Expression and function of chicken integrin beta 1 subunit and its cytoplasmic domain mutants in mouse NIH 3T3 cells.

Abstract

Abstract. Chicken integrin B1 eDNA and its sitedirected mutants were cloned into a mammalian expression vector and introduced into mouse NIH 3T3 cells. Stable transfectants expressing the chicken/31 subunit or its site-directed mutants were identified by immunostaining with antibodies specific for the chicken integrin/31 subunit. The chicken/~1 proteins were expressed predominately in the endoplasmic reticulum of transfectants and to a lesser degree in the plasma membrane. Immunoblots and immunoprecipitations, using anti-chicken integrin antibodies, revealed three different sizes of the chicken subunit (90, 95, and 120 kD) and a mouse 140-kD cz subunit. Immunoprecipitations of the cell surface receptors showed only two peptides, an 120-kD/31 and an 140-kD ot subunit. Antibodies perturbing mouse and chicken integrin-specific cell adhesions were used to demonstrate that the chimeric receptors functioned in adhesion to both laminin and fibronectin. Immunofluorescent staining with antibodies specific for either the chicken or mouse receptors showed that both the wild type and the chimeric receptors localized in focal contacts. Several mutations in the cytoplasmic domain were synthesized and used in the transfection experiments. In one mutant the tyrosine (Tyr 788) in the consensus sequence for phosphorylation was replaced by a phenylalanine. In another the lysine (Lys 757) at the end of the membrane spanning region was replaced by a leucine. Both of these mutants formed dimers with mouse ~ subunits, participated in adhesion, localized in focal contacts, and displayed biological properties indistinguishable from the wild-type transfection. In contrast, mutants containing deletions greater than 5-15 amino acids nearest the carboxyl end in the cytoplasmic domain neither promoted adhesion nor localized in focal contacts. They did, however, form heterodimers that were expressed on the cell surface.

Polarized expression of integrin β1 in diencephalic roof plate during chick development, a possible receptor for SCO‐spondin

“…1B, lane 2), coinciding with previous reports on chick SCO extracts (Didier et al, 1995;Cifuentes et al, 1996). Bands with expected Mr were also obtained with the antibodies against integrin ␣6 (160 kDa; de Cur- tis et al, 1991) and integrin ␤1 (a 120 and 95 kDa doublet; Hayashi et al, 1990).…”

The Protein-tyrosine Phosphatase SHP-2 Associates with Tyrosine-phosphorylated Adhesion Molecule PECAM-1 (CD31)

“…The cytoplasmic domain of adhesion molecules can control their function (52)(53)(54)(55)(56)(57). RBL-2H3 cells bind to fibronectin through integrin receptors resulting in cell spreading and a redistribution of the granules to the periphery of the cells (30,58).…”

Optimised retroviral infection of human epidermal keratinocytes: long-term expression of transduced integrin gene following grafting on to SCID mice

“…Transfer to Immobilon PVDF membrane (Millipore, Harlow, UK) was performed at 200 mA for 1 h using a semidry Milliblot graphite electroblotter I system (Millipore). Membranes were blocked with 5% skimmed milk powder (Marvel, Premier Brands UK, Stafford, UK), 1% bovine serum albumin in PBS/0.05% Tween-20 (PBS/Tween) overnight at 4°C and incubated with the W1B10 antichick ␤ 1 integrin antibody 37 (50 g/ml) diluted in 5% skimmed milk in PBS/Tween for 1 h. After four washes in PBS/Tween, the membranes were incubated for 1 h with horseradish peroxidase-conjugated anti-mouse IgG (DAKO, High Wycombe, UK) diluted in PBS/Tween. The membranes were washed in PBS/Tween and immunoreactive proteins were visualised by chemiluminescence (ECL, Amersham International, Amersham, UK).…”