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• W2000263240 abstract "We examined over 50 mutations in the Drosophila βPS integrin subunit that alter integrin function in situ for their ability to bind a soluble monovalent ligand, TWOW-1. Surprisingly, very few of the mutations, which were selected for conditional lethality in the fly, reduce the ligand binding ability of the integrin. The most prevalent class of mutations activates the integrin heterodimer. These findings emphasize the importance of integrin affinity regulation and point out how molecular interactions throughout the integrin molecule are important in keeping the integrin in a low affinity state. Mutations strongly support the controversial deadbolt hypothesis, where the CD loop in the β tail domain acts to restrain the I domain in the inactive, bent conformation. Site-directed mutations in the cytoplasmic domains of βPS and αPS2C reveal different effects on ligand binding from those observed for αIIbβ3 integrins and identify for the first time a cytoplasmic cysteine residue, conserved in three human integrins, as being important in affinity regulation. In the fly, we find that genetic interactions of the βPS mutations with reduction in talin function are consistent with the integrin affinity differences measured in cells. Additionally, these genetic interactions report on increased and decreased integrin functions that do not result in affinity changes in the PS2C integrin measured in cultured cells. We examined over 50 mutations in the Drosophila βPS integrin subunit that alter integrin function in situ for their ability to bind a soluble monovalent ligand, TWOW-1. Surprisingly, very few of the mutations, which were selected for conditional lethality in the fly, reduce the ligand binding ability of the integrin. The most prevalent class of mutations activates the integrin heterodimer. These findings emphasize the importance of integrin affinity regulation and point out how molecular interactions throughout the integrin molecule are important in keeping the integrin in a low affinity state. Mutations strongly support the controversial deadbolt hypothesis, where the CD loop in the β tail domain acts to restrain the I domain in the inactive, bent conformation. Site-directed mutations in the cytoplasmic domains of βPS and αPS2C reveal different effects on ligand binding from those observed for αIIbβ3 integrins and identify for the first time a cytoplasmic cysteine residue, conserved in three human integrins, as being important in affinity regulation. In the fly, we find that genetic interactions of the βPS mutations with reduction in talin function are consistent with the integrin affinity differences measured in cells. Additionally, these genetic interactions report on increased and decreased integrin functions that do not result in affinity changes in the PS2C integrin measured in cultured cells. Integrin cell surface receptors are important for morphogenetic events in normal development and are also critical for adult and many disease-related processes as well (1Hynes R.O. Cell. 2002; 110: 673-687Abstract Full Text Full Text PDF PubMed Scopus (6955) Google Scholar). The integrin αβ heterodimer is a dynamic receptor, whose activity is regulated by interactions with extracellular, intracellular, and other transmembrane proteins. This regulation can change the affinity of individual integrin molecules or alter the clustering of integrins such that their avidity is altered. Experiments utilizing conformation-specific antibodies, high resolution x-ray, NMR, small angle neutron scattering, molecular dynamics, steered molecular dynamics, and electron microscopy are elucidating how the α and β subunits are organized and change in response to activating conditions and ligand binding. One goal of the current work on integrin biology is to understand how the structural information gained on isolated integrins relates to integrin function in a cellular context. Therefore, models for integrin activation are being tested by site-directed mutagenesis (reviewed in Refs. 2Shattil S.J. Kim C. Ginsberg M.H. Nat. Rev. Mol. Cell Biol. 2010; 11: 288-300Crossref PubMed Scopus (756) Google Scholar, 3Luo B.H. Carman C.V. Springer T.A. Annu. Rev. Immunol. 2007; 25: 619-647Crossref PubMed Scopus (1262) Google Scholar, 4Arnaout M.A. Goodman S.L. Xiong J.P. Curr. Opin. Cell Biol. 2007; 19: 495-507Crossref PubMed Scopus (323) Google Scholar). We have used a complementary approach of first identifying amino acids in the Drosophila integrin βPS subunit that alter its function in the whole organism and then examining the effects of these mutations at the cellular and molecular level. Previous work demonstrated that myospheroid (mys, encoding βPS integrin) hypomorphic mutations displayed stronger phenotypes at higher temperatures (5Bunch T.A. Salatino R. Engelsgjerd M.C. Mukai L. West R.F. Brower D.L. Genetics. 1992; 132: 519-528Crossref PubMed Google Scholar). Therefore, we screened for mutations in mys that produce viable flies at low temperatures but lethality at high temperatures. Because null alleles of mys are completely lethal at all temperatures, we know that these mutations do not completely inactivate the function of the integrin but rather compromise its function. Due to unique properties of one of the mutants, mysb58, we examined it in detail and found that it is an activating mutation that results in an integrin with higher affinity for a soluble extracellular matrix ligand (6Jannuzi A.L. Bunch T.A. West R.F. Brower D.L. Mol. Biol. Cell. 2004; 15: 3829-3840Crossref PubMed Scopus (32) Google Scholar, 7Bunch T.A. Helsten T.L. Kendall T.L. Shirahatti N. Mahadevan D. Shattil S.J. Brower D.L. J. Biol. Chem. 2006; 281: 5050-5057Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). At that time, we suspected that mysb58 was unique among the mutants in our screen. We have now tested the ligand binding ability of all 50 of the mutants generated in our previous screen for function-altering alleles as well as a number of lethal mutants that produce mutant integrin subunits that are expressed on the cell surface and site-directed mutations suggested by our initial studies or results obtained in work on vertebrate integrins. The ligand binding assay that we used measures the ability of the cell surface integrin to bind to a monovalent ligand and therefore probes only the affinity of integrins for one ligand. The assay reports on the effect a mutation has on the regulation of integrin affinity, presumably by altering an amino acid contacting the ligand or one regulating integrin conformation. This is in contrast to the testing of numerous mutations in the human integrin β3 subunit generated by site-directed mutagenesis or identified in Glanzmann thrombasthenia patients. Typically, these mutations were tested for their ability to bind soluble multivalent ligands like PAC-1 IgM, PAC-1 Fab fragments in combination with polyclonal secondary antibodies, or fibrinogen. The activity of these mutant integrins as measured by these multivalent ligands did not distinguish between effects on affinity or clustering. Our results, specifically addressing affinity changes, do generally support the conclusions drawn from the use of multivalent ligands. To confirm that affinity changes measured in our cell culture assay have similar effects in the whole organism, we tested their effects in flies that expressed reduced levels of talin. Because talin increases integrin activity in flies (8Brown N.H. Gregory S.L. Rickoll W.L. Fessler L.I. Prout M. White R.A. Fristrom J.W. Dev. Cell. 2002; 3: 569-579Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 9Tanentzapf G. Brown N.H. Nat. Cell Biol. 2006; 8: 601-606Crossref PubMed Scopus (107) Google Scholar), reducing talin levels would be predicted to increase phenotypes (lethality in our tests) of mutations that we find reduce integrin affinity and rescue phenotypes of mutations that increase integrin affinity. In general this is what we find. Using this genetic interaction test, we also tested those mutants that did not show an alteration in affinity of PS2C integrins for soluble ligand. Most of these mutants showed increased integrin function in the organism. The surprising finding from our work is that most of our integrin mutations do not reduce the ability to bind ligand but actually enhance it. They are activating mutations. Expression, in flies, of activating deletion mutants of the αPS2 subunit was previously found to be unable to rescue the lethality of a null mutation in that subunit, pointing out in a different context the importance of proper regulation of integrin activity (10Martin-Bermudo M.D. Dunin-Borkowski O.M. Brown N.H. J. Cell Biol. 1998; 141: 1073-1081Crossref PubMed Scopus (51) Google Scholar). We discuss our mutant results in the context of current structures available for the human αIIbβ3 integrin. We have additionally used site-directed mutagenesis to test the deadbolt hypothesis of integrin affinity regulation (11Arnaout M.A. Mahalingam B. Xiong J.P. Annu. Rev. Cell Dev. Biol. 2005; 21: 381-410Crossref PubMed Scopus (424) Google Scholar). This hypothesis predicts that the CD loop in the βTD (tail domain) contacts the β I domain in the bent conformation and restrains it in a low affinity state. Work on αIIbβ3 and αVβ3 integrins has yielded contradictory results (12Zhu J. Boylan B. Luo B.H. Newman P.J. Springer T.A. J. Biol. Chem. 2007; 282: 11914-11920Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 13Xiong J.P. Mahalingham B. Alonso J.L. Borrelli L.A. Rui X. Anand S. Hyman B.T. Rysiok T. Müller-Pompalla D. Goodman S.L. Arnaout M.A. J. Cell Biol. 2009; 186: 589-600Crossref PubMed Scopus (146) Google Scholar). Our results on the Drosophila PS2C integrin strongly support the deadbolt hypothesis. Finally, site-directed mutagenesis suggests that a conserved cysteine in the cytoplasmic domain of αPS2C that is palmitoylated in human α3 and α6 is involved in affinity regulation. Drosophila S2/M3 cells were cultured in Shields and Sang M3 medium supplemented with 12% heat-inactivated fetal calf serum as described previously (14Bunch T.A. Brower D.L. Development. 1992; 116: 239-247Crossref PubMed Google Scholar). Cells were co-transfected with plasmids expressing an αPS2C subunit and a βPS subunit, both under the regulation of the heat shock protein 70 promoter, and the bacterial dihydrofolate reductase selectable marker as described previously (15Jannuzi A.L. Bunch T.A. Brabant M.C. Miller S.W. Mukai L. Zavortink M. Brower D.L. Mol. Biol. Cell. 2002; 13: 1352-1365Crossref PubMed Scopus (29) Google Scholar). Transformed cells were selected in 2 × 10−7 m methotrexate. Prior to performing TWOW-1 assays, cells were treated with dsRNA targeting the 3′-untranslated sequence of mys for 3 days to remove expression of endogenous βPS integrin as described earlier (7Bunch T.A. Helsten T.L. Kendall T.L. Shirahatti N. Mahadevan D. Shattil S.J. Brower D.L. J. Biol. Chem. 2006; 281: 5050-5057Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). This 3′-untranslated sequence is not present in the βPS transgenes. This assay has been described in detail (7Bunch T.A. Helsten T.L. Kendall T.L. Shirahatti N. Mahadevan D. Shattil S.J. Brower D.L. J. Biol. Chem. 2006; 281: 5050-5057Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). TWOW-1 is an Fab molecule whose heavy chain complementarity-determining region 3 was replaced with a sequence encoding 53 amino acids found in Drosophila Tiggrin. These 53 amino acids contain the Tiggrin RGD sequence and 24 amino acids prior to and 26 amino acids following the RGD (7Bunch T.A. Helsten T.L. Kendall T.L. Shirahatti N. Mahadevan D. Shattil S.J. Brower D.L. J. Biol. Chem. 2006; 281: 5050-5057Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). To remove preexisting integrins and matrix and to induce high expression from the heat shock-regulated integrin transgenes, cells were incubated at 36 °C with collagenase/dispase. Cells were washed and allowed to recover for 4 h at 23 °C. Cells were incubated with TWOW-1 for 10 min at room temperature. This was done in the presence of Ca2+ and Mg2+ (standard binding); Ca2+, Mg2+, and Mn2+ (Mn2+-activated binding); or EDTA (non-binding). Buffer containing formaldehyde was added to fix the bound TWOW-1 to the cells. Bound TWOW-1 was detected with AlexaFluor488-labeled secondary antibody. Levels of PS2C integrin on the surface of cells were determined by staining with a biotin-labeled anti-αPS2 antibody (CF2C7) followed by detection with phycoerythrin-streptavidin. Fluorescence levels for both TWOW-1 and PS2 levels were analyzed by flow cytometry. Specific TWOW-1 binding is the mean fluorescence intensity (MFI) 2The abbreviations used are: MFImean fluorescence intensityβTDβ tail domainI-EGFintegrin-EGFPSIplexin-semaphorin-integrinMIDASmetal ion-dependent adhesion siteADMIDASadjacent to MIDASSyMBSsynergistic metal binding site. of TWOW-1 binding minus the MFI of the same cells in the presence of EDTA. Typically, specific binding was greater than 90% of total binding. For our analyses, this specific binding is expressed relative to surface integrin expression (TWOW-1 MFI/anti-αPS2 MFI). Finally, because assays were conducted over a period of more than 2 years, expression and TWOW-1 MFI/anti-αPS2 MFI were normalized to a control cell line (untagged wild type line 1 or WT1) expressing wild type PS2C integrins that was included in every assay. Z scores are the number of S.D. values from the average binding of wild type PS2C integrins taken from 10 independent lines expressing Myc-tagged βPS and αPS2C. mean fluorescence intensity β tail domain integrin-EGF plexin-semaphorin-integrin metal ion-dependent adhesion site adjacent to MIDAS synergistic metal binding site. We discuss the Drosophila βPS functional mutants as they relate to crystal structures of β3 integrins. Therefore, the mutants generated in fly genetic screens are described as follows: mysallele(single letter designation of the amino acid change in βPS; corresponding amino acid in β3; z score for TWOW-1 binding). Site-directed mutations not obtained from genetic screens are designated as follows: βPS amino acid change (corresponding amino acid in β3). The βPS numbering begins with the start methionine as in our previous descriptions of these mutants (6Jannuzi A.L. Bunch T.A. West R.F. Brower D.L. Mol. Biol. Cell. 2004; 15: 3829-3840Crossref PubMed Scopus (32) Google Scholar). The β3 numbering system begins with the first amino acid (G) in the mature, processed subunit and corresponds to the common β3 numbering used in the structure, Protein Data Bank entry 3FCS (16Zhu J. Luo B.H. Xiao T. Zhang C. Nishida N. Springer T.A. Mol. Cell. 2008; 32: 849-861Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar), that we have used as a reference to assess likely consequences of our mutations. Unless otherwise noted, amino acid interactions are discussed using this structure as a reference, and the αIIb and β3 amino acids are presented in boldface type. To test mutant mys alleles for genetic interactions with talin, w mys/w mys (or w mys/FM7c);rhea+/rhea+ females were crossed to w mys+; rhea2 (w+)/TM3 Ser males. The mys alleles, located on the X chromosome, were generated in previous screens (6Jannuzi A.L. Bunch T.A. West R.F. Brower D.L. Mol. Biol. Cell. 2004; 15: 3829-3840Crossref PubMed Scopus (32) Google Scholar, 17Wright, T. R. (1968) in Proceedings of the 12th Annual Congress on Genetics, Tokyo, Japan, Vol. 1, p. 41.Google Scholar). rhea2, located on chromosome 3, is a strong allele of the gene encoding talin (8Brown N.H. Gregory S.L. Rickoll W.L. Fessler L.I. Prout M. White R.A. Fristrom J.W. Dev. Cell. 2002; 3: 569-579Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 18Prout M. Damania Z. Soong J. Fristrom D. Fristrom J.W. Genetics. 1997; 146: 275-285Crossref PubMed Google Scholar). Crosses were done at a temperature (18, 22, 25, 28, or 31 °C) that resulted in reduced viability in mutant mys male progeny. This temperature differed for different mys mutant alleles and is indicated in supplemental Table S5. Due to sterility at 31 °C, tests at this temperature involved egg laying at 28 °C for 24–48 h, and then eggs and larvae were transferred to 31 °C. Progeny bearing one mutant copy of rhea were identified by the presence of a w+ transgene on the rhea2 chromosome (and the absence of Ser in the case of mysb3, mysb4, and mysts2, which contained a w+ gene on the chromosome containing the mys mutation). All male progeny were mutant for the mys allele, and females were heterozygous for the mys mutation (Fm7c males and females were discarded). Flies were raised on cornmeal, sugar, and yeast medium. Marker mutations are described in FlyBase (available on the World Wide Web) and described by Lindsley and Zimm (19Lindsley D.L. Zimm G.G. Annu. Rev. Genomics Hum. Genet. 1992; 4: 1133Google Scholar). We have generated stable cell lines expressing more than 50 different function-altering mutations in the βPS integrin subunit. All of the mutant βPS integrin subunits were labeled with a Myc tag inserted into the large serine-rich loop (present only in the Drosophila βPS subunit) that is located between the X and A strands of the Hybrid domain. To control for experiment-to-experiment variability over the 2 years that these experiments were performed, each group of cell lines being assayed for integrin expression and TWOW-1 binding always included line WT1 (wild type 1; expressing untagged βPS and αPS2C), and values were normalized to its levels. The level of TWOW-1 used in all binding assays was 40 μg/ml. For wild type PS2C integrins, this is a subsaturating concentration resulting in ∼50% of the binding levels seen for integrins activated with Mn2+ (7Bunch T.A. Helsten T.L. Kendall T.L. Shirahatti N. Mahadevan D. Shattil S.J. Brower D.L. J. Biol. Chem. 2006; 281: 5050-5057Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar) (Fig. 1B). At this concentration, we are able to detect both increases and decreases in affinity. Variability in integrin expression levels and their ability to bind TWOW-1 is due to either the specific βPS mutant subunit being expressed or cell line variability. To determine cell line variability, we examined the integrin expression levels and TWOW-1 binding of 17 transformed cell lines expressing wild type PS2C integrins. Seven lines expressed untagged βPS, and 10 expressed Myc-tagged βPS. All lines also expressed the αPS2C integrin subunit. Fig. 1A demonstrates that independent cell lines transfected with wild type PS2C integrin transgenes express variable levels of cell surface integrin. The average levels are not significantly different between those lines expressing untagged or Myc-tagged βPS (p = 0.94). In contrast to the variable integrin expression levels, the amount of TWOW-1 bound per integrin shows much less variability (Fig. 1B). The range of TWOW-1 binding varies between 0.81 and 1.25 (mean = 1.08 ± 0.15) for the lines expressing untagged integrins and between 1.01 and 1.33 (mean = 1.15 ± 0.11) for tagged integrins, relative to that observed for WT1. Mn2+ activates the affinity of PS2C integrins for TWOW-1 (7Bunch T.A. Helsten T.L. Kendall T.L. Shirahatti N. Mahadevan D. Shattil S.J. Brower D.L. J. Biol. Chem. 2006; 281: 5050-5057Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar) (Fig. 1B). For untagged and tagged integrins, levels of TWOW-1 binding in the absence of Mn2+ were 44 ± 10 and 42 ± 14%, respectively, of that measured in the presence of Mn2+. These data demonstrate that the presence of the 15-amino acid Myc-tag in the serine-rich loop does not alter the ligand binding ability of the PS2C integrin or its affinity modulation by Mn2+. Second, the data demonstrate that line-to-line variability is to be expected for expression levels but not for TWOW-1 binding normalized to PS2C integrin expression levels. As with different cell lines expressing wild type PS2C integrins, expression levels of cell lines expressing mutant integrins vary widely (Fig. 2 and supplemental Table S1). Only three mutants, mysXN101(C629S;C549), mysb47(A293T;A225), and mysG12(D356N;D288), expressed levels of integrins 2 S.D. values below the average of the wild type lines. mysXN101 was already known to be poorly expressed in flies, whereas mysb47 and mysG12 did not show reduction in expression upon limited examination in either wing discs or muscle attachment sites (5Bunch T.A. Salatino R. Engelsgjerd M.C. Mukai L. West R.F. Brower D.L. Genetics. 1992; 132: 519-528Crossref PubMed Google Scholar, 6Jannuzi A.L. Bunch T.A. West R.F. Brower D.L. Mol. Biol. Cell. 2004; 15: 3829-3840Crossref PubMed Scopus (32) Google Scholar, 15Jannuzi A.L. Bunch T.A. Brabant M.C. Miller S.W. Mukai L. Zavortink M. Brower D.L. Mol. Biol. Cell. 2002; 13: 1352-1365Crossref PubMed Scopus (29) Google Scholar). To ensure that low expression of mysb47(A293T;A225) and mysG12(D356N;D288) was not due to line-to-line variability, a second stably transformed line was obtained and tested. Again, both showed dramatically reduced PS2 integrin staining (supplemental Table S1). The extremely low levels of expression of the lethal mutation mysG12(D356N;D288) and the viable mysb47(A293T;A225) are consistent with observations of mutations of β1 amino acids Asp-295 (Asp-288) and Asp-233 (adjacent to β1 Ala-234; β3 Ala-225). Both of these were poorly expressed on the surface of the cell and appeared to be defective in α/β association (20Puzon-McLaughlin W. Takada Y. J. Biol. Chem. 1996; 271: 20438-20443Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). In the crystal structure (supplemental Fig. S1), Asp-288 hydrogen-bonds with Leu-258, which is itself hydrogen-bonded to αIIb Tyr-237. Leu-258 is in the 310 helix that contains other residues interacting with the α subunit, including Asp-259, Arg-261, and Leu-262. Mutation of Asp-288 may therefore affect not only Leu-258 but other nearby α/β contacts as well. Finally, Asp-288 is immediately followed by helix α5, which contains Ser-291 and Glu-297, which are within 3 Å of αIIb. The effect of mysb47(A293T;A225) is less direct. Ala-225 is closest to (within 3.5 Å of) Leu-117, which contacts (within 3 Å) Val-247 and Thr-249 in β4, which precedes the β4–310 helix loop containing the close α/β contact Lys-253. Mutation of alanine to threonine probably alters the positioning of β4-310 helix loop and α/β contacts. Mutation of Cys-549 in β3 integrin (corresponding to mysXN101) showed reduced expression (21Mor-Cohen R. Rosenberg N. Peretz H. Landau M. Coller B.S. Awidi A. Seligsohn U. Thromb. Haemost. 2007; 98: 1257-1265Crossref PubMed Scopus (37) Google Scholar). Cys-549 stabilizes a loop in I-EGF3 that contacts the Hybrid domain in the closed conformation of β3 integrin (supplemental Fig. S1C), and its mutation activates the integrin. This activation probably alters the position of the Hybrid domain and breaks the hydrogen bond between α subunit (Asp-319) and the β Hybrid domain (Leu-362). Additional separation of the α and β leg regions due to Hybrid swing-out may also be an additional consequence (22Zhu J. Luo B.H. Barth P. Schonbrun J. Baker D. Springer T.A. Mol. Cell. 2009; 34: 234-249Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Failure of these associations are the likely causes of poor α/β association. Ligand binding assays with the lethal mysG12 mutant were inconsistent between separate lines, due to the extremely low levels of expression, and we therefore do not report this. mysb47(A293T;A225) and mysXN101(C629S;C549), although also expressed at very low levels, gave very consistent ligand binding data, and these are presented but should also be viewed with more caution than the data from the other mutants. Finally, we have done duplicate transformations of some mutant integrins that expressed at or more than 2 S.D. values lower than wild type levels, and most again expressed at levels between 1 and 2 S.D. values below wild type, which suggests that they too may affect expression levels to some extent (supplemental Table S1). The affinity for the soluble monovalent ligand, TWOW-1, of PS2C integrins with different mutant βPS subunits was examined and compared with that of wild type integrins expressed on 10 independent cell lines. Results of this assay are arranged in order of binding ability in Fig. 3A (and supplemental Table S2). Of the 51 mutants generated in genetic screens, five displayed severely reduced binding levels that were more than 8 S.D. values below the mean of the wild type integrins (z score <−8). Seventeen lines showed no significant changes in TWOW-1 binding. Twenty-two of the mutant integrins displayed increased binding with z scores greater than 5, and an additional seven bound with z scores of 2.4–5.0. In addition to comparing ligand binding levels of mutant integrins normalized to the amount of integrin on the surface, we also compared their activation indexes (supplemental Table S2, column I). The activation index is the binding of TWOW-1 in the absence of Mn2+ divided by the binding in the presence of activating Mn2+. Although this approach removes the dependence on an antibody to measure surface integrin levels, it has its own limitations, including the observation that effects of mutants and Mn2+ can be additive (7Bunch T.A. Helsten T.L. Kendall T.L. Shirahatti N. Mahadevan D. Shattil S.J. Brower D.L. J. Biol. Chem. 2006; 281: 5050-5057Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar), and mutants may influence the ability of Mn2+ to activate the integrins. For the mutants we analyzed, the activation index data give almost identical results as binding normalized to integrin levels. Of the 22 strongly activating mutants (z scores above 5), only two did not give similarly significant z scores when normalized by Mn2+-activated values (supplemental Table S2). The remaining two strongly activating mutants and six of the seven moderately activating (z scores 2.4–4.9) did not show significant activation index increases due to the mutations increasing binding both in the absence and in the presence of Mn2+ to similar extents. Examination of the overall distribution of the mutations within the βPS subunit and their effects on ligand binding is interesting (Fig. 3B and summarized in Table 1). All mutations that reduce ligand binding are found in the I domain and specifically in the metal ion-dependent adhesion site (MIDAS) and synergistic metal binding site (SyMBS). This confirms the previously recognized importance of these domains in ligand binding. Mutations that increase ligand binding are distributed in all domains, illustrating the importance of all domains in restraining the integrin in the low affinity (bent, head closed) conformation (Fig. 3C). Activating mutations are postulated to disrupt this conformation and push the equilibrium toward the unbent and/or head open conformations that result in increased affinity for ligand (Fig. 3C; reviewed in Refs. 2Shattil S.J. Kim C. Ginsberg M.H. Nat. Rev. Mol. Cell Biol. 2010; 11: 288-300Crossref PubMed Scopus (756) Google Scholar, 3Luo B.H. Carman C.V. Springer T.A. Annu. Rev. Immunol. 2007; 25: 619-647Crossref PubMed Scopus (1262) Google Scholar, 4Arnaout M.A. Goodman S.L. Xiong J.P. Curr. Opin. Cell Biol. 2007; 19: 495-507Crossref PubMed Scopus (323) Google Scholar).TABLE 1Summary of mys mutant TWOW-1 binding, expression, and genetic interactions with rhea Open table in a new tab Detailed data describing TWOW-1 binding arranged by mutant position in the βPS integrin, including references to similar mutations found in other integrins, is shown in supplemental Table S3. In the following sections, we group the mutations by the domains they are likely to affect and discuss the probable consequences for integrin structures. Supplemental figures are provided for each set of mutants that focus on their positions and interactions with important structural components (supplemental Figs. S1–S9). The three mutations in the PSI domain that disrupt disulfide bridges, mysb3(C58S;C26;z = 6.7), mysb68(C58Y;C26;z = 7.9), mysb41(C40Y;C5;z = 8.7) activate the integrins. mysb3 and mysb68 both mutate the same cysteine residue and have similar effects on activating the integrin. Cys-26 is located between Trp-25 and Ser-27 that along with Glu-29 and Arg-37 are at the interface with the I-EGF1 domain residues Gly-458 and Cys-457 (supplemental Fig. S2). One of these I-EGF1 residues is mutated in mysb63(G531D;G458;z = 4.6) and is also activating (see “I-EGF Domain Mutants”). It is likely that defects in all three of these mutants perturb the PSI-I-EGF1 interaction. Activating mysb41(C40Y;C5;z = 8.7) probably disrupts PSI-Hybrid interactions. In β2 integrin, the Cys-3 residue (Cys-5 in β3) is adjacent to Thr-4 (Thr-6 in β3) that has been implicated in restraining the activation state of the integrin (23Zang Q. Springer T.A. J. Biol. Chem. 2001; 276: 6922-6929Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Also in β2, Cys-3 (Cys-5) forms a disulfide bridge with Cys-21 (Cys-23), which is part of the PSI-Hybrid interaction domain (24Shi M. Sundramurthy K. Liu B. Tan S.M. Law S.K. Lescar J. J. Biol. Chem. 2005; 280: 30586-30593Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). In β3, Cys-23 forms a hydrogen bond with Hybrid residue Arg-93 (supplemental Fig. S2). Breaking of this disulfide bridge in mysb41 probably alters the position of Thr-6 and Cys-23 and thereby weakens the PSI-Hybrid interaction. Integrin crystal structures suggest that the PSI domain is important in stabilizing the Hybrid-I-EGF1 connections. This could be important for restraining the" @default.
• W2000263240 created "2016-06-24" @default.
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• W2000263240 date "2011-09-01" @default.
• W2000263240 modified "2023-09-30" @default.
• W2000263240 title "Identification of Integrin β Subunit Mutations That Alter Affinity for Extracellular Matrix Ligand" @default.
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