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Transmembrane Domain Helix Packing Stabilizes Integrin αIIbβ3 in the Low Affinity State

2005; Elsevier BV; Volume: 280; Issue: 8 Linguagem: Inglês

10.1074/jbc.m412701200

1083-351X

Anthony W. Partridge, Shouchun Liu, Sanguk Kim, James U. Bowie, Mark H. Ginsberg,

Monoclonal and Polyclonal Antibodies Research

Regulated changes in the affinity of integrin adhesion receptors (“activation”) play an important role in numerous biological functions including hemostasis, the immune response, and cell migration. Physiological integrin activation is the result of conformational changes in the extracellular domain initiated by the binding of cytoplasmic proteins to integrin cytoplasmic domains. The conformational changes in the extracellular domain are likely caused by disruption of intersubunit interactions between the α and β transmembrane (TM) and cytoplasmic domains. Here, we reasoned that mutation of residues contributing to α/β interactions that stabilize the low affinity state should lead to integrin activation. Thus, we subjected the entire intracellular domain of the β3 integrin subunit to unbiased random mutagenesis and selected it for activated mutants. 25 unique activating mutations were identified in the TM and membrane-proximal cytoplasmic domain. In contrast, no activating mutations were identified in the more distal cytoplasmic tail, suggesting that this region is dispensable for the maintenance of the inactive state. Among the 13 novel TM domain mutations that lead to integrin activation were several informative point mutations that, in combination with computational modeling, suggested the existence of a specific TM helix-helix packing interface that maintains the low affinity state. The interactions predicted by the model were used to identify additional activating mutations in both the α and β TM domains. Therefore, we propose that helical packing of the α and β TM domains forms a clasp that regulates integrin activation. Regulated changes in the affinity of integrin adhesion receptors (“activation”) play an important role in numerous biological functions including hemostasis, the immune response, and cell migration. Physiological integrin activation is the result of conformational changes in the extracellular domain initiated by the binding of cytoplasmic proteins to integrin cytoplasmic domains. The conformational changes in the extracellular domain are likely caused by disruption of intersubunit interactions between the α and β transmembrane (TM) and cytoplasmic domains. Here, we reasoned that mutation of residues contributing to α/β interactions that stabilize the low affinity state should lead to integrin activation. Thus, we subjected the entire intracellular domain of the β3 integrin subunit to unbiased random mutagenesis and selected it for activated mutants. 25 unique activating mutations were identified in the TM and membrane-proximal cytoplasmic domain. In contrast, no activating mutations were identified in the more distal cytoplasmic tail, suggesting that this region is dispensable for the maintenance of the inactive state. Among the 13 novel TM domain mutations that lead to integrin activation were several informative point mutations that, in combination with computational modeling, suggested the existence of a specific TM helix-helix packing interface that maintains the low affinity state. The interactions predicted by the model were used to identify additional activating mutations in both the α and β TM domains. Therefore, we propose that helical packing of the α and β TM domains forms a clasp that regulates integrin activation. Integrin heterodimers are essential for the development and functioning of multicellular animals, because they mediate cell migration and cell adhesion and can influence gene expression and cell proliferation (1Hynes R.O. Cell. 2002; 110: 673-687Abstract Full Text Full Text PDF PubMed Scopus (6951) Google Scholar). All of the integrin heterodimers are composed of single pass Type I transmembrane (TM) 1The abbreviations used are: TM, transmembrane; CHO, Chinese hamster ovary; FACS, fluorescence-activated cell sorter; TMD, transmembrane domain; LIBS, ligand-induced binding site. protein subunits α and β. A central feature of these receptors is their capacity for rapid changes in their adhesive function mediated by changes in their ligand binding affinity, operationally defined here as “activation.” The prototypical integrin, platelet αIIbβ3, is activated through interactions of the cytoplasmic integrin tails (∼20 and 47 residues for α and β tails, respectively) with intracellular proteins such as talin (2Campbell I.D. Ginsberg M.H. Trends Biochem. Sci. 2004; 29: 429-435Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). These interactions initiate a long-range conformational change in the large extracellular domains (>700 residues each), resulting in high affinity binding of fibrinogen, von Willebrand factor, and fibronectin and consequently platelet aggregation and adherence to the vessel wall (1Hynes R.O. Cell. 2002; 110: 673-687Abstract Full Text Full Text PDF PubMed Scopus (6951) Google Scholar). Initial mutational studies suggested that a salt bridge between αIIbArg995 and β3Asp723 helps maintain the integrin in the low affinity state by forming part of an interactive face between α and β subunit cytoplasmic domains (3Hughes P.E. Diaz-Gonzalez F. Leong L. Wu C. McDonald J.A. Shattil S.J. Ginsberg M.H. J. Biol. Chem. 1996; 271: 6571-6574Abstract Full Text Full Text PDF PubMed Scopus (511) Google Scholar). Protein engineering studies from Springer laboratory have further advanced the idea that specific integrin α/β interactions maintain the low affinity conformation of the receptor. In particular, enforced association of either the C-terminal region of the extracellular domains (4Takagi J. Erickson H.P. Springer T.A. Nat. Struct. Biol. 2001; 8: 412-416Crossref PubMed Scopus (227) Google Scholar) or that of the cytoplasmic domains (5Lu C. Takagi J. Springer T.A. J. Biol. Chem. 2001; 276: 14642-14648Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar) leads to expression of an inactive integrin. Furthermore, during physiological integrin activation, changes in fluorescence resonance energy transfer between fluorophores joined to the αL and β2 cytoplasmic domains are consistent with cytoplasmic domain separation (6Kim M. Carman C.V. Springer T.A. Science. 2003; 301: 1720-1725Crossref PubMed Scopus (646) Google Scholar). Finally, constraining the integrin α and β transmembrane domains with intersubunit disulfide bonds blocks integrin activation from inside the cell. However, this constraint does not prevent activation by divalent cations and antibodies that activate by binding to the extracellular domain (7Luo B.H. Springer T.A. Takagi J. PLoS Biol. 2004; 2: 776-786Crossref Scopus (159) Google Scholar). Taken together, these data suggest that default low affinity state of integrins is maintained by interactions between integrin α and β subunits and that physiological activation occurs when cytoplasmic domain ligands, such as talin, disrupt these interactions. Support for the idea that an Arg995-Asp723 salt bridge is an important constraint for the low affinity state comes from a NMR spectroscopy study (8Vinogradova O. Velyvis A. Velyviene A. Hu B. Haas T. Plow E. Qin J. Cell. 2002; 110: 587-597Abstract Full Text Full Text PDF PubMed Scopus (451) Google Scholar). Specifically, in isolated αIIb and β3 cytoplasmic domain peptides, the salt bridge was identified as part of a helical interface between the membrane-proximal regions of α and β subunits. Furthermore, this interaction was disrupted by talin, supporting the notion that disruption of this salt bridge is involved in integrin activation (8Vinogradova O. Velyvis A. Velyviene A. Hu B. Haas T. Plow E. Qin J. Cell. 2002; 110: 587-597Abstract Full Text Full Text PDF PubMed Scopus (451) Google Scholar). However, other groups have failed to observe intersubunit interactions in the membrane-proximal region, suggesting that it is of relatively low affinity (9Ulmer T.S. Yaspan B. Ginsberg M.H. Campbell I.D. Biochemistry. 2001; 40: 7498-7508Crossref PubMed Scopus (107) Google Scholar, 10Li R. Babu C.R. Lear J.D. Wand A.J. Bennett J.S. DeGrado W.F. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12462-12467Crossref PubMed Scopus (159) Google Scholar). Therefore, additional intracellular regions of the receptor could contribute α/β interactions to “clasp” it into the low affinity state. Indeed, in vitro model systems identified heterodimeric interactions between integrin α and β TM domains (11Schneider D. Engelman D.M. J. Biol. Chem. 2004; 279: 9840-9846Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar) and such interactions have also been suggested by molecular modeling (12Gottschalk K.E. Adams P.D. Brunger A.T. Kessler H. Protein Sci. 2002; 11: 1800-1812Crossref PubMed Scopus (82) Google Scholar, 13Adair B.D. Yeager M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14059-14064Crossref PubMed Scopus (145) Google Scholar) and disulfide crosslinking approaches (7Luo B.H. Springer T.A. Takagi J. PLoS Biol. 2004; 2: 776-786Crossref Scopus (159) Google Scholar). Mutation of residues contributing to α/β interactions that stabilize the low affinity state should lead to integrin activation. Thus, we subjected the entire intracellular domain (cytoplasmic plus TM domain in Fig. 1) of the β3 integrin subunit to unbiased random mutagenesis and selected it for activated mutants. Through this analysis, we have confirmed the importance of the membrane-proximal domain in maintenance of the low affinity state. In contrast, no activating mutations were identified in the more distal cytoplasmic tail suggesting that this region is dispensable for the maintenance of the inactive state. This approach also identified 13 novel TM domain mutations that lead to integrin activation. Among these were several informative point mutations that suggested the existence of a TM helix-packing interface that maintains the low affinity state. Computational modeling indicates that these mutations disrupted intersubunit interactions either directly or indirectly by altering helical length/tilt angle. The interactions predicted in the model were used to create additional activating mutations in both the α and β TM domains. Therefore, we propose that α and β TM regions interact to form a clasp that constrains integrin activation. Cell Culture, Cell Lines, and Reagents—Chinese hamster ovary (CHO) cells were obtained from American Type Culture Collection (ATCC) and cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum, 1% non-essential amino acids (Sigma), 50 units of penicillin/ml, and 50 μg of streptomycin sulfate/ml in a 37 °C tissue culture incubator. αIIbC5 cells from an αIIb integrin subunit-expressing CHO cell line were created by transfecting CHO cells with CDM8 vectors encoding the αIIb integrin subunit along with CD-neomycin plasmid. Stable colonies were selected with neomycin for 2 weeks. The pooled stable colonies were subsequently infected with an adenovirus encoding β3 integrin subunit and sorted for single cells expressing αIIbβ3 integrin. Each individual stable clone was then examined for the absence of β3 subunit to make sure that the adenovirus had not integrated into the genome. The clones that do not express β3 subunit were then transiently transfected with a plasmid that encodes β3 and tested for expression of αIIbβ3. The clones that revealed normal expression of αIIbβ3 after transient expression of β3 subunit were then used for further studies. The anti-αIIbβ3 antibodies D57, PAC1, and anti-LIBS6 as well as Ro43-5054, an αIIbβ3-specifc peptide-mimetic competitive inhibitor, have been described previously (14O'Toole T.E. Mandelman D. Forsyth J. Shattil S.J. Plow E.F. Ginsberg M.H. Science. 1991; 254: 845-847Crossref PubMed Scopus (275) Google Scholar, 15Shattil S.J. Hoxie J.A. Cunningham M. Brass L.F. J. Biol. Chem. 1985; 260: 11107-11114Abstract Full Text PDF PubMed Google Scholar, 16Frelinger III, A.L. Du X. Plow E.F. Ginsberg M.H. J. Biol. Chem. 1991; 266: 17106-17111Abstract Full Text PDF PubMed Google Scholar, 17Kouns W.C. Kirchhofer D. Hadvary P. Edenhofer A. Weller T. Pfenninger G. Baumgartner H.R. Jennings L.K. Steiner B. Blood. 1992; 80: 2539-2547Crossref PubMed Google Scholar). The D57 antibody was biotinylated with biotin-N-hydroxy-succinimide (B-D57, Sigma) according to the manufacturer's instructions. Construction of Random Mutagenic cDNA Libraries of β3 Integrin Subunit—To facilitate construction of random mutagenic cDNA libraries of transmembrane and cytoplasmic domains of β3 integrin subunit, a SphI restriction enzyme site was created via a silent mutation at nucleotide 1899 (C→ A) of β3 cDNA sequence using site-directed mutagenesis. The SphI site-containing full-length β3 cDNA was then subcloned into a hygromycin-resistant derivative of CDM8 expression vector (Invitrogen), CDHYG, to create a wide-type β3-expressing vector, CDHYG3A.SphI. Transiently transfection of this construct in a αIIb-expressing CHO cell line, αIIbC5, or co-transfection with wild-type αIIb cDNA in CHO cells resulted in a protein product with properties identical to those of wild-type αIIbβ3. Specifically, the mutant species was immunoprecipitated by D57 (data not shown), an antibody against the extracellular domains of αIIbβ3, and was found to be in the low affinity state because it failed to bind PAC1, a monoclonal antibody specific for the high affinity state of αIIbβ3. Furthermore, the SphI construct gained PAC1 binding ability with treatment of an αIIbβ3-activating antibody anti-LIBS6 but failed to bind PAC1 in the presence of Ro43-5054, an αIIbβ3-specifc peptide-mimetic competitive inhibitor (data not shown). Thus, this silent mutation does not interfere with the normal expression or function of β3 integrin subunit. The spiked megaprimer method was chosen to generate random mutagenic cDNA libraries of β3 cytoplasmic and transmembrane domains (18Hermes J.D. Parekh S.M. Blacklow S.C. Koster H. Knowles J.R. Gene (Amst.). 1989; 84: 143-151Crossref PubMed Scopus (104) Google Scholar). Specifically, the β3 transmembrane and cytoplasmic domains were divided into four “windows” of 66 nucleotides, each with a nine-nucleotide overlap between windows (Fig. 1). Spiked oligonucleotide primers corresponding to each window (Fig. 1) were synthesized with a contamination level of 1.5% incorrect phosphoramidites, i.e. 0.5% of each of the three other bases. PCR was performed using the spiked primer (Fig. 2A, S.M.P.) for each window and a 3′-reverse primer that contains an XbaI restriction enzyme site (Fig. 2A, R.P.). For a second round of PCR reactions (Fig. 2A, PCR-II), the PCR-I product (megaprimer) was used as a reverse primer. PCR reaction was performed with the megaprimer and a forward primer that contains the SphI site (Fig. 2A, F.P.). Products from PCR-II were then subcloned into SphI-XbaI sites of CDHYG3A.SphI. The efficiency of random mutagenesis was assessed by cDNA sequencing of 10 randomly picked transformants from each window. cDNA sequencing indicated that ∼70% inserts in each window contained 1, 2, or 3 point mutations, which is in the reasonable range of efficiency for random mutagenesis. Theoretically, for a window of 66 nucleotides, 150,000 transformants should cover 99% of the possible one-base changes and ∼75% of the possible two-base changes. Therefore, a random mutagenic cDNA library containing ∼200,000 transformants for each window of transmembrane and cytoplasmic domains of β3 subunit was constructed by large-scale preparation of transformants and used for subsequent transfection into αIIbC5 cells and identification of activating mutations in the β3 subunit. Site-directed Mutagenesis—Site-directed mutations in both the αIIb and β3 subunits were generated using the QuikChange mutagenesis kit (Stratagene) and pCDM8 vectors containing the integrin subunits. Mutants were confirmed by DNA sequencing. Flow Cytometry—Random mutagenic cDNA library corresponding to each window of β3 transmembrane and cytoplasmic domains was transfected into αIIC5 cells by electroporation. Stable colonies were selected for 2 weeks in the presence of both hygromycin (750 μg/ml) and neomycin (750 μg/ml). Approximately 5 × 106 cells from pooled stable colonies from each window were then individually sorted on a FACStar Plus (BD Biosciences) using two-color flow cytometry. The biotinylated monoclonal antibody, B-D57, was used to detect the expression of αIIbβ3, whereas PAC1, an activation-specific monoclonal IgM antibody was used to assess the activation state of the αIIbβ3 integrin. For single cell sorting, the pooled stable colonies from each window (∼5 × 106 cells/each window) were resuspended by treatment with trypsin and double-stained as described previously (19O'Toole T.E. Katagiri Y. Faull R.J. Peter K. Tamura R.N. Quaranta V. Loftus J.C. Shattil S.J. Ginsberg M.H. J. Cell Biol. 1994; 124: 1047-1059Crossref PubMed Scopus (581) Google Scholar, 20Hughes P.E. O'Toole T.E. Ylanne J. Shattil S.J. Ginsberg M.H. J. Biol. Chem. 1995; 270: 12411-12417Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). Rare cells that exhibited both bright phycoerythrin staining (D57) and fluorescein isothiocyanate staining (PAC1) were individually sorted into 96-well plates. FACS analysis of isolated clonal cell lines was performed on a FACScan using both B-D57 and PAC1 antibodies as described previously (19O'Toole T.E. Katagiri Y. Faull R.J. Peter K. Tamura R.N. Quaranta V. Loftus J.C. Shattil S.J. Ginsberg M.H. J. Cell Biol. 1994; 124: 1047-1059Crossref PubMed Scopus (581) Google Scholar, 20Hughes P.E. O'Toole T.E. Ylanne J. Shattil S.J. Ginsberg M.H. J. Biol. Chem. 1995; 270: 12411-12417Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). PAC1 staining in the presence of Ro43-5054 (2 μm) was used to estimate nonspecific binding. In some cases, treatment with anti-LIBS6 was used to estimate maximal PAC1 binding because anti-LIBS6 directly induces αIIbβ3 binding to PAC1 regardless of the status of cellular activation mechanism (15Shattil S.J. Hoxie J.A. Cunningham M. Brass L.F. J. Biol. Chem. 1985; 260: 11107-11114Abstract Full Text PDF PubMed Google Scholar). For retransfection analysis, αIIbC5 cells were transfected with plasmid CDHYG3A.SphI, encoding wild-type or mutant β3, using Lipofectamine (Invitrogen) following the manufacturer's instructions. 48 h after transfection, the transfected cells were stained with B-D57 and PAC1 in the presence and absence of Ro43-5054 and FACS analysis was performed as described above. Cytometric analysis of the site-directed mutations was done by cotransfection of the pCDM8 plasmids containing the αIIb and β3 subunits with Plus reagent and Lipofectamine. 48 h after transfection, the transfected cells were stained with B-D57 and PAC1 in the presence and absence of Ro43-5054 and anti-LIBS6 and FACS analysis was performed as described above. Activation index was calculated by using the formula (F - F0)/(Fmax - F0), where F = PAC1 binding, F0 = PAC1 binding in the presence of Ro43-5054, and Fmax = PAC1 binding in the presence of anti-LIBS6. Reverse Transcriptase-PCR, Subcloning, and Sequencing of β3 Integrin Subunit—Total cellular RNA from each individual cell line was isolated using TRIzol (Invitrogen). cDNA was synthesized using oligo(dT) primer and the cDNA Cycle kit (Invitrogen), and PCR was performed following the manufacturer's instructions. PCR products were digested with restriction enzymes SphI and XbaI to create a fragment of ∼550 bp. This SphI-XbaI fragment was then subcloned into the SphI-XbaI sites of CDHYG3A. SphI vector and sequenced using primers derived from CDHYG3A.SphI plasmid but outside of the SphI-XbaI region. To eliminate possible PCR errors, at least four clones were sequenced for each mutant cell line. Computer Simulations—The modeling procedures for TM helix oligomerization were described elsewhere (21Kim S. Chamberlain A.K. Bowie J.U. J. Mol. Biol. 2003; 329: 831-840Crossref PubMed Scopus (67) Google Scholar). The TMD sequences of integrin αIIb and β3 subunits were built into uniform α-helices having the backbone torsion angles of φ = -65° and Ψ = -40°. Their TMD sequences of αIIb and β3 were aligned based on the glycosylmappingdata (22Armulik A. Nilsson I. von Heijne G. Johansson S. J. Biol. Chem. 1999; 274: 37030-37034Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar,23Stefansson A. Armulik A. Nilsson I. von Heijne G. Johansson S. J. Biol. Chem. 2004; 279: 21200-21205Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Side chain rotamers were chosen using the backbone-dependent rotamer library program SCWRL (24Bower M.J. Cohen F.E. Dunbrack Jr., R.L. J. Mol. Biol. 1997; 267: 1268-1282Crossref PubMed Scopus (488) Google Scholar). Four hundred potential helix packings were first generated using a Monte Carlo search procedure as described previously (21Kim S. Chamberlain A.K. Bowie J.U. J. Mol. Biol. 2003; 329: 831-840Crossref PubMed Scopus (67) Google Scholar). The αIIb and β3 TMD dimeric structures then were filtered to remove the structures incompatible with the bilayer constraints. We then clustered the remaining structures by Cα root mean square distances using NMR CLUSTER (25Kelley L.A. Gardner S.P. Sutcliffe M.J. Protein Eng. 1996; 9: 1063-1065Crossref PubMed Scopus (419) Google Scholar), which resulted in two equally populated clusters: one with a crossing point near the N terminus and the other with a crossing point close to C terminus. Both models were evaluated for consistency with the experimental results (see below). Random Mutagenesis Identifies Novel Integrin Activating Mutations in the TM and Membrane-proximal Cytoplasmic Regions of β3—To generate the β3 random mutants, we used CDHYG3A.SphI as a template and spiked oligonucleotide primers corresponding to four overlapping windows that cover the entire β3 intracellular domain (Fig. 1). Using the protocol outlined (see “Experimental Procedure” and Fig. 2), a cDNA library containing ∼200,000 transformants for each window was constructed by large-scale preparation of transformants and used for subsequent transfection into an αIIb-expressing cell line (αIIbC5 cells) and identification of activating mutations in the β3 subunit. We developed a library of stable cell lines and selected activated αIIbβ3 integrin mutants by their binding to PAC1, an antibody specific for activated αIIbβ3 (Fig. 3A, R2) (15Shattil S.J. Hoxie J.A. Cunningham M. Brass L.F. J. Biol. Chem. 1985; 260: 11107-11114Abstract Full Text PDF PubMed Google Scholar). Using this approach, we isolated 91 and 192 cell lines bearing activated αIIbβ3 from windows I and II, respectively (Table I). In contrast, only five cell lines were isolated from either window III or window IV (Table I). Thus, mutations in the membrane-distal segments of the β3 cytoplasmic domain encoded by regions III and IV were less likely to activate αIIbβ3 integrin.Table IClonal cell lines with activated αIIβ3WindowCell lines isolatedActivation reconfirmedUnique mutations identifiedI288917II46819222III6550IV3000 Open table in a new tab The cell lines expressing activated integrin αIIbβ3 could have arisen as a consequence of mutations within the target window from adventitious mutations elsewhere in the integrin or from mutations in genes that indirectly control integrin activation. To identify mutations in the target window that activated αIIbβ3, we sequenced cDNA clones obtained from β3 reverse transcriptase-PCR amplicons spanning this region. To confirm that sequenced mutations were responsible for integrin activation, these amplicons were used to replace this region in wild-type β3. The resulting plasmids were transfected into αIIbC5 cells and tested for PAC1 binding in the presence and absence of Ro43-5054. When the transiently expressed mutant integrin was able to bind PAC1 and this PAC1 binding was inhibited by Ro43-5054, we concluded that the mutation was responsible for the activation of αIIbβ3. A total of 25 unique mutations in the transmembrane and membrane-proximal region of cytoplasmic domain of β3 subunit thus were identified, and there were multiple examples of the same mutations present in different clonal cell lines (Fig. 4). 13 of them were found N-terminal of Lys716 in the presumptive transmembrane domain, and 12 were in the membrane-proximal region of cytoplasmic domain (Fig. 5). No mutation was identified that only affected the region of the cytoplasmic domain C-terminal of Asp723 (Fig. 5). The absence of activating mutations C-terminal of Asp723 indicates that the C terminus of β3 is not involved in the maintenance of the low affinity state of the integrin. The presence of such mutations in the membraneproximal and transmembrane domains suggests that these sites are important in regulating integrin activation.Fig. 5Frequency of activating mutations in the β3 transmembrane and cytoplasmic domains. Frequency of activating mutations is plotted against each amino acid residue in the β3 transmembrane and cytoplasmic domains. Amino acid sequence of the β3 transmembrane and cytoplasmic domains is illustrated.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Activating Mutations Are Predicted to Alter the TM Helix Length—We had previously established the importance of the membrane-proximal domains of the α and β subunits in regulating integrin activation (20Hughes P.E. O'Toole T.E. Ylanne J. Shattil S.J. Ginsberg M.H. J. Biol. Chem. 1995; 270: 12411-12417Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). Consequently, we focused our attention on the numerous mutations in the transmembrane domain. Many of these mutations would be predicted to shorten the β-subunit TM helix (i.e. fewer residues embedded in the membrane) by deletion of one or more residues or by introduction of a charged residue (Table II). Indeed, the majority of the mutations (9 of 13) would be expected to disrupt or shorten the TM helix. Previous glycosylation-mapping studies (22Armulik A. Nilsson I. von Heijne G. Johansson S. J. Biol. Chem. 1999; 274: 37030-37034Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 23Stefansson A. Armulik A. Nilsson I. von Heijne G. Johansson S. J. Biol. Chem. 2004; 279: 21200-21205Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar) indicated that activating mutations in the membrane-proximal domain can shorten the TM helix. The present results extend those findings by showing that such mutations throughout the TM helix activate the integrin.Table IIActivating β3 transmembrane mutationsMutations that directly shorten the TM helix (loop-out and truncation mutations)Δ(Val695Val696)Leu697 → Pro and Δ(Leu698-Ser699-Val700)Δ(Ser699-Val700)Leu709→His & Δ(Leu697-Leu698)Trp715 → StopMutations that are predicted to shorten the TM helix (apolar to charged mutations)Leu697 → ArgLeu712 → ArgLeu713 → HisTrp715 → ArgMutations that are not predicted to shorten the TM helixGly708 → SerAla711 → Thr & Thr720→AlaIle714 → ThrIle714 → Thr & Δ(Ile721) Open table in a new tab Mutagenesis of Predicted TM Packing Residues Activates αIIbβ3—A subset (4 of 13) of the membrane-embedded activating mutations had no obvious effect on TM helical length. This finding suggests that the TM segments help stabilize the inactive state through sequence-specific interactions. To investigate this possibility, we used a Monte Carlo simulation method (21Kim S. Chamberlain A.K. Bowie J.U. J. Mol. Biol. 2003; 329: 831-840Crossref PubMed Scopus (67) Google Scholar) to produce a first approximation of the intersubunit packing of integrin TM domains. We caution that this method assumes idealized rigid α helices and disregards potential changes in membrane insertion. However, several reports indicate that this protocol does yield models that conform well to known structural and functional data (21Kim S. Chamberlain A.K. Bowie J.U. J. Mol. Biol. 2003; 329: 831-840Crossref PubMed Scopus (67) Google Scholar, 27Melnyk R.A. Kim S. Curran A.R. Engelman D.M. Bowie J.U. Deber C.M. J. Biol. Chem. 2004; 279: 16591-16597Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 28Kim S. Chamberlain A.K. Bowie J.U. Biophys. J. 2004; 87: 792-799Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 29Kim S. Chamberlain A.K. Bowie J.U. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 5988-5991Crossref PubMed Scopus (70) Google Scholar). Here, the resulting output predicted two alternative structures, one with the TM helices packing near the C termini (Fig. 6, Structure A) and another with the helices packing close to the N termini (Structure B). The random mutagenesis data focused our attention on Structure A, because all of the uncharged activating point mutations clustered in the C-terminal region of the β3 TM domain (G708S, A711T,T720A, and I714T) (Fig. 5). Indeed, each of these mutations affected a residue predicted to be a helical contact in Structure A (Fig. 6B). Three additional sitedirected mutants were employed to test the hypothesis that the interhelical interface predicted in Structure A functioned as a clasp to maintain the integrin in the low affinity state (vida infra). The β3(G708S) mutation was strongly activating. Previous studies (30Li R. Mitra N. Gratkowski H. Vilaire G. Litvinov R. Nagasami C. Weisel J.W. Lear J.D. DeGrado W.F. Bennett J.S. Science. 2003; 300: 795-798Crossref PubMed Scopus (258) Google Scholar) have shown that the introduction of an Asn at this position activated αIIbβ3 and suggested that it did so by forming hydrogen bonds that favor β3 homo-oligomerization. The Ser substitution could also, in principle, lead to enhanced β3-β3 interactions through such a mechanism. However, the weakly polar nature of the Ser side chain coupled with the observation that insertion of the bulky aliphatic Ile residue at this position (β3(G708I) mutation) was strongly activating suggests that a Gly residue is strictly required at this position for efficient α/β TM packing (Fig. 7, A–C). The packing motif in Structure A allowed us to identify an additional Van der Waals packing residue that helps stabilize the inactive state, because the apolar to apolar I704A mutation resulted in an activated integrin (Fig. 7, B and C). Also, the Structure A model predicted that αIIb TM residues would pack against the identified β3 residues. Accordingly, we substituted a bulky Ile residue for αIIb Thr981, the residue predicted to pack against β3 Gly708 (Fig. 6B). The observation that this mutant activated αIIbβ3 supports the packing of Thr981 against Gly708, an interaction that would be stabilized by both Van der Waals packing and a potential Cα/hydroxyl hydrogen bond. Overall, both random mutagenesis and site-directed mutagenesis (Fig. 7C) support the hypothesis that the specific packing of the C-terminal portions of αIIb and β3 transmembrane helices against each other maintains the low affinity state of integrin αIIbβ3. Activation by mutagenically dissociating the integrin TM helices is consistent with reports that suggest that activation is associated with separation of the cytoplasmic domains (2Campbell I.D. Ginsberg M.H. Trends Biochem. Sci. 2004; 29: 429-435Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 6Kim M. Carman C.V. Springer T.A. Science. 2003; 301: 1720-1725Crossref PubMed Scopus (646) Google Scholar). However, the unbiased random mutagenesis approach identified a preponderance of activating TM domain mutations predicted to shorten the β3 TM domain, indicating that TM helix shortening can also lead to integrin activation. In agreement with this mechanism, previous glycosylation mapping studies suggested that the membrane-proximal domains of the α and β subunits can reside with the membrane bilayers and that certain activating mutations in this region (22Armulik A. Nilsson I. von Heijne G. Johansson S. J. Biol. Chem. 1999; 274: 37030-37034Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 23Stefansson A. Armulik A. Nilsson I. von Heijne G. Johansson S. J. Biol. Chem. 2004; 279: 21200-21205Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar) result in a shortened TM domain. How might shortening the TM domain lead to disruption of intracellular α/β interactions and consequent integrin activation? To avoid hydrophobic mismatch with the fixed width of the membrane bilayer, a shortened TM helix would change its membrane tilt angle and associated register with neighboring helices (31de Planque M.R. Killian J.A. Mol. Membr. Biol. 2003; 20: 271-284Crossref PubMed Scopus (262) Google Scholar). Because helix-helix packing is dependent on specific crossing angles and specific in-register side chain arrays, changes to TM helical length would break the proposed clasp. Previous observations showing that αIIb sequences with a shortened TM segment (7Luo B.H. Springer T.A. Takagi J. 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Ginsberg M.H. Campbell I.D. Biochemistry. 2003; 42: 8307-8312Crossref PubMed Scopus (73) Google Scholar). This interaction could contribute to the physiological activation of integrins in two separate but related ways. First, one consequence of talin binding to this region would be to displace the membrane-proximal domain from the bilayer, thereby shortening the TM domain. As noted above, this process would probably lead to separation of the intracellular domains. Talin binding could also directly disrupt the cooperative membrane proximal/TM clasp by breaking the Arg995-Asp723 salt bridge (8Vinogradova O. Velyvis A. Velyviene A. Hu B. Haas T. Plow E. Qin J. Cell. 2002; 110: 587-597Abstract Full Text Full Text PDF PubMed Scopus (451) Google Scholar) and associated intersubunit interactions. Our findings also have implications for integrin-mediated biochemical signals that control cell shape, cell migration, proliferation, and survival. The capacity of integrins to deliver such signals depends on their occupancy with resultant conformational change in the integrin (34Diaz-Gonzalez F. Forsyth J. Steiner B. Ginsberg M.H. Mol. Biol. Cell. 1996; 7: 1939-1951Crossref PubMed Scopus (155) Google Scholar) in combination with receptor clustering (35Guan J.-L. Trevithick J.E. Hynes R.O. Cell Regul. 1991; 2: 951-964Crossref PubMed Scopus (474) Google Scholar, 36Miyamoto S. Akiyama S.K. Yamada K.M. Science. 1995; 267: 883-885Crossref PubMed Scopus (791) Google Scholar). These conformational changes are associated with a dramatic change in the quaternary structure of the integrin, resulting in a switch from a “bent” conformation observed in the crystal structure (37Xiong J.P. Stehle T. Diefenbach B. Zhang R. Dunker R. Scott D.L. Joachimiak A. Goodman S.L. Arnaout M.A. Science. 2001; : 1064535Google Scholar) to an extended one (38Takagi J. Strokovich K. Springer T.A. Walz T. 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A. 2001; 98: 12462-12467Crossref PubMed Scopus (159) Google Scholar) shows that isolated integrin α and β TM peptides homo-oligomerize, a process that could contribute to integrin clustering. This finding suggests a sequential model in which α/β transmembrane separation might then be followed by homo-oligomerization to favor receptor clustering (10Li R. Babu C.R. Lear J.D. Wand A.J. Bennett J.S. DeGrado W.F. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12462-12467Crossref PubMed Scopus (159) Google Scholar, 26Hynes R.O. Science. 2003; 300: 755-756Crossref PubMed Scopus (31) Google Scholar). Strikingly, the mutational studies of Li et al. (30Li R. Mitra N. Gratkowski H. Vilaire G. Litvinov R. Nagasami C. Weisel J.W. Lear J.D. DeGrado W.F. Bennett J.S. Science. 2003; 300: 795-798Crossref PubMed Scopus (258) Google Scholar) identify β3 Gly708 as an important packing residue for β3 homo-oligomerization and suggest that homo-oligomerization may occur after TM separation. Our findings indicating that Gly708 also participates in an α/β interaction that regulates activation lends additional credence to this hypothesis and suggests that the TM helix packing interface proposed here is a nexus for bidirectional transmembrane signaling through integrins. Note Added in Proof—Recently, two other groups (41Li W. Metcalf D. Gorelik R. Li R. Mitra N. Nanda V. Law P.B. Lear J.D. DeGrado W.F. Bennett J.S. Proc. Natl. Acad, Sci. U. S. A. 2005; (in press)Google Scholar, 42Luo B.-H. Carman C.V. Takagi J. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 2005; (in press)Google Scholar) have reported studies supporting the hypothesis that heterodimeric transmembrane domain-packing interactions stabilize the integrin-inactive state.