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Fig. 3. Annexin 2 binds directly to PtdIns(4,5)P₂. (A) Co-sedimentation assays, employing the annexin 2-p11 heterotetramer and phospholipid liposomes. Annexin 2-p11 complex was mixed with brain-extract liposomes containing no (–) or 0.5% (w/w) of the indicated phosphoinositides in the presence of 1 mM Ca²⁺ (+) or in the presence of 1 mM EGTA (–). Liposomes were collected by ultracentrifugation and bound annexin 2-p11 complex was detected by immunoblotting with an antibody specific for the annexin 2 subunit. Experiments were carried out several times and a representative blot is shown. Signal intensities of blots of three independent experiments were quantified by densitometric scanning. Relative intensities are presented as fold-binding (mean value±s.e.m.) over control, i.e. brain-extract liposomes without added phosphoinositides, in the diagramm below the blot. Note the increase in co-pelleted annexin 2 in the case of liposomes containing PtdIns(4,5)P₂ which is seen in the absence of Ca²⁺. (B) Lipid-plate binding-assays. Microtiter wells were coated with the indicated lipids and blocked with BSA. Purified annexin 2-p11 complex was added in the presence of Ca²⁺ or EGTA as indicated; (–) denotes reactions carried out in buffer alone. The amount of bound complex was determined by a colorimetric reaction using annexin 2-specific antibodies and peroxidase-coupled secondary antibodies. In the absence of phosphoinositides (control), a weak background signal was detected. Binding assays were performed at least three times and the bar graphs give the mean value ± s.e.m. calculated from triplicate samples of a representative individual experiment. (C) The annexin 2-p11 complex was dissociated and the binding of the individual subunits to both, immobilized PtdIns(4,5)P₂ and PtdIns(3,4,5)P₃ was investigated with the experimental setup described in (B), using either annexin 2- or p11-specific antibodies. (D) The binding of monomeric annexin 2 to immobilized PtdIns(4,5)P₂ was compared with that of the recombinantly expressed PH domain of human PLC-1. Experiments were carried out using increasing molar ratios of PHD-PLC to annexin 2 and bound annexin 2 was determined as described in (B).

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