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.

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Figures

Figure 1

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

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

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

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

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

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

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

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

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

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

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

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.

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