassociated alpha M precursor (alpha 'M). All mAbs immunoprecipitated the mature alpha M subunit with molecular size of about 170 kDa from the lysate of cells pulse-labeled with [35S]methionine for 30 min and chased for 16 h (Fig. 2, lower panel). The alpha M subunit was complexed with the beta 2 subunit as shown by co-immunoprecipitation of the beta 2 subunit with the alpha M subunit. However, mAbs differentially precipitated the alpha 'M precursor, which is slightly smaller than the mature alpha M subunit from the pulse-labeled cells (Fig. 2, upper panel). There was little or no alpha 'M precursor associated with the beta 2 precursor (beta '2) in the pulse-labeled cells, since no detectable beta '2 over background was co-precipitated by mAbs to the alpha M subunit, but beta '2 was precipitated with mAb CBRLFA-1/2 to the beta 2 subunit (upper panel, lane 18). All mAbs to the I-domain precipitated alpha 'M (upper panel, lanes 2-6). By contrast, three mAbs (CBRN1/6, CBRN3/4, and CBRM1/20) to the beta -propeller domain did not precipitate alpha 'M (upper panel, lanes 7, 8, and 21). mAb CBRM1/32 to the beta -propeller domain did not precipitate the alpha M/beta 2 complex or alpha 'M from cell lysates (data not shown), suggesting that its epitope is sensitive to detergent extraction. Five mAbs (OKM1, CBRM1/10, CBRM1/23, CBRM1/25, and CBRM1/26) to the C-terminal region precipitated alpha 'M (upper panel, lanes 9 and 10 and 14-16), whereas four other mAbs (CBRM1/16, CBRM1/17, CBRM1/18, and CBRM1/30) precipitated no to very little alpha 'M (upper panel, lanes 11-13 and 17). Thus, epitopes of mAbs to the I-domain are expressed on the unassociated alpha M precursor, whereas epitopes of beta -propeller domain mAbs and a subset of mAbs to the C-terminal region are not.


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Fig. 1.   Schematic diagram of the integrin alpha M subunit and monoclonal antibody epitope localization. Numbers are positions of amino acid residues at the putative boundaries of different regions. The Ws are beta -sheets of the beta -propeller domain. The I-domain is inserted between beta -sheets 2 and 3 of the beta -propeller domain. The transmembrane domain (TM) is shown in black. mAbs and their epitope localization are shown below alpha M. Mapping of mAbs to different regions in the alpha M subunit has been described previously (29).2


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Fig. 2.   Immunoprecipitation of the unassociated alpha M precursor (alpha 'M) and the alpha M/beta 2 complex. PMA-induced U937 cells were pulse-labeled with [35S]methionine for 30 min and chased with unlabeled methionine for 16 h. The alpha M and beta 2 precursors (alpha 'M and beta '2, respectively) and the alpha M/beta 2 complex were immunoprecipitated from lysates of pulse-labeled cells (upper panel) and pulse-chased cells (lower panel). Immunoprecipitates were subjected to 7.5% SDS-polyacrylamide gel electrophoresis and fluorographed. The nonbinding mAb, X63, was used as negative control. CBRLFA-1/2 is specific for the beta 2 subunit, and all other mAbs are against the alpha M subunit (see Fig. 1). In lanes 20-22, cell lysis and immunoprecipitation were carried out in the presence of 1 mM Ca2+.

To examine alpha  subunit structure independently of maturation events occurring during biosynthesis, we examined mAb reactivity with the unassociated alpha M subunit expressed on the surface of COS cells. COS cells were transfected with cDNA for alpha M alone or for both alpha M and beta 2 subunits, and mAb reactivity with the uncomplexed alpha M or the alpha M/beta 2 complex expressed on the surface of COS transfectants was determined by immunofluorescent flow cytometry (Fig. 3). All mAbs to the I-domain (OKM9, TGM-65, CBRM1/1, CBRM1/2, and CBRM1/29) reacted with the unassociated alpha M subunit as well as with the alpha M/beta 2 complex expressed on the COS cell surface. By contrast, all mAbs to the beta -propeller domain (CBRN1/6, CBRN3/4, CBRM1/32, and CBRM1/20) reacted with COS cells expressing the alpha M/beta 2 complex but not with COS cells expressing alpha M alone. Four mAbs to the C-terminal region (CBRM1/16, CBRM1/17, CBRM1/18, and CBRM1/30) did not stain COS cells expressing the alpha M subunit alone, whereas five other mAbs did (OKM1, CBRM1/10, CBRM1/23, CBRM1/25, and CBRM1/26).


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Fig. 3.   Flow cytometry of COS cells expressing the alpha M subunit or the alpha M/beta 2 complex. COS cells were either mock-transfected, transfected with the alpha M cDNA alone, or co-transfected with the alpha M and beta 2 cDNAs. The transfectants were stained with mAbs to different regions in the alpha M subunit as indicated. The flow cytometry histogram of COS cells transfected with alpha M alone or co-transfected with alpha M and beta 2, as indicated on the right, was overlaid on that of the mock-transfected COS cells (shown in gray) stained with the same mAb.

The data obtained by immunofluorescent flow cytometry were confirmed by immunoprecipitation. COS transfectants expressing the alpha M subunit alone or the alpha M/beta 2 complex were surface-iodinated, and the labeled proteins were immunoprecipitated from cell lysates. All mAbs precipitated the alpha M/beta 2 complex from COS cells co-transfected with alpha M and the beta 2 (Fig. 4, lower panel). mAbs to the I-domain precipitated the alpha M subunit expressed alone on the COS cell surface (upper panel, lanes 3-5). By contrast, beta -propeller domain mAbs (upper panel, lanes 6, 7 and 15) and a subset of mAbs to the C-terminal region (upper panel, lanes 10, 11 and 14) failed to precipitate the uncomplexed alpha M subunit. Thus, expression of epitopes of the beta -propeller domain mAbs and a subset of mAbs to the C-terminal region is dependent on alpha M and beta 2 heterodimer formation.


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Fig. 4.   Immunoprecipitation of the alpha M subunit and the alpha M/beta 2 complex from surface-labeled COS transfectants. COS transfectants expressing alpha M alone or the alpha M/beta 2 complex were surface-labeled with 125I. Immunoprecipitates from lysates of COS transfectants were subjected to 7.5% SDS-polyacrylamide gel electrophoresis and autoradiography. Upper panel, COS cells transfected with the alpha M cDNA alone; lower panel, COS cells co-transfected with the alpha M and beta 2 cDNAs. The nonbinding mAb X63 was used as negative control. TS1/18 recognizes the complexed beta 2 subunit, and all other mAbs are against the alpha M subunit. In lanes 15 and 16, cell lysis and immunoprecipitation were carried out in the presence of 1 mM Ca2+.

To test the possibility that the beta 2 subunit may directly contribute to the epitopes of the mAbs that did not react with alpha M in the absence of the beta 2 subunit, we expressed human alpha M in association with the mouse beta 2 subunit or the chicken beta 2 subunit on the surface of COS cells and human 293 cells. mAb reactivity with the transfectants was determined by immunofluorescent flow cytometry. mAbs CBRN1/6, CBRN3/4, CBRM1/20, CBRM1/32, CBRM1/16, CBRM1/17, CBRM1/18, and CBRM1/30 that did not react with the unassociated alpha M subunit reacted with the human alpha M/mouse beta 2 and human alpha M/chicken beta 2 complexes as well as with the human alpha M/human beta 2 complex (data not shown). These results suggest that the beta 2 subunit does not directly contribute to the epitopes of these mAbs.

Epitope Mapping of mAbs to the C-terminal Region and to the beta -Propeller Domain of the alpha M Subunit-- The finding that a subset of mAbs to the C-terminal region does not react with the unassociated alpha M subunit suggests that the structures of certain subregion(s) in this C-terminal 493-amino acid segment may be dependent on association with beta 2. To localize such subregion(s), as well as subregion(s) that fold independently of beta 2 association, epitopes of the nine mAbs to the C-terminal region were mapped using human-mouse alpha M chimeras. The chimeras were generated by progressively replacing the human sequences from the C terminus with the corresponding sequences from mouse alpha M (Fig. 5) and were co-expressed with human beta 2 in COS cells. mAb reactivity with chimeric alpha M/beta 2 was determined by immunofluorescent flow cytometry (Table I). All chimeras were expressed on the surface in association with the human beta 2 subunit, with levels of cell-surface chimeric alpha M/beta 2 complex comparable with that of wild-type alpha M/beta 2 complex. In addition, all chimeric alpha M/beta 2 complexes were stained with mAbs to the beta -propeller domain (Table I and data not shown), showing structural integrity of the beta -propeller domain despite the C-terminal region swapping. The results from epitope mapping are summarized in Table I and Fig. 6. A 41-amino acid sequence (residues 718-759) was required for epitopes of the four mAbs (CBRM1/16, CBRM1/17, CBRM1/18, and CBRM1/30) that did not react with the unassociated alpha M subunit. The epitopes of five mAbs that reacted with the unassociated alpha M subunit were mapped to three other subregions as follows: OKM1 to a region immediately following the beta -propeller domain (residues 599-679); CBRM1/10, CBRM1/25, and CBRM1/26 to a region from residues 820 to 882; and CBRM1/23 to a region from residues 943 to 1047. Thus, mAb epitopes that map to one subregion (residues 718-759) require association of alpha M with beta 2, whereas epitopes localized in three other subregions (residues 599-679, 820-882, and 943-1047) are independent of the beta 2 subunit.


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Fig. 5.   Schematic representation of human-mouse alpha M chimeras. Human alpha M sequences (open bar) were progressively replaced from the C terminus with the corresponding sequences from the mouse alpha M subunit (hatched bar) as described under "Materials and Methods." Amino acid residues at the boundaries between human and mouse sequences are indicated above human alpha M (hualpha M).

                              
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Table I
mAb reactivity with COS cells expressing the human or chimeric alpha M subunit complexed with human beta 2
The human (hu) wild-type or human-mouse chimeric alpha M subunit was expressed in association with human beta 2 on the surface of transfected COS cells. mAb reactivity was determined by immunofluorescence flow cytometry. +, positive staining with mean fluorescence intensity comparable to human wild-type alpha M/beta 2 stained with the same mAb; -, staining was not significantly different from mock-transfected cells stained with the same mAb; ND, not determined. Epitopes of CBRM1/1 and CBRM1/32 were previously mapped (29). Of note, TS1/18 and CBRM1/32 are specific for complexed beta 2 and alpha M, respectively.


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Fig. 6.   mAb reactivity with the alpha M and alpha L subunits in the absence of the beta 2 subunit. Schematic diagrams of the alpha M and alpha L subunits are shown. W1 to W7 are beta -sheets 1-7 of the beta -propeller domain. Numbers are positions of amino acid residues at the boundaries between domains and between subregions in the alpha M C-terminal region. mAbs, except for those to the I-domain, and their epitope localization are shown under alpha M and alpha L. + indicates mAbs that react with the alpha M or alpha L in the absence of the beta 2 subunit; - indicates mAbs that do not react with the alpha M or alpha L in the absence of the beta 2 subunit; -/+ refers to weak reactivity with the unassociated alpha  subunit compared with mAbs to the I-domain. All tested mAbs to the I-domain react with the unassociated alpha  subunits. The original data on the alpha L subunit was reported elsewhere (24).

The region from residues 718 to 759 contains eight amino acid differences between the human and mouse sequences (Fig. 7). To identify individual amino acid residues in this region that are required for epitopes of mAbs CBRM1/16, CBRM1/17, CBRM1/18, and CBRM1/30, single or double amino acid residues in the human alpha M sequence were replaced with corresponding residues from mouse alpha M. The mutants were co-expressed with the beta 2 subunit in 293 cells, and mAb reactivity was determined by immunofluorescent flow cytometry (Table II). Substitution of Thr725 to Glu (mutant T725E) completely abolished binding of all four mAbs, whereas substitution of Ser728 and Ala729 to Arg and Ser, respectively (mutant S728R/A729S), completely abrogated binding of mAbs CBRM1/16, CBRM1/17, and CBRM1/30 and decreased binding of CBRM1/18. All other substitutions did not affect binding of all four mAbs. Thus, mAbs CBRM1/16, CBRM1/17, CBRM1/18, and CBRM1/30 recognize overlapping epitopes.


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Fig. 7.   Sequence alignment and secondary structure prediction of the C-terminal regions of representative integrin alpha  subunits. The sequence alignments are condensed from a master alignment of 36 integrin alpha  subunits ("Materials and Methods"). Cysteines known to be disulfide bonded in alpha IIb (48) are connected by solid lines; one disulfide that may differ in alpha M (see text) is shown with a dashed line. The arrow points at the main chymotryptic (CT) cleavage site (around Asn570) in alpha IIb (47, 48, 50). The region preceding the chymotryptic cleavage site and following the beta -propeller domain that contains the OKM1 epitope is predicted to fold into a domain (see text). The regions to which mAb epitopes localize are shown above the human alpha M sequence. Two different sequence alignments containing separate branches of the integrin alpha  subunit evolutionary tree were used to predict the secondary structure of human alpha M and alpha IIb with PHD (42). These predictions are independent of one another (see "Materials and Methods"). E, beta -sheet; H, alpha -helix.

                              
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Table II
mAb reactivity with human alpha M subunit mutants carrying human-to-mouse substitutions in the region from residues 718 to 759 
293 cells were transiently co-transfected with cDNAs for human beta 2 and the wild-type or mutated human alpha M subunit containing human-to-mouse single or double amino acid residue substitution. mAb binding to the transfected cells was determined by immunofluorescence flow cytometry. +++, binding comparable to human wild type; +, binding decreased to less than 30% of human wild type; and -, binding completely abolished.

To map mAb epitopes in the beta -propeller domain, 32 different alpha M chimeras were constructed, in which a short segment of mouse sequence comprising a predicted loop or a strand was inserted in the human sequence, and mAbs were tested for reactivity with the alpha M chimeras co-expressed with the human beta 2 subunit in COS cells. Substitution of the 2-3 loop of W6 (mutant hu(W6L2-3)mo) or mutation of Arg534 to Gln in this loop (mutant R534Q) completely abolished binding of mAb CBRM1/32, whereas binding of three other beta -propeller domain mAbs (CBRN1/6, CBRN3/4, and CBRM1/20) and the I-domain mAb CBRM1/29 to these two mutants was not affected (Table III). Arg534 is predicted to be on the upper, outer edge of the beta -propeller domain in W6 (Fig. 8). Substitution of the loop 3-4 of W4 (mutant hu(W4L3-4)mo) completely abrogated mAb CBRN1/6 binding and decreased CBRN3/4 binding. This substitution did not affect CBRM1/32 and CBRM1/20 binding. The three human residues substituted in this mutant, Gln421, Thr423, and Met425, are predicted to be in the lower outer edge of the beta -propeller domain in W4 (Fig. 8). All other substitutions in 30 different segments of the beta -propeller domain had no effect on binding of mAbs CBRM1/32, CBRN1/6, and CBRN3/4. mAb CBRM1/20 was mapped to five amino acid residues in the 1-2 loop of W5 and the 3-4 loop of W63 on the bottom of the beta -propeller domain (Fig. 8).

                              
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Table III
mAb reactivity with human alpha M subunit mutants containing human-to-mouse substitutions in the beta -propeller domain
32 different chimeric alpha M subunits were made in which a short segment of mouse sequence comprising a predicted loop was inserted in the human sequence (see "Materials and Methods"). Mutants were named after the sheet (W) and the loop (L) that was exchanged, e.g. hu(W4L3-4)mo has mouse sequence in the loop between strands 3 and 4 of W4. The wild-type or mutated human alpha M subunit was transiently co-expressed in COS cells with human beta 2. mAb binding to the transfected cells was determined by immunofluorescence flow cytometry. +++, binding comparable to human wild type; +, binding decreased to less than 30% of human wild type; and -, binding completely abolished. Only the mutants that affected binding of mAbs CBRM1/32, CBRN1/6 and CBRN3/4 were listed in the table.


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Fig. 8.   mAb epitope localization in the beta -propeller domain of the alpha M subunit. This stereoview of the side of the putative beta -propeller domain with the upper surface on the top shows a C-alpha trace, with all atoms shown for residues involved in antigenic epitopes. They are R534 for CBRM1/32 (magenta), V450, N453, D457, G545, and G547 for CBRM1/20 (red), and Q421, T423, and M425 for CBRN1/6 and CBRN3/4 (purple). All of the latter three residues were substituted together, and the epitope may require only a subset of these three residues. The beta -sheets (W) are shown in different colors, with W1 in cyan, W2 in orange, W3 in yellow, W4 in olive, W5 in green, W6 in aquamarine, and W7 in turquoise. Ca2+ ions are gray spheres. The beta -propeller domain of the Mac-1 alpha M subunit was modeled using the G-protein beta  subunit beta -propeller domain as template, as described.3 This figure was made with Look of GeneMineTM (Molecular Applications Group, CA).

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

By using mAbs as probes, we have examined the structure of different regions in the Mac-1 alpha M subunit during biosynthesis and alpha M/beta 2 heterodimer assembly and after expression on the cell surface. All five different mAbs to the I-domain reacted with the unassociated alpha M subunit, confirming that the folding of the I-domain does not require the beta 2 subunit. By contrast, four mAbs (CBRN1/6, CBRN3/4, CBRM1/20, and CBRM1/32) that map to three different subregions in the beta -propeller domain did not react with the unassociated alpha M subunit (Fig. 6). CBRN1/6 and CBRN3/4 mapped to one or more of three residues in the 3-4 loop of W4 (residues 421-425) (Fig. 8). CBRM1/20 is specific for three amino acid residues in the 1-2 loop of W5 and two residues in the 3-4 loop of W63 (Fig. 8). The epitope for CBRM1/20 includes two residues, Asn453 and Asp457, that are predicted to coordinate with Ca2+ in the 1-2 loop of W5, and binding of this mAb requires Ca2+ with an EC50 of 0.2 mM.3 These mAbs did not immunoprecipitate the unassociated alpha M precursor or react with the alpha M subunit expressed alone on the surface of COS cells. mAb CBRM1/32 reacted with the alpha M/beta 2 complex expressed on the cell surface but did not react with the alpha M subunit expressed alone on the cell surface. The epitope of CBRM1/32 requires residue Arg-534 in the 2-3 loop of W6 (Fig. 8). One possible interpretation of our results is that all three epitopes in the beta -propeller domain require the presence of the beta  subunit because the alpha  and beta  subunits associate with one another in each of these regions, and each antibody binding site includes contacts with both the beta  subunit and alpha  subunit. If so, the contacts with the beta  subunit do not include any antigenic residues, because all mAb reacted equally well whether the human or murine beta  subunit was associated with human alpha M. Furthermore, we tested the chicken beta 2 subunit, because 35% of the residues in the human and chicken beta 2 subunits differ, as opposed to only 18% between the human and the mouse (45). Amino acid differences between species are preferentially found on the surface of proteins rather than buried. Although a substantial portion of surface residues are expected to differ on the chicken and human beta 2 subunits, whether the chicken or human beta 2 subunit was present did not affect mAb reactivity. The epitopes that were localized include some that are quite distant. The Arg-534 residue recognized by CBRM1/32 mAb is on the upper surface of the beta -propeller, whereas residues recognized by the CBRM1/20 and the CBRN1/6 mAb are on the lower surface and point in opposite directions from one another. The C-alpha carbon of the Arg-534 residue is predicted to be 30 ± 3 Å and 41 ± 3 Å distant from residues recognized by the CBRM1/20 and the CBRN1/6 mAb, respectively, and the C-alpha carbons of residues recognized by the CBRM1/20 and the CBRN1/6 mAb are 23 ± 7 Å distant from one another. The probability that three out of three different epitopes would include surfaces from both the alpha  and beta  subunits, even though some epitopes are quite distant from one another, would appear to be low. Because of this, and the lack of effect of the species origin of the beta  subunit on mAb reactivity, we favor the interpretation that association between the alpha  subunit and beta  subunit is required for the beta -propeller domain to assume its final three-dimensional structure, i.e. to assume the correct fold. Our data are consistent with the idea that there is an interface between the alpha  subunit beta -propeller domain and the beta  subunit, although we believe that the interface is not necessarily associated with any of the epitopes we have mapped. Conversely, a number of mAb to different epitopes in the conserved domain of the integrin beta  subunit are not reactive in the absence of the alpha  subunit (23). Thus, the conserved domain of the beta  subunit is a candidate for association with the putative beta -propeller domain of the alpha  subunit. Analogously, the G-protein beta  subunit beta -propeller domain is not properly folded in the absence of association with the G-protein gamma  subunit (46).

A previous study on the LFA-1 beta -propeller domain used two mAbs (S6F1 and TS2/4) that map to the alpha L subunit N-terminal 57 amino acids, i.e. to part of beta -sheets W7 and W1, and one mAb (G-25.2) that maps to a 212-amino acid region spanning W5-7 of the beta -propeller domain and part of the C-terminal region. These mAbs did not react with the unassociated alpha L subunit (24) (Fig. 6). Another mAb (CBRLFA-1/1) that overlaps the I-domain and W3 of the beta -propeller domain showed weak reactivity in the absence of the beta 2 subunit. It is not known whether this mAb recognizes a boundary region between the I and beta -propeller domains. Taken together, the findings on LFA-1 and Mac-1 demonstrate that multiple mAbs to different regions in the beta -propeller domain do not react with the alpha  subunit in the absence of the beta  subunit and suggest that the beta -propeller domain folds as a unit and that this folding depends on association with the beta  subunit.

mAbs to the C-terminal region of the alpha M extracellular domain differentially reacted with the unassociated alpha M subunit. Five mAbs (OKM1, CBRM1/10, CBRM1/25, CBRM1/26, and CBRM1/23) reacted with both the unassociated and the complexed alpha M subunit and were mapped to three subregions. OKM1 mapped to a subregion immediately following the beta -propeller domain, residues 599-679. CBRM1/10, CBRM1/25, and CBRM1/26 mapped to amino acids 820-882, and CBRM1/23 mapped to residues 943-1047. Within each of these subregions, there are multiple differences between the mouse and human sequences (Fig. 7). Whether the multiple mAbs that react with residues 820-882 recognize one or more epitopes within this subregion is not known. Minimally, these data show that three epitopes in three different subregions of the C-terminal segment are independent of the beta 2 subunit. By contrast, four other mAbs to the C-terminal region (CBRM1/16, CBRM1/17, CBRM1/18, and CBRM1/30) only reacted with the alpha M/beta 2 complex. These mAbs did not react with the unassociated alpha M precursor or with the uncomplexed alpha M subunit expressed on the COS cell surface. All four mAbs were mapped to residues Thr725 and, additionally, Ser728 and/or Ala729. Although CBRM1/16, CBRM1/17, CBRM1/18, and CBRM1/30 did not react with the unassociated alpha M subunit, they reacted with the human alpha M/mouse beta 2 and human alpha M/chicken beta 2 complexes as well as with the human alpha M/human beta 2 complex (data not shown). Thus, association with the beta 2 subunit may be required for this region to assume its final structure. Although we believe that the interpretation that the alpha  and beta  subunits both contribute to the antibody-binding site is less likely, either interpretation shows an important interaction with the beta  subunit for the region of residues 725-729. Overall, the results show that three out of four epitopes in the C-terminal region of Mac-1 alpha M subunit are intact in the absence of association with the beta 2 subunit. If these results are representative of the C-terminal region as a whole, our data would suggest that much of this region folds independently of the beta 2 subunit. This is in marked contrast to the beta -propeller domain.

To place our results on the C-terminal region within a structural framework, we predicted its secondary structure using the PHD program (42) (Fig. 7). By using a phylogenetic tree based on an iteratively refined alignment (41) of 36 alpha  subunit C-terminal region sequences, two subfamilies were identified. These subfamilies were large and contained members that were 1) sufficiently similar to one another to allow accurate alignment and to not be too divergent in tertiary structure, and 2) were sufficiently different from one another to contain a large amount of sequence information, and hence optimize prediction accuracy (42). An alignment of 11 subunits was used to predict the secondary structure of human alpha M, and an alignment of 17 other alpha  subunits was used to predict the structure of human alpha IIb (Fig. 7). Since no sequences were shared between the two alignments, and between the two groups there is only 16-21% sequence identity, the predictions for alpha M and alpha IIb are largely independent of one another.

In the C-terminal segment, a total of 30-34 beta -strands were predicted. Of these, 22 were independently predicted in both alpha M and alpha IIb. Only 5 alpha -helices were predicted, and in each case these were predicted in only one of the two alpha  subunits. Thus, the C-terminal region is predicted to form domains of the all beta  class. In this respect, it is similar to the beta -propeller domain (9) and different from the I-domain which is of the alpha /beta class (14, 16).

The disulfide bond topology of alpha IIb has been chemically determined (47, 48). The conservation of cysteines suggest that 5 of 6 disulfide bonds are conserved in human alpha M, whereas one differs (Fig. 7). The first disulfide in this region, alpha IIb C473-C484, is confirmed by the sequence alignment of 36 integrin alpha  subunits, since these two cysteines are selectively absent in the chicken alpha 6 subunit, and the cysteines and the loop in between them are absent in alpha 2 subunits and alpha E subunits. The cysteines corresponding to the last disulfide bond in alpha IIb, Cys885-Cys890, are missing from alpha L subunits. Otherwise, there is only one predicted difference between disulfide bonds in alpha IIb and the leukocyte integrin alpha  subunits. The cysteine corresponding to alpha IIb Cys484 is missing in all leukocyte integrin alpha  subunits, and all leukocyte integrin alpha  subunits contain a cysteine with no equivalent residue in alpha IIb, i.e. Cys706 in alpha M. We predict that the cysteines at alpha IIb position 473, although aligned by sequence, are non-equivalent, i.e. that the cysteine in alpha M is involved in a different disulfide bond, to Cys706 (dashed line in Fig. 7).

Folds of the all-beta class as a general rule contain anti-parallel beta -sheets (49). The vast majority but not all of the predicted beta -strands in the alpha IIb and alpha M C-terminal regions are markedly amphipathic with alternating hydrophobic and hydrophilic residues. We therefore predict that the C-terminal region folds into 2-layer, anti-parallel beta -sheet structures, i.e. beta -sandwich or beta -barrel domains of which the Ig fold is one of many representatives. The total length of the C-terminal region of about 500 residues, the number of predicted beta -strands, and the overall number and location of disulfide bonds are appropriate for approximately four to six beta -sandwich domains.

In alpha IIb, a main chymotryptic cleavage site is located around Asn570 (47, 48). Cleavage of cell-surface alpha IIbbeta 3 releases a ligand binding complex containing an N-terminal fragment of alpha IIbeta of 55 kDa ending at approximately Asn570, and an 85-kDa N-terminal fragment of beta 3 (50). This suggests that the region around Asn570 is well exposed and may represent a domain boundary region. It is interesting that four mAbs dependent on beta  subunit association map to essentially the same site in alpha M (Fig. 7). The region preceding the chymotryptic cleavage site and following the beta -propeller domain in alpha IIb contains one long range disulfide bond (Cys490-Cys545), and six predicted beta -strands. In alpha M, the corresponding region contains two predicted long range disulfide bonds (Cys639-Cys696, Cys623-Cys706), and seven predicted beta -strands. Based on these features, we predict that this region of about 120 amino acids following the beta -propeller domain, residues 599 to about 718 for alpha M, and 450 to about 570 for alpha IIb, folds into a structurally independent domain. Consistent with this prediction, this region in alpha M appears to fold independently of association with the beta  subunit, as shown with the OKM1 mAb. This contrasts with the flanking N-terminal beta -propeller domain and the flanking C-terminal region from residues 725 to 729, to which mAbs CBRM1/16, CBRM1/17, CBRM1/18, and CBRM1/30 map.

Our results together with other recent studies provide new insight into how different domains in the integrin alpha  and beta  subunits may associate. The I-domain is predicted to be connected to the upper surface of the beta -propeller domain (9). The alpha  subunit beta -propeller domain and the beta  subunit conserved domain may associate, since both are dependent on alpha  and beta  subunit association for folding (23, 24) (this study). The predicted beta -sandwich/beta -barrel domain that follows the beta -propeller domain and contains the OKM1 epitope, residues 599-718, is connected to the C terminus of strand 3 of W7 of the predicted beta -propeller domain and hence to the bottom of the beta -propeller domain. The following subregion of the alpha M subunit, from residues 725 to 729, may directly associate with the beta 2 subunit, or its structure may be indirectly dependent on associations elsewhere with the beta  subunit. Other subregions in the C-terminal portions of alpha  and beta  subunits might also participate in alpha  and beta  subunit association as proposed for the alpha IIbbeta 3 integrin (32, 51), while retaining similar conformations in the unassociated and complexed forms.

In summary, the results from this study suggest that proper folding of the beta -propeller domain of the integrin alpha M subunit requires association with the beta 2 subunit, whereas the I-domain folds independently of the beta 2 subunit. Much of the region C-terminal to the beta -propeller domain folds prior to beta  subunit association. Our results further advance the understanding of integrin structure and provide information that will be useful in guiding studies leading to the characterization of integrin three-dimensional structure.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant CA31799, a fellowship from The Cancer Research Institute (to C. L.), and a fellowship from The Danish Natural Science Research Council (to C. O.).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.

Dagger To whom correspondence should be addressed: The Center for Blood Research and Harvard Medical School, Dept. of Pathology, 200 Longwood Ave., Boston, MA 02115. Tel.: 617-278-3200; Fax: 617-278-3232; E-mail: springer{at}sprsgi.med.harvard.edu.

1 The abbreviations used are: mAb, monoclonal antibody; FBS, fetal bovine serum; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; hu, human; mo, mouse.

2 S. Q. Na and T. A. Springer, unpublished data.

3 C. Oxvig and T. A. Springer (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 4870-4875.

4 Available on-line at the following address: http://www.embl-heidelberg.de./predict protein/.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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