|Divalent cations regulate the folding and activation status of integrins during their intracellular trafficking. |
Tiwari, Shweta, et al.
J. Cell. Sci., 124: 1672-80 (2011)
Integrins are divalent cation-dependent, αβ heterodimeric adhesion receptors that control many fundamental aspects of cell behaviour by bi-directional signalling between the extracellular matrix and intracellular cytoskeleton. The activation state of cell surface integrins is tightly regulated by divalent cation occupancy of the ligand-binding pocket and by interaction with cytoplasmic adaptor proteins, such as talin. These agents elicit gross conformational changes across the entire molecule, which specify the activation state. Much less is known about the activation state of newly synthesised integrins or the role of cations during the early folding and trafficking of integrins. Here we use a number of well-characterised, conformation-specific antibodies to demonstrate that β1-integrins adopt the bent, inactive conformation after assembly with α-integrins in the endoplasmic reticulum. Folding and assembly are totally dependent on the binding of Ca(2+) ions. In addition, Ca(2+) binding prevents integrin activation before its arrival at the cell surface. Activation at the cell surface occurs only following displacement of Ca(2+) with Mg(2+) or Mn(2+). These results demonstrate the essential roles played by divalent cations to facilitate folding of the β-integrin subunit, to prevent inappropriate intracellular integrin signalling, and to activate ligand binding and signalling at the cell surface.
|Integrin activation in bovine placentomes and in caruncular epithelial cells isolated from pregnant cows. |
Bridger, PS; Haupt, S; Leiser, R; Johnson, GA; Burghardt, RC; Tinneberg, HR; Pfarrer, C
Biology of reproduction
In the bovine synepitheliochorial placenta, restricted trophoblast invasion requires complex interactions of integrin receptors with proteins of the extracellular matrix (ECM) and integrin receptors of neighboring cells. Activated integrins assemble to focal adhesions and are linked to the actin cytoskeleton via signaling molecules including alpha-actinin (ACTN), focal adhesion kinase (PTK2 or FAK), phosphotyrosine, and talin (TLN1). Aims of this study were to assess integrin activation and focal adhesion assembly within epithelial cells of bovine placentomes and low-passage (not transformed) placentomal caruncular epithelial cells cultured on dishes coated with ECM proteins. Immunofluorescence analysis was performed to colocalize the signaling molecules ACTN, PTK2, phosphotyrosine, and TLN1 with each other and with beta(1)-integrin (ITGB1) in placentomal cryosections throughout pregnancy and in caruncular epithelial cells in vitro. Antibody specificity was confirmed by Western blot. Cells were cultured on uncoated dishes, and the dishes were coated with fibronectin (FN), laminin (LAMA), and collagen type IV (COL4), thereby statistically assessing cell number and qualitatively assessing the expression pattern of ITGB1, phosphotyrosine, and TLN1. Results demonstrated integrin activation and focal adhesion assembly in the placentome and that low-passage caruncular epithelial cells maintain integrin-associated properties observed in vivo. Expression and/or colocalization of signaling molecules with ITGB1 confirmed, for the first time, integrin activation and participation in "outside-in" and "inside-out" signaling pathways. The prominent role of ECM, and FN in particular, in integrin signaling is supported by the in vitro enhancement of proliferation and focal adhesion expression. Thus, this in vitro model provides excellent potential for further mechanistic studies designed to elucidate feto-maternal interactions in the bovine placentome.
|Evidence that monoclonal antibodies directed against the integrin beta subunit plexin/semaphorin/integrin domain stimulate function by inducing receptor extension. |
Mould, A Paul, et al.
J. Biol. Chem., 280: 4238-46 (2005)
The overall structure of integrins is that of a ligand-binding head connected to two long legs. The legs can exhibit a pronounced bend at the "knees," and it has been proposed that the legs undergo a dramatic straightening when integrins transit from a low affinity to a high affinity state. The knee region contains domains from both alpha and beta subunits, including the N-terminal plexin/semaphorin/integrin (PSI) domain of the beta subunit. The role played by the knee domains in the regulation of integrin-ligand binding is uncertain. Here we show that: (i) monoclonal antibodies (mAbs) N29 and 8E3 have epitopes in the beta(1) subunit PSI domain and stimulate ligand binding to alpha(5)beta(1); (ii) N29 and 8E3 cause long range conformational changes that alter the ligand binding activity of the head region; (iii) the stimulatory action of these mAbs is dependent on the calf-1 domain, which forms part of the alpha subunit knee; and (iv) the epitopes of 8E3 and N29 map close to the extreme N terminus of the PSI and are likely to lie on the side of this domain that faces the alpha subunit. Taken together, our data suggest that the binding of these mAbs results in a levering apart of the PSI and calf-1 domains, and thereby causes the alpha and beta subunit knees to separate. Several major inferences can be drawn from our findings. First, the PSI domain appears to form part of an interface with the alpha subunit that normally restrains the integrin in a bent state. Second, the PSI domain is important for the transduction of conformational changes from the knee to head. Third, unbending is likely to provide a general mechanism for control of integrin-ligand recognition.