The Structure of the β-Propeller Domain and C-terminal Region of the Integrin αM Subunit
1998; Elsevier BV; Volume: 273; Issue: 24 Linguagem: Inglês
10.1074/jbc.273.24.15138
ISSN1083-351X
AutoresChafen Lu, Claus Oxvig, Timothy A. Springer,
Tópico(s)Monoclonal and Polyclonal Antibodies Research
ResumoThe αM subunit of integrin Mac-1 contains several distinct regions in its extracellular segment. The N-terminal region has been predicted to fold into a β-propeller domain composed of seven β-sheets each about 60 amino acid residues long, with the I-domain inserted between β-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 αM subunit on association with the β2 subunit in the αM/β2 heterodimer. All of the mAbs to the I-domain immunoprecipitated the unassociated αM precursor and reacted with the αM subunit expressed alone on the surface of COS cells. By contrast, four mAbs to the β-propeller domain did not react with the unassociated αM precursor nor with the uncomplexed αM subunit expressed on COS cell surface. The four mAbs were mapped to three subregions in three different β-sheets, making it unlikely that each recognized an interface between the α and β subunits. These results suggest that folding of different β-propeller subregions is coordinate and is dependent on association with the β2 subunit. The segment C-terminal to the β-propeller domain, residues 599–1092, was studied with nine mAbs. A subset of four mAbs that reacted with the αM/β2 complex but not with the unassociated αM subunit were mapped to one subregion, residues 718–759, and five other mAbs that recognized both the unassociated and the complexed αM 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 β-propeller domain folds independently of association with the β2 subunit. Our data provide new insights into how different domains in the integrin α and β subunits may interact. The αM subunit of integrin Mac-1 contains several distinct regions in its extracellular segment. The N-terminal region has been predicted to fold into a β-propeller domain composed of seven β-sheets each about 60 amino acid residues long, with the I-domain inserted between β-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 αM subunit on association with the β2 subunit in the αM/β2 heterodimer. All of the mAbs to the I-domain immunoprecipitated the unassociated αM precursor and reacted with the αM subunit expressed alone on the surface of COS cells. By contrast, four mAbs to the β-propeller domain did not react with the unassociated αM precursor nor with the uncomplexed αM subunit expressed on COS cell surface. The four mAbs were mapped to three subregions in three different β-sheets, making it unlikely that each recognized an interface between the α and β subunits. These results suggest that folding of different β-propeller subregions is coordinate and is dependent on association with the β2 subunit. The segment C-terminal to the β-propeller domain, residues 599–1092, was studied with nine mAbs. A subset of four mAbs that reacted with the αM/β2 complex but not with the unassociated αM subunit were mapped to one subregion, residues 718–759, and five other mAbs that recognized both the unassociated and the complexed αM 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 β-propeller domain folds independently of association with the β2 subunit. Our data provide new insights into how different domains in the integrin α and β subunits may interact. The integrin family of adhesion molecules participate in important cell-cell and cell-extracellular matrix interactions in a diverse range of biological processes (1Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (9014) Google Scholar). Integrins are noncovalently associated α/β heterodimers, with each subunit consisting of a large extracellular domain (>100 kDa for α subunits and >75 kDa for β subunits), a single transmembrane region, and a short cytoplasmic tail (50 amino acids or less, except for the β4 subunit) (1Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (9014) Google Scholar). The adhesiveness of integrins is dynamically regulated in response to cytoplasmic signals, termed "inside-out" signaling (2Sastry S.K. Horwitz A.F. Curr. Opin. Cell Biol. 1993; 5: 819-831Crossref PubMed Scopus (410) Google Scholar, 3Diamond M.S. Springer T.A. Curr. Biol. 1994; 4: 506-517Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar, 4Ginsberg M.H. Biochem. Soc. Trans. 1995; 23: 439-446Crossref PubMed Scopus (20) Google Scholar). The leukocyte integrin subfamily consists of four members that share the common β2 subunit (CD18) but have distinct α subunits, αL (CD11a), αM (CD11b), αX (CD11c), and αd for LFA-1, Mac-1, p150, 95, and αd/β2, respectively (5Springer T.A. Nature. 1990; 346: 425-433Crossref PubMed Scopus (5852) Google Scholar, 6Larson R.S. Springer T.A. Immunol. Rev. 1990; 114: 181-217Crossref PubMed Scopus (518) Google Scholar, 7Van der Vieren M. Le Trong H. Wood C.L. Moore P.F. St. John T. Staunton D.E. Gallatin W.M. Immunity. 1995; 3: 683-690Abstract Full Text PDF PubMed Scopus (231) Google Scholar). The leukocyte integrins mediate a range of adhesive interactions that are essential for normal immune and inflammatory responses (5Springer T.A. Nature. 1990; 346: 425-433Crossref PubMed Scopus (5852) Google Scholar). Although the overall structure of integrins is unknown, several structurally distinct domains in the extracellular portions of both α and β subunits have been predicted or identified. The N-terminal region of the integrin α subunits contains seven repeats of about 60 amino acids each (8Corbi A.L. Miller L.J. O'Connor K. Larson R.S. Springer T.A. EMBO J. 1987; 6: 4023-4028Crossref PubMed Scopus (180) Google Scholar) and has recently been predicted to fold into a β-propeller domain that consists of seven β-sheets, with each β-sheet containing four anti-parallel β-strands (9Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 65-72Crossref PubMed Scopus (390) Google Scholar). The leukocyte integrin α subunits (10Larson R.S. Corbi A.L. Berman L. Springer T.A. J. Cell Biol. 1989; 108: 703-712Crossref PubMed Scopus (206) Google Scholar), the α1 (11Briesewitz R. Epstein M.R. Marcantonio E.E. J. Biol. Chem. 1993; 268: 2989-2996Abstract Full Text PDF PubMed Google Scholar) and α2 (12Takada Y. Hemler M.E. J. Cell Biol. 1989; 109: 397-407Crossref PubMed Scopus (252) Google Scholar) subunits of the β1 subfamily, and the αE subunit (13Shaw S.K. Cepek K.L. Murphy E.A. Russell G.J. Brenner M.B. Parker C.M. J. Biol. Chem. 1994; 269: 6016-6025Abstract Full Text PDF PubMed Google Scholar) of the β7 subfamily contain an inserted domain or I-domain of about 200 amino acids that is predicted to be inserted between β-sheets 2 and 3 of the β-propeller domain (9Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 65-72Crossref PubMed Scopus (390) Google Scholar). The three-dimensional structure of the I-domain from the Mac-1, LFA-1, and α2β1 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 (14Lee J.-O. Rieu P. Arnaout M.A. Liddington R. Cell. 1995; 80: 631-638Abstract Full Text PDF PubMed Scopus (805) Google Scholar, 15Lee J.-O. Bankston L.A. Arnaout M.A. Liddington R.C. Structure. 1995; 3: 1333-1340Abstract Full Text Full Text PDF PubMed Scopus (362) Google Scholar, 16Qu A. Leahy D.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10277-10281Crossref PubMed Scopus (290) Google Scholar, 17Emsley J. King S.L. Bergelson J.M. Liddington R.C. J. Biol. Chem. 1997; 272: 28512-28517Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar). The integrin β 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 (14Lee J.-O. Rieu P. Arnaout M.A. Liddington R. Cell. 1995; 80: 631-638Abstract Full Text PDF PubMed Scopus (805) Google Scholar, 18Puzon-McLaughlin W. Takada Y. J. Biol. Chem. 1996; 271: 20438-20443Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 19Tuckwell D.S. Humphries M.J. FEBS Lett. 1997; 400: 297-303Crossref PubMed Scopus (100) Google Scholar). Very little is known about the structure of the C-terminal half of the extracellular portions of both α and β subunits. Electron microscopic images of integrins reveal that the N-terminal portions of the α and β 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 α and β extracellular domains (20Nermut M.V. Green N.M. Eason P. Yamada S.S. Yamada K.M. EMBO J. 1988; 7: 4093-4099Crossref PubMed Scopus (195) Google Scholar, 21Weisel J.W. Nagaswami C. Vilaire G. Bennett J.S. J. Biol. Chem. 1992; 267: 16637-16643Abstract Full Text PDF PubMed Google Scholar, 22Wippler J. Kouns W.C. Schlaeger E.-J. Kuhn H. Hadvary P. Steiner B. J. Biol. Chem. 1994; 269: 8754-8761Abstract Full Text PDF PubMed Google Scholar). This would suggest that the C-terminal portions of both subunits are quite extended. Previous studies using mAbs 1The abbreviations used are: mAb, monoclonal antibody; FBS, fetal bovine serum; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; hu, human; mo, mouse. as probes have shown that the structure of specific domains in LFA-1 requires association of the αL and β2 subunits. mAbs to the β2 subunit conserved domain do not react with the unassociated β2 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 αL subunit (23Huang C. Lu C. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3156-3161Crossref PubMed Scopus (60) Google Scholar). mAbs to the I-domain react with the unassociated αL subunit (24Huang C. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3162-3167Crossref PubMed Scopus (66) Google Scholar). This finding together with the fact that the I-domain can be expressed as an isolated domain (14Lee J.-O. Rieu P. Arnaout M.A. Liddington R. Cell. 1995; 80: 631-638Abstract Full Text PDF PubMed Scopus (805) Google Scholar, 16Qu A. Leahy D.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10277-10281Crossref PubMed Scopus (290) Google Scholar, 25Zhou L. Lee D.H.S. Plescia J. Lau C.Y. Altieri D.C. J. Biol. Chem. 1994; 269: 17075-17079Abstract Full Text PDF PubMed Google Scholar, 26Kubota Y. Kleinman H.K. Martin G.R. Lawley T.J. J. Cell. Biol. 1988; 107: 1589-1598Crossref PubMed Scopus (984) Google Scholar) show that the I-domain assumes a native structure independently of the β2 subunit. By contrast, two mAbs (S6F1 and TS2/4) mapped to the N-terminal region of the β-propeller domain, and one mAb (G-25.2) that maps to a region of 212 amino acids with 159 amino acids located in the β-propeller domain and the remainder in the C-terminal region, do not recognize the αL subunit in the absence of association with the β2 subunit (24Huang C. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3162-3167Crossref PubMed Scopus (66) Google Scholar). Another mAb (CBRLFA-1/1) that maps to a region overlapping the I-domain and β-propeller domain reacts weakly with the uncomplexed αL subunit. These results indicate that at least one region in the β-propeller domain is dependent on association with the β2 subunit for mAb reactivity, and it has been suggested that the most likely explanation is that folding of the β-propeller domain is not completed until after association with the β subunit (24Huang C. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3162-3167Crossref PubMed Scopus (66) Google Scholar). Since mAbs specific for the region of the αL subunit C-terminal to the β-propeller domain have not been described, it is not known whether folding of this region is dependent on association with the β subunit. In this study, we have used mAb probes to study the structure of the Mac-1 α subunit in the presence and absence of association with the β2 subunit. We have studied the β-propeller domain, the I-domain, and the extensive region C-terminal to the β-propeller domain. Compared with the previous studies on LFA-1, our studies on the β-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 β-propeller structure all show a dependence on β subunit association for reactivity. Furthermore, we employ a panel of mAbs that defines four different subregions within the C-terminal region of the α subunit. The results show that epitopes in three of these regions have a native structure in the absence of β subunit association, whereas a fourth epitope is dependent on association with the β subunit. Thus, much of the C-terminal region of the αM subunit appears to assume a native fold independently of association with the β2 subunit. 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. The following murine mAbs against the αM subunit of human Mac-1 were previously described: OKM1, OKM9 (27Wright S.D. Rao P.E. Van Voorhis W.C. Craigmyle L.S. Iida K. Talle M.A. Westberg E.F. Goldstein G. Silverstein S.C. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 5699-5703Crossref PubMed Scopus (475) Google Scholar), TGM-65 (28Uciechowski P. Schmidt R. Knapp W. Dorken B. Gilks W.R. Rieber E.P. Schmidt R.E. Stein H. Von dem Borne Jr., A.E.G. Leucocyte Typing IV: White Cell Differentiation Antigens. Oxford University Press, Oxford1989: 543-551Google Scholar), 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 (29Diamond M.S. Garcia-Aguilar J. Bickford J.K. Corbi A.L. Springer T.A. J. Cell Biol. 1993; 120: 1031-1043Crossref PubMed Scopus (469) Google Scholar). 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 αM subunit of Mac-1 2S. Q. Na and T. A. Springer, unpublished data. were used as hybridoma supernatant. TS1/18 and CBRLFA-1/2 against human leukocyte integrin β2 subunit were described previously (30Sanchez-Madrid F. Krensky A.M. Ware C.F. Robbins E. Strominger J.L. Burakoff S.J. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 7489-7493Crossref PubMed Scopus (587) Google Scholar, 31Petruzzelli L. Maduzia L. Springer T. J. Immunol. 1995; 155: 854-866PubMed Google Scholar) and used as purified IgG. The human wild-type αM subunit cDNA was subcloned in the expression vector pCDNA3.1+ (Invitrogen, Carlsbad, CA) as described. 3C. Oxvig and T. A. Springer (1998)Proc. Natl. Acad. Sci. U. S. A. 95,4870–4875. For generating human-mouse αM chimeras, a Sac II 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 αM cDNA (33Pytela R. EMBO J. 1988; 7: 1371-1378Crossref PubMed Scopus (96) Google Scholar) 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 αM cDNA fragment covering nucleotides from the Sfi I site (nucleotide 1688) to the stop codon and having a Sac II site immediately after the stop codon was made. This mouse αM Sfi I-Sac II fragment was used to replace the corresponding human αMSfi I-Sac II fragment to generate the initial chimeric αM 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 αM chimeras with a variable mouse C-terminal portion were generated by overlap extension PCR (34Horton R.M. Hunt H.D. Ho S.N. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 61-68Crossref PubMed Scopus (2641) Google Scholar, 35Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 51-59Crossref PubMed Scopus (6833) Google Scholar). Briefly, outer primers for overlap PCR were just 5′ to the Sfi I site and 3′ to the Sac II site, and the first set of reactions was carried out using the human wild-type αM and the initial chimeric construct as templates. After the overlap extension reaction, the chimeric products were digested with Sfi I and Sac II, and theSfi I-Sac II fragments were swapped into the human wild-type αM in vector pCDNA3.1+. Human to mouse individual amino acid substitutions in the region from amino acids 718–759 of human αM were made by overlap extension PCR (34Horton R.M. Hunt H.D. Ho S.N. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 61-68Crossref PubMed Scopus (2641) Google Scholar, 35Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 51-59Crossref PubMed Scopus (6833) Google Scholar). Briefly, the overlapping primers contained the desired mutations, and the outer primers were 5′ to the Sfi I site and 3′ to theNde I site, respectively. The overlap extension PCR products were digested with Sfi I and Nde I and swapped into human wild-type αM in expression vector pEFpuro (36Lu C. Springer T.A. J. Immunol. 1997; 159: 268-278PubMed Google Scholar). For mapping mAb epitopes in the β-propeller domain of the human αM subunit, 32 different chimeric αM 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 αM cDNA fragments that included the Not I site 5′ to the coding region and theBsp EI site at amino acid residue 180 or included theBsp EI-Bbs I fragment from 180 to 672, as described elsewhere.3 The mutated cDNA fragments were excised with Not I and Bsp EI or Bsp EI andBbs I and swapped into wild-type αM 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 α 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. COS cells were transfected by the DEAE-dextran method (36Lu C. Springer T.A. J. Immunol. 1997; 159: 268-278PubMed Google Scholar) with the αM cDNA alone or were co-transfected with the wild-type or chimeric αM and β2 cDNA. The wild-type and chimeric αM cDNA were in plasmid pCDNA3.1+, and the β2 cDNA was contained in plasmid pEF-BOS (36Lu C. Springer T.A. J. Immunol. 1997; 159: 268-278PubMed Google Scholar). 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 (37Heinzel S.S. Krysan P.J. Calos M.P. DuBridge R.B. J. Virol. 1988; 62: 3738-3746Crossref PubMed Google Scholar, 38DuBridge R.B. Tang P. Hsia H.C. Leong P.M. Miller J.H. Calos M.P. Mol. Cell. Biol. 1987; 7: 379-387Crossref PubMed Scopus (915) Google Scholar). Briefly, 7.5 μg of wild-type or mutant αM cDNA in plasmid pEFpuro and 7.5 μg of β2 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. 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 mmCa2+. After washing, cells were resuspended in cold PBS and analyzed on a FACScan (Becton Dickinson, San Jose, CA). For metabolic labeling, U937 cells were plated in four 10-cm Petri dishes and induced with PMA for 3 days as described previously (39Miller L.J. Springer T.A. J. Immunol. 1987; 139: 842-847PubMed Google Scholar). 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 mmTris-HCl, pH 7.5, 150 mm NaCl, 2 mmMgCl2, 1 mm iodoacetamide, 1 mmphenylmethylsulfonyl 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 × 106cells) 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 (40Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207233) Google Scholar). The gels were processed for fluorography for [35S]methionine-labeled proteins or autoradiography for 125I-labeled proteins. The amino acid sequences between the β-propeller domain and the transmembrane segment of 36 integrin α subunits (9Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 65-72Crossref PubMed Scopus (390) Google Scholar) 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 (41Gotoh O. J. Mol. Biol. 1996; 264: 823-838Crossref PubMed Scopus (268) Google Scholar). The αM and αIIb subunits fall in different branches of this tree, each of which is well populated. One branch containing 11 subunits most closely related to human αM,i.e. murine αM, human αD, and αX, murine and human αL, human and rat α1, and bovine, human, and mouse α2, were realigned with one another using PRRP. They are 21–70%,x̄ = 34% identical to human αM. Another branch containing the 17 subunits most closely related to human αIIb,i.e. hamster, human, and mouse α3, human andXenopus α5, chicken and human α6, mouse α7, chicken and human α8, and chicken, human, mouse, and Pleurodes αV, and YMA1 of Caenorhabditis elegans , were realigned in a separate group. They are 20–38%, x̄ = 28% identical to human αIIb. The alignments in MSF format, with gaps in human αM and human αIIb removed to increase prediction accuracy, were separately submitted for secondary structure prediction to PHD (42Rost B. Methods Enzymol. 1996; 266: 525-539Crossref PubMed Google Scholar). 4Available on-line at the following address:http://www.embl-heidelberg.de./predict protein/. Smaller subgroups containing a higher degree of relationship to αM (6 α subunits, with 27–70% identity to αM) or to αIIb (9 α subunits, with 33–38% identity to αIIb) gave very similar predictions but with a slightly lower correlation between the αM and αIIb predictions. To study whether folding of the αM subunit is dependent on association with the β2 subunit, we examined the expression of mAb epitopes on the unassociated αM subunit. Eighteen mAbs that have previously been mapped to different regions in the αM subunit were used (29Diamond M.S. Garcia-Aguilar J. Bickford J.K. Corbi A.L. Springer T.A. J. Cell Biol. 1993; 120: 1031-1043Crossref PubMed Scopus (469) Google Scholar)2 (Fig. 1). Previous studies on leukocyte integrin biosynthesis have shown that the α and β 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 α and β complex (39Miller L.J. Springer T.A. J. Immunol. 1987; 139: 842-847PubMed Google Scholar, 43Ho M.-K. Springer T.A. J. Biol. Chem. 1983; 258: 2766-2769Abstract Full Text PDF PubMed Google Scholar, 44Sanchez-Madrid F. Nagy J. Robbins E. Simon P. Springer T.A. J. Exp. Med. 1983; 158: 1785-1803Crossref PubMed Scopus (611) Google Scholar). We therefore examined whether mAbs to the I-domain, to the β-propeller domain, and to the C-terminal region immunoprecipitated the unassociated αM precursor (α′M). All mAbs immunoprecipitated the mature αM 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 αM subunit was complexed with the β2 subunit as shown by co-immunoprecipitation of the β2 subunit with the αM subunit. However, mAbs differentially precipitated the α′M precursor, which is slightly smaller than the mature αM subunit from the pulse-labeled cells (Fig. 2, upper panel ). There was little or no α′M precursor associated with the β2 precursor (β′2) in the pulse-labeled cells, since no detectable β′2 over background was co-precipitated by mAbs to the αM subunit, but β′2 was precipitated with mAb CBRLFA-1/2 to the β2 subunit (upper panel, lane 18 ). All mAbs to the I-domain precipitated α′M (upper panel, lanes 2–6 ). By contrast, three mAbs (CBRN1/6, CBRN3/4, and CBRM1/20) to the β-propeller domain did not precipitate α′M (upper panel, lanes 7, 8 , and 21 ). mAb CBRM1/32 to the β-propeller domain did not precipitate the αM/β2 complex or α′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 α′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 α′M (upper panel, lanes 11–13 and 17 ). Thus, epitopes of mAbs to the I-domain are expressed on the unassociated αM precursor, whereas epitopes of β-propeller domain mAbs and a subset of mAbs to the C-terminal region are not.Figure 2Immunoprecipitation of the unassociated αM precursor (α′M) and the αM/β2 complex. PMA-induced U937 cells were pulse-labeled with [35S]methionine for 30 min and chased with unlabeled methionine for 16 h. The αM and β2 precursors (α′M and β′2, respectively) and the αM/β2 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
Referência(s)