Modulation of beta1A integrin functions by tyrosine residues in the beta1 cytoplasmic domain - PubMed (original) (raw)
Modulation of beta1A integrin functions by tyrosine residues in the beta1 cytoplasmic domain
T Sakai et al. J Cell Biol. 1998.
Abstract
beta1A integrin subunits with point mutations of the cytoplasmic domain were expressed in fibroblasts derived from beta1-null stem cells. beta1A in which one or both of the tyrosines of the two NPXY motifs (Y783, Y795) were changed to phenylalanines formed active alpha5 beta1 and alpha6 beta1 integrins that mediated cell adhesion and supported assembly of fibronectin. Mutation of the proline in either motif (P781, P793) to an alanine or of a threonine in the inter-motif sequence (T788) to a proline resulted in poorly expressed, inactive beta1A. Y783,795F cells developed numerous fine focal contacts and exhibited motility on a surface. When compared with cells expressing wild-type beta1A or beta1A with the D759A activating mutation of a conserved membrane-proximal aspartate, Y783, 795F cells had impaired ability to transverse filters in chemotaxis assays. Analysis of cells expressing beta1A with single Tyr to Phe substitutions indicated that both Y783 and Y795 are important for directed migration. Actin-containing microfilaments of Y783,795F cells were shorter and more peripheral than microfilaments of cells expressing wild-type beta1A. These results indicate that change of the phenol side chains in the NPXY motifs to phenyl groups (which cannot be phosphorylated) has major effects on the organization of focal contacts and cytoskeleton and on directed cell motility.
Figures
Figure 1
Deduced amino acid sequence (in single letter code) of the intracellular domain of murine β1A and comparable domains of β1D, β2, β3, β5, β6, and β7. The sequences are aligned with lysine (K) at position 752 of mature β1A. The residues mutated in β1A and homologous residues in other β subunits are shown in bold. The mutations are depicted above the β1A sequence.
Figure 2
Expression of β1A and associated α5 and α6 subunits in β1-deficient GD25, wild-type, and mutant β1A cells by flow cytometry. Control, no primary antibody; β1(9EG7), and β1(MB1.2), antibodies to β1; α5(MFR5), antibody to α5; α6(GoH3), antibody to α6; GD25, β1-deficient cells; β1GD25, GD25 cells expressing wild-type β1A; other cells are designated by mutation(s).
Figure 3
Attachment of wild-type and mutant β1A cells on vitronectin, fibronectin, and laminin-1. Bars represent the mean of attachment activity quantified by spectrophotometric analysis at OD = 595 nm after staining of adherent cells with bromphenol blue. Error bars represent ±SD of quadruplicate experiments. Absorbance resulting from nonspecific cell adhesion as measured on BSA-coated wells was ∼0.05, and has been subtracted. GD25, β1-deficient cells; β1GD25, GD25 cells expressing wild-type β1A; other cells are designated by mutation(s).
Figure 4
Immunofluorescence of fibronectin matrix after 3 d of culture in serum-containing medium. GD25, β1-deficient cells; β1GD25, GD25 cells expressing wild-type β1A; other cells are designated by mutation(s). Bar, 60 μm.
Figure 5
Assembly of exogenous fibronectin in short-term assay. Assembly of FITC–fibronectin during a 1-h period began 4 h after seeding of cells on surfaces coated with vitronectin or laminin-1. The cells used for analysis are indicated in each figure. Bar, 60 μm.
Figure 6
Binding of the 70-kD NH2-terminal fragment of fibronectin to cells seeded on surfaces coated with vitronectin, fibronectin, or laminin-1. Symbols represent the mean of specific binding. Bars represent mean ± SD of triplicate experiments with duplicate determinations in each experiment (n = 6). GD25, β1-deficient cells; β1GD25, GD25 cells expressing wild-type β1A; other cells are designated by mutation(s). *, β1-deficient GD25 cells could not be studied on laminin-1 coating because too few cells adhered (see Fig. 3).
Figure 7
Double fluorescence of β1 integrin (red) and FITC-labeled fibronectin (green). Cells were incubated for 4 h on a fibronectin-, laminin-, or vitronectin-coated substratum and additional 1 h with FITC–fibronectin. After fixation with paraformaldehyde, staining for β1 integrin was performed using β1 antibody MB1.2 and LRSC-labeled anti–rat IgG. Samples were viewed with an emission filter that allowed visualization of both fluorochromes and photographed focusing on the interface of cells and substrate. GD25, β1-deficient cells; β1GD25, GD25 cells expressing wild-type β1A; other cells are designated by mutation(s). Bar, 25 μm.
Figure 8
Cell motility as assessed by clearing of beads layered on vitronectin-, fibronectin-, or laminin-1–coated surfaces. Cells were added in the presence of PDGF, 10 ng/ ml. Wells were fixed 16 hours after seeding and photographed by phase microscopy. Cells, which were covered with beads, appear as dark specks in cleared-out areas. GD25, β1-deficient cells; β1GD25, GD25 cells expressing wild-type β1A; other cells are designated by mutation(s). Bar, 240 μm.
Figure 9
Cell migration through vitronectin-, fibronectin-, or laminin-1–coated filters in response to EGF or PDGF. EGF (100 ng/ml) or PDGF (10 ng/ml), was in the lower chamber. Each bar represents the mean of cell number per 0.16-mm2 field. Error bars indicate ± SD of quadruplicate determinations. GD25, β1-deficient cells; β1GD25, GD25 cells expressing wild-type β1A; other cells are designated by mutation(s).
Figure 10
Visualization of focal contacts and associated structures in β1-deficient GD25, wild-type or mutant β1A cells. (A) Immunofluorescent detection of paxillin in focal contacts of cells expressing wild-type β1A, or D759A, or Y783,795F mutations. Cells were cultured for 4 h on a fibronectin-coated substratum. (B) Double fluorescence of vinculin (green) and rhodamine–phallondin (red). Cells were incubated for 4 h on a fibronectin-, laminin-1–, or vitronectin-coated substratum. GD25, β1-deficient cells; β1GD25, GD25 cells expressing wild-type β1A; other cells are designated by mutation(s). Bar, 25 μm.
Figure 10
Visualization of focal contacts and associated structures in β1-deficient GD25, wild-type or mutant β1A cells. (A) Immunofluorescent detection of paxillin in focal contacts of cells expressing wild-type β1A, or D759A, or Y783,795F mutations. Cells were cultured for 4 h on a fibronectin-coated substratum. (B) Double fluorescence of vinculin (green) and rhodamine–phallondin (red). Cells were incubated for 4 h on a fibronectin-, laminin-1–, or vitronectin-coated substratum. GD25, β1-deficient cells; β1GD25, GD25 cells expressing wild-type β1A; other cells are designated by mutation(s). Bar, 25 μm.
Figure 11
Models whereby directed cell migration may be accomplished by phosphorylation/dephosphorylation of tyrosines in the cytoplasmic domain of β1A. The integrin heterodimer is depicted as being in an equilibrium between inactive and active forms. Upon binding of extracellular ligand (triangle), the integrin complexes with cytoplasmic components of focal contacts (various boxes). Phosphorylation (P) of β1A by a tyrosine kinase (Y Kinase) causes the integrin to dissociate from both extracellular ligand and focal contact components. Dephosphorylation by a tyrosine phosphatase (Y P'tase) regenerates the integrin in an inactive form. Alternatively or additionally, binding of adapter molecule(s) to phosphorylated NPXY motifs may initiate a pathway leading to cytoskeletal rearrangement, polarization, and directional integrin cycling.
Similar articles
- The cytoplasmic tyrosines of integrin subunit beta1 are involved in focal adhesion kinase activation.
Wennerberg K, Armulik A, Sakai T, Karlsson M, Fässler R, Schaefer EM, Mosher DF, Johansson S. Wennerberg K, et al. Mol Cell Biol. 2000 Aug;20(15):5758-65. doi: 10.1128/MCB.20.15.5758-5765.2000. Mol Cell Biol. 2000. PMID: 10891511 Free PMC article. - Restoration of beta1A integrins is required for lysophosphatidic acid-induced migration of beta1-null mouse fibroblastic cells.
Sakai T, Peyruchaud O, Fässler R, Mosher DF. Sakai T, et al. J Biol Chem. 1998 Jul 31;273(31):19378-82. doi: 10.1074/jbc.273.31.19378. J Biol Chem. 1998. PMID: 9677354 - Role of the cytoplasmic tyrosines of beta 1A integrins in transformation by v-src.
Sakai T, Jove R, Fässler R, Mosher DF. Sakai T, et al. Proc Natl Acad Sci U S A. 2001 Mar 27;98(7):3808-13. doi: 10.1073/pnas.240456398. Epub 2001 Mar 20. Proc Natl Acad Sci U S A. 2001. PMID: 11259684 Free PMC article. - The integrin beta1 subunit transmembrane domain regulates phosphatidylinositol 3-kinase-dependent tyrosine phosphorylation of Crk-associated substrate.
Armulik A, Velling T, Johansson S. Armulik A, et al. Mol Biol Cell. 2004 Jun;15(6):2558-67. doi: 10.1091/mbc.e03-09-0700. Epub 2004 Mar 19. Mol Biol Cell. 2004. PMID: 15034138 Free PMC article. - beta1-integrin cytoplasmic subdomains involved in dominant negative function.
Retta SF, Balzac F, Ferraris P, Belkin AM, Fässler R, Humphries MJ, De Leo G, Silengo L, Tarone G. Retta SF, et al. Mol Biol Cell. 1998 Apr;9(4):715-31. doi: 10.1091/mbc.9.4.715. Mol Biol Cell. 1998. PMID: 9529373 Free PMC article.
Cited by
- The dipeptide prolyl-hydroxyproline promotes cellular homeostasis and lamellipodia-driven motility via active β1-integrin in adult tendon cells.
Ide K, Takahashi S, Sakai K, Taga Y, Ueno T, Dickens D, Jenkins R, Falciani F, Sasaki T, Ooi K, Kawashiri S, Mizuno K, Hattori S, Sakai T. Ide K, et al. J Biol Chem. 2021 Jul;297(1):100819. doi: 10.1016/j.jbc.2021.100819. Epub 2021 May 23. J Biol Chem. 2021. PMID: 34029590 Free PMC article. - Transcription factor scleraxis vitally contributes to progenitor lineage direction in wound healing of adult tendon in mice.
Sakabe T, Sakai K, Maeda T, Sunaga A, Furuta N, Schweitzer R, Sasaki T, Sakai T. Sakabe T, et al. J Biol Chem. 2018 Apr 20;293(16):5766-5780. doi: 10.1074/jbc.RA118.001987. Epub 2018 Mar 5. J Biol Chem. 2018. PMID: 29507095 Free PMC article. - Protein 4.1G Regulates Cell Adhesion, Spreading, and Migration of Mouse Embryonic Fibroblasts through the β1 Integrin Pathway.
Chen L, Wang T, Wang Y, Zhang J, Qi Y, Weng H, Kang Q, Guo X, Baines AJ, Mohandas N, An X. Chen L, et al. J Biol Chem. 2016 Jan 29;291(5):2170-80. doi: 10.1074/jbc.M115.658591. Epub 2015 Dec 7. J Biol Chem. 2016. PMID: 26644476 Free PMC article. - The interaction of Gα13 with integrin β1 mediates cell migration by dynamic regulation of RhoA.
Shen B, Estevez B, Xu Z, Kreutz B, Karginov A, Bai Y, Qian F, Norifumi U, Mosher D, Du X. Shen B, et al. Mol Biol Cell. 2015 Oct 15;26(20):3658-70. doi: 10.1091/mbc.E15-05-0274. Epub 2015 Aug 26. Mol Biol Cell. 2015. PMID: 26310447 Free PMC article. - Contributions of the integrin β1 tail to cell adhesive forces.
Elloumi-Hannachi I, García JR, Shekeran A, García AJ. Elloumi-Hannachi I, et al. Exp Cell Res. 2015 Mar 15;332(2):212-22. doi: 10.1016/j.yexcr.2014.11.008. Epub 2014 Nov 25. Exp Cell Res. 2015. PMID: 25460334 Free PMC article.
References
- Albelda SM, Buck CA. Integrins and other cell adhesion molecules. FASEB (Fed Am Soc Exp Biol) J. 1990;4:2868–2880. - PubMed
- Amano M, Chihara K, Kimura K, Fukata Y, Nakamura N, Matsuura Y, Kaibuchi K. Formation of actin stress fibers and focal adhesions enhanced by Rho kinase. Science. 1997;275:1308–1311. - PubMed
- Bansal A, Gierasch LM. The NPXY internalization signal of the LDL receptor adopts a reverse turn conformation. Cell. 1991;67:1195–1201. - PubMed
- Bazzoni G, Shib DT, Buck CA, Hemler ME. Monoclonal antibody 9EG7 defines a novel β1integrin epitope induced by soluble ligand and manganese, but inhibited by calcium. J Biol Chem. 1995;270:25570–25577. - PubMed