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

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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 in a CHO cell or with αIIb cDNA in CHO cells in a protein with to of αIIbβ3. Specifically, the was by D57 not an antibody the extracellular domains of αIIbβ3, and was to in the low affinity state because it failed to PAC1, a antibody specific for the high affinity state of αIIbβ3. Furthermore, the SphI binding with of an antibody anti-LIBS6 failed to in the of Ro43-5054, an αIIbβ3-specifc peptide-mimetic competitive not Thus, this silent mutation does not with the normal expression or function of β3 integrin The was to random mutagenic cDNA libraries of β3 cytoplasmic and transmembrane domains J.D. H. PubMed Scopus Google Scholar). Specifically, the β3 transmembrane and cytoplasmic domains were into of with a between to 1) were with a of of of the other was using the for and a that an restriction enzyme site a of the was used as a was with the and a that the SphI site from were then subcloned into of CDHYG3A.SphI. The of random mutagenesis was by cDNA of from cDNA that in or point is in the of for random mutagenesis. for a of should of the changes and of the Therefore, a random mutagenic cDNA for of transmembrane and cytoplasmic domains of β3 subunit was by of and used for transfection into αIIbC5 cells and of activating mutations in the β3 mutations in both the αIIb and β3 subunits were using the mutagenesis and vectors the integrin subunits. were confirmed by mutagenic cDNA to of β3 transmembrane and cytoplasmic domains was transfected into cells by Stable colonies were selected for 2 in the of both and neomycin cells from pooled stable colonies from were then sorted a using The biotinylated was used to the expression of αIIbβ3, PAC1, an antibody was used to the activation state of the αIIbβ3 integrin. single cell the pooled stable colonies from were by with and as described previously T.E. Shattil S.J. Ginsberg M.H. J. Cell Biol. PubMed Scopus Google Scholar, P.E. T.E. J. Shattil S.J. Ginsberg M.H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). cells that both and were sorted into of isolated cell was a using both and antibodies as described previously T.E. Shattil S.J. Ginsberg M.H. J. Cell Biol. PubMed Scopus Google Scholar, P.E. T.E. J. Shattil S.J. Ginsberg M.H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). in the of was used to In with anti-LIBS6 was used to binding because anti-LIBS6 directly αIIbβ3 binding to of the of activation S.J. Hoxie J.A. Cunningham M. Brass L.F. J. Biol. Chem. 1985; 260: 11107-11114Abstract Full Text PDF PubMed Google Scholar). analysis, αIIbC5 cells were transfected with plasmid encoding or using the manufacturer's instructions. after the transfected cells were with and in the and absence of and was as described of the site-directed mutations was by of the the αIIb and β3 subunits with and after the transfected cells were with and in the and absence of and anti-LIBS6 and was as described was by using the binding in the of Ro43-5054, and binding in the of and of β3 Integrin from individual cell line was isolated using cDNA was using and the cDNA (Invitrogen), and was the manufacturer's instructions. were with restriction SphI and to create a of This was then subcloned into the of SphI vector and using from plasmid of the at clones were for cell modeling for TM were described S. J. Biol. 2003; PubMed Scopus Google Scholar). The of integrin αIIb and β3 subunits were into the of and of αIIb and β3 were the A. von G. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google A. A. von G. S. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). were using the J. Biol. PubMed Scopus Google Scholar). were using a as described previously S. J. Biol. 2003; PubMed Scopus Google Scholar). The αIIb and β3 then were to the with the then the by using NMR Protein 1996; PubMed Scopus Google in with a point the and the other with a point to were for with the Random Integrin in the TM and of the β3 random we used as a and to that the entire β3 intracellular domain the and Fig. a cDNA for was by of and used for transfection into an cell line and of activating mutations in the β3 a of stable cell and selected activated αIIbβ3 integrin by their binding to PAC1, an antibody specific for activated αIIbβ3 S.J. Hoxie J.A. Cunningham M. Brass L.F. J. Biol. Chem. 1985; 260: 11107-11114Abstract Full Text PDF PubMed Google Scholar). this we isolated and cell activated αIIbβ3 from I and In contrast, cell were isolated from either or Thus, mutations in the of the β3 cytoplasmic domain by regions and were likely to activate αIIbβ3 cell with activated mutations in a The cell expressing activated integrin αIIbβ3 could have as a of mutations the from mutations in the integrin or from mutations in that indirectly integrin activation. identify mutations in the that activated αIIbβ3, we cDNA clones obtained from β3 this that mutations were for integrin activation, these were used to this region in The resulting were transfected into αIIbC5 cells and tested for binding in the and absence of the transiently integrin was to and this binding was by Ro43-5054, we that the mutation was for the activation of αIIbβ3. A of 25 unique mutations in the transmembrane and membrane-proximal region of cytoplasmic domain of β3 subunit were and were of the mutations in cell 13 of were of in the transmembrane and were in the membrane-proximal region of cytoplasmic domain mutation was identified that the region of the cytoplasmic domain C-terminal of The absence of activating mutations C-terminal of indicates that the of β3 is not involved in the maintenance of the low affinity state of the integrin. The of such mutations in the and transmembrane domains that these are important in integrin of activating mutations in the β3 transmembrane and cytoplasmic domains. of activating mutations is amino in the β3 transmembrane and cytoplasmic domains. sequence of the β3 transmembrane and cytoplasmic domains is to the TM had previously the importance of the membrane-proximal domains of the α and β subunits in integrin activation P.E. T.E. J. Shattil S.J. Ginsberg M.H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). we the numerous mutations in the transmembrane domain. of these mutations predicted to the TM residues in the by of or more residues or by of a Indeed, the of the mutations of to disrupt or the TM studies A. von G. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A. A. von G. S. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar) that activating mutations in the membrane-proximal domain can the TM The by that such mutations the TM activate the β3 transmembrane that directly the TM and and that are predicted to the TM to that are not predicted to the TM in a of TM of of the activating mutations had no TM helical This that the TM stabilize the inactive state through interactions. this we used a S. J. Biol. 2003; PubMed Scopus Google Scholar) to a of the intersubunit packing of integrin TM domains. that this α and changes in However, several that this does that well to and data S. J. Biol. 2003; PubMed Scopus Google Scholar, S. Engelman D.M. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar, S. J. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, S. Proc. Natl. Acad. Sci. U. S. A. 2004; PubMed Scopus Google Scholar). Here, the resulting predicted with the TM packing the A) and with the packing to the The random mutagenesis data because of the activating point mutations in the C-terminal region of the β3 TM domain and Indeed, of these mutations a predicted to a helical in A additional were to the that the interface predicted in A as a clasp to maintain the integrin in the low affinity state The mutation was studies R. H. G. R. C. Lear J.D. DeGrado W.F. Bennett J.S. Science. 2003; PubMed Scopus Google Scholar) have that the of an at this activated αIIbβ3 and suggested that it by forming bonds that β3 The could in lead to interactions through such a However, the of the with the that of the at this was activating that a is at this for α/β TM packing The packing in A to identify an additional packing that helps stabilize the inactive because the to mutation in an activated integrin and the A model predicted that αIIb TM residues the identified β3 we a for αIIb the predicted to β3 The that this activated αIIbβ3 the packing of an interaction that by both packing and a both random mutagenesis and site-directed mutagenesis the that the specific packing of the C-terminal of αIIb and β3 transmembrane other maintains the low affinity state of integrin αIIbβ3. by the integrin TM is consistent with that suggest that activation is 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, 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 of activating TM domain mutations predicted to the β3 TM that TM can also lead to integrin activation. In with this studies suggested that the membrane-proximal domains of the α and β subunits can with the and that activating mutations in this region A. von G. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A. A. von G. S. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar) result in a TM domain. the TM domain lead to disruption of intracellular α/β interactions and integrin with the of the a TM change and with J.A. Biol. 2003; PubMed Scopus Google Scholar). helix-helix packing is specific and specific changes to TM helical the that αIIb with a TM (7Luo B.H. Springer T.A. Takagi J. PLoS Biol. 2004; 2: 776-786Crossref Scopus (159) Google Scholar) the to a disulfide of the αIIb and β3 transmembrane this In the inactive state intersubunit interactions at membrane-proximal could with the TM packing to maintain the α/β talin been to to the membrane-proximal region (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, S. A. J.D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, T.S. Ginsberg M.H. Campbell I.D. Biochemistry. 2003; PubMed Scopus Google an that to important for αIIbβ3 activation T.S. Ginsberg M.H. Campbell I.D. Biochemistry. 2003; PubMed Scopus Google Scholar). This interaction could contribute to the physiological activation of integrins in of talin binding to this region to the membrane-proximal domain from the the TM domain. this lead to separation of the intracellular domains. binding could also directly disrupt the clasp by 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 intersubunit interactions. also have for that cell cell and The capacity of integrins to such their with conformational change in the integrin F. Forsyth J. Steiner B. Ginsberg M.H. Biol. Cell. 1996; PubMed Scopus Google Scholar) in combination with receptor R.O. Cell 1991; 2: PubMed Scopus Google Scholar, S. Science. PubMed Scopus Google Scholar). These conformational changes are with a change in the of the integrin, resulting in a from a conformation in the T. B. R. R. A. Science. 2001; Scholar) to an J. Springer T.A. T. J. 2003; PubMed Scopus Google Scholar) that a C-terminal separation C. M. Biochemistry. PubMed Scopus Google Scholar) that disrupt the TM helical packing This disruption could lead to the changes in the intracellular interactions of integrins by adhesion and F. Forsyth J. Steiner B. Ginsberg M.H. Biol. Cell. 1996; PubMed Scopus Google Scholar, J. Cell Biol. 1992; PubMed Scopus Google Scholar). Furthermore, the of 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) that isolated integrin α and β TM a that could contribute to integrin This a model in α/β transmembrane separation then by to receptor 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, R.O. Science. 2003; PubMed Scopus Google Scholar). the mutational studies of R. H. G. R. C. Lear J.D. DeGrado W.F. Bennett J.S. Science. 2003; PubMed Scopus Google Scholar) identify β3 as an important packing for β3 and suggest that after TM that also in an α/β interaction that regulates activation additional to this and that the TM packing interface here is a for transmembrane through in other groups D. R. R. Lear J.D. DeGrado W.F. Bennett J.S. Proc. Natl. Sci. U. S. A. Scholar, Carman C.V. Takagi J. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. Scholar) have studies supporting the that heterodimeric transmembrane interactions stabilize the state.