Figure 3.

Involvement of G_αq in inter-receptor signaling. (a) Effect of G-QL IgG on [³H]SQ29,548 binding to solubilized platelet membranes. Platelet membranes were solubilized in CHAPS (15) and incubated with preimmune IgG (PI IgG; 150 μg/ml) or an antibody raised against the C terminus of G_αq (G-QL IgG; 150 μg/ml) in the presence of 1 unit/ml thrombin. [³H]SQ29,548 binding was performed as described (15), and results are expressed as the percentage increase in [³H]SQ29,548-specific binding relative to unstimulated solubilized platelet membranes. All experiments were done in triplicate at least four times, and the values represent means ± SEM of all results. The average specific binding in the absence of agonist pretreatment was 138 ± 15 fmol/mg protein. Statistical analysis measuring the effect of G-QL IgG on thrombin-induced ligand binding was performed by using a two-sample Student’s t test (∗, P < 0.05). Addition of G-QL IgG or PI IgG did not significantly affect baseline [³H]SQ29,548 binding (data not shown). For comparison with thrombin treatment alone see Fig. 2b. (b) Effect of TRAP treatment on G_αq association with TXA₂ receptors. Solubilized platelet membranes were subjected to immunoaffinity chromatography purification (24) in the presence or absence of TRAP (fraction 1 = last wash; fractions 2–6 = elution fractions). Results of one representative experiment are expressed in counts per minute (cpm) of specifically eluted [¹²⁵I]G-QL IgG. (Inset) Average TRAP-induced percentage increase in [¹²⁵I]G-QL IgG counts relative to vehicle (n = 9). TRAP treatment had no effect on the amount of TXA₂ receptor purified (data not shown). Statistical significance was evaluated by using a two-sample Student’s t test (∗, P < 0.05).