CaM is required for thrombin-induced activation of Rac1 in CHRF-288-11 cells and EGF-induced activation of Rac1 in HeLa cells.

<p>(A) CHRF-288-11 cells or (B) HeLa cells were serum starved for 12 h and incubated with W7 (150 μM) for 10 min followed by addition of thrombin to CHRF-288-11 cells and EGF to HeLa cells for 1 min or 3 min. At the end of the incubation cells were lysed using RIPA buffer. After centrifugation, 60 µl of the supernatant was suspended in 20 µl 4X Laemmli's sample buffer to determine level of endogenous Rac1 in various samples by western blotting. The rest of the supernatant was incubated with GST-PAK1 for 2 h at 4°C. After incubation, the beads were washed three times with Rac1 washing buffer. The final bead pellet was suspended in 30 µl of Laemmli's sample buffer and heated at 100°C for 5 min. Western blotting was performed using mouse anti-Rac1 antibody. Quantification (adjusted for endogenous level of Rac1) was carried out using Bio-Rad “quantity one” program and *p<0.05 were considered significantly different. #p<0.05 was considered significantly different compared with corresponding thrombin or EGF treatment. The experiments were repeated a minimum of three times. In part (C), an equal amount of lysate (500 µg) from HeLa cells transiently expressing wild type HA-Rac1 and stimulated for various times with EGF was incubated for 2 hrs at 4°C with anti-HA antibody coupled to agarose beads. At the end of incubation beads were washed and bound proteins analyzed using SDS-PAGE and western blotting using anti-CaM antibody. Quantification was carried out using Bio-Rad “quantity one” program and *p<0.05 were considered significantly different when compared to EGF at 0 min.</p

Predicted model for the interaction between various forms of Rac1 and CaM.

<p>The Apo-CaM (PDB id: 1CFD) was docked with Rac1 151–164 amino acids of Rac1 WT (PDB id: 1FOE), Rac1 K153A, Rac1 R163A, and K153A/R163A using ZDock server (<a href="http://zdock.bu.edu/" target="_blank">http://zdock.bu.edu/</a>). The docking calculations were carried out using Fast fourier transform based protein docking method using ZDock. All possible binding modes in the translational and rotational space between two proteins were searched and each was evaluated by an energy scoring function. The poses with the best energy scores were chosen for further analysis. The models were visualized using PyMol (<a href="http://www.pymol.org/" target="_blank">http://www.pymol.org/</a>). CaM is shown in green and Rac1 is in red. The figure above represents interaction between: (A) WT Rac1 and CaM; (B) Rac1 K153A and CaM; (C) Rac1 R163A and CaM and (D) Rac1 K153A/R163A and CaM.</p

Binding of pure CaM to Rac1(K153A), Rac1(R163A), and Rac1(K153A/R163A).

<p>Equal amount (20 µg) of wild type (WT) GST-Rac1 and different GST-Rac1 mutants were incubated with purified CaM (20 µg) in MOPS buffer and allowed to shake for 2 h at 4°C. GST beads were used as negative control. The incubation conditions were beads containing WT GST-Rac1 or different GST-Rac1 mutants with buffer alone, buffer plus 5 mM Ca²⁺ or buffer plus 10 mM EGTA. At the end of the incubation, beads were washed three times and bound proteins were eluted using Laemmli's sample buffer. Western blot analysis was carried out using anti-CaM antibodies. Quantification was carried out using Bio-Rad Quantity one program and *p<0.05 values were considered significantly different (n = 3). In the figure above double refers to Rac1(K153A/R163A).</p

Characterization and Functional Analysis of the Calmodulin-Binding Domain of Rac1 GTPase

<div><p>Rac1, a member of the Rho family of small GTPases, has been shown to promote formation of lamellipodia at the leading edge of motile cells and affect cell migration. We previously demonstrated that calmodulin can bind to a region in the C-terminal of Rac1 and that this interaction is important in the activation of platelet Rac1. Now, we have analyzed amino acid residue(s) in the Rac1-calmodulin binding domain that are essential for the interaction and assessed their functional contribution in Rac1 activation. The results demonstrated that region 151–164 in Rac1 is essential for calmodulin binding. Within the 151–164 region, positively-charged amino acids K153 and R163 were mutated to alanine to study impact on calmodulin binding. Mutant form of Rac1 (K153A) demonstrated significantly reduced binding to calmodulin while the double mutant K153A/R163A demonstrated complete lack of binding to calmodulin. Thrombin or EGF resulted in activation of Rac1 in CHRF-288-11 or HeLa cells respectively and W7 inhibited this activation. Immunoprecipitation studies demonstrated that higher amount of CaM was associated with Rac1 during EGF dependent activation. In cells expressing mutant forms of Rac1 (K153A or K153A/R163A), activation induced by EGF was significantly decreased in comparison to wild type or the R163A forms of Rac1. The lack of Rac1 activation in mutant forms was not due to an inability of GDP-GTP exchange or a change in subcelllular distribution. Moreover, Rac1 activation was decreased in cells where endogenous level of calmodulin was reduced using shRNA knockdown and increased in cells where calmodulin was overexpressed. Docking analysis and modeling demonstrated that K153 in Rac1 interacts with Q41 in calmodulin. These results suggest an important role for calmodulin in the activation of Rac1 and thus, in cytoskeleton reorganization and cell migration.</p> </div

Effect of CaM over-expression or down-regulation on activation of Rac1.

<p>(A) Control HeLa cells and HeLa cells transiently over-expressing HA-CaM were serum starved for 12 h and stimulated with EGF for 3 min and analyzed for level of Rac1-GTP using GST-PAK1 pull-down assay. The total lysates were subjected to SDS-PAGE and immunoblotting performed using anti-Rac1 antibody. Anti-HA antibody was used to detect HA-CaM expression. β-actin antibody was used to establish equal loading of protein in different samples. Data presented is a representative immunoblot of at least three independent experiments. Quantification was carried out using Bio-Rad “quantity one” program and *p<0.05 were considered significantly different. #p<0.05 was considered significantly different compared with corresponding non-treatment samples. (B) HeLa cells were stably co-transfected with shRNAs targeting human CaM2 and human CaM3 or non-target shRNA as a negative control. HeLa cells were serum starved for 12 h and stimulated with EGF for 3 min. The level of Rac1-GTP was assessed using GST-PAK1 pull down assay. The total lysates were subjected to SDS-PAGE and immunoblotting using anti-Rac1 antibody or anti-CaM antibody or β-actin antibody. Data presented is one representative immunoblot of at least three independent experiments. Quantification was carried out using Bio-Rad “quantity one” program and *p<0.05 were considered significantly different. #p<0.05 was considered significantly different compared with corresponding non-treatment sample.</p

Activation of HA-Rac1 mutants is induced by EGF in HeLa cells.

<p>(A) Various HA-Rac1 mutants were transiently expressed in HeLa cells. 48 h post transfection cells were serum starved for 12 h and stimulated for 3 min with EGF and lysed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042975#s2" target="_blank">Materials & Methods</a>. After centrifugation, the supernatant was incubated with GST-PAK1 for 2 h at 4°C and the beads were washed three times with washing buffer. The final bead pellet was suspended in 30 µl of Laemmli's sample buffer and heated at 100°C for 5 min. Western blotting was performed using mouse anti-HA antibody. Data presented is a representative immunoblot of at least three independent experiments. Quantification was carried out using Bio-Rad “quantity one” program and key * p<0.05 were considered significantly different. (B) GTP loading of WT Rac1 and mutants of Rac1 was tested by the GST-PAK1 pull-down assay. HeLa cells expressing various forms of HA-Rac1 were lysed in buffer as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042975#s2" target="_blank">Materials & Methods</a>. After centrifugation, guanine nucleotides (100 µM GTPγS or 100 µM GDPβS) plus 10 mM EGTA (final concn.) were added to the supernatant and the mixture was incubated at 30°C for 15 min. At the end of the incubation, magnesium chloride (MgCl₂) was added to a final concentration of 60 mM to lock in nucleotides. The mixture was incubated with 100 µl GST-PAK1 beads for 2 h at 4°C. Unbound proteins were removed by washing three times with binding buffer. 30 µl of Laemmli's sample buffer was added to beads and heated at 100°C for 5 min. Western blotting was performed using mouse anti-HA antibody. In the figure above double refers to the Rac1 (K153A/R163A) mutant.</p

Effect of deletion of Rac1 putative CaM binding domain on interaction with purified bovine brain CaM.

<p>Equal amount (20 µg) of wild type (WT) GST-Rac1 and GST-Rac1 mutant (amino acids 151 to 164 deleted) were incubated with purified CaM (20 µg) in MOPS buffer and allowed to shake for 2 h at 4°C. GST beads were used as negative control. The incubation conditions were WT GST-Rac1 or GST-Rac1 mutant beads with buffer alone, buffer plus 5 mM Ca²⁺ or buffer plus 10 mM EGTA. At the end of the incubation, beads were washed three times and bound proteins were eluted using Laemmli's sample buffer. Western blot analysis was carried out using anti-CaM antibodies. A representative autoradiograph and quantitation is shown above. The experiment was repeated a minimum of three times.</p

Effect of TP ligands on intracellular Ca²⁺ signaling.

<p>Concentration dependent changes in calcium mobilization of cells expressing WT-TP (A) and A160T (B) after application of different ligands. Ca<b>²⁺</b> levels were measured as described in materials and methods. Results are presented as % RFU of the maximal response obtained with after stimulation with 10 µM of TP agonist U46619. Data are represented as mean± SD and are from at least three independent experiments done in duplicate.</p

Effect of TP antagonists “In Platelet” Functional Analysis.

<p>The bar plot represents FACS analysis of P selectin (CD62P) on the surface PLPs liberated from cultured Meg-01 cells. Activity or response under basal conditions were measured. A160T showed a considerable higher basal activity compared to that of WT-TP, which was decreased by addition of each of 1 µM SQ 29,548 or Ramatroban. A one way ANOVA with <i>tukey's post hoc test</i> between WT-TP and SQ 29,548 as well as A160T pretreated with different compounds SQ 29548, and Ramatroban showed a significant decrease in basal activity at p<0.05 and p<0.01 respectively. The results are from a minimum of 3 independent experiments and are represented as Mean ± SD.</p

Inverse Agonism of SQ 29,548 and Ramatroban on Thromboxane A2 Receptor

<div><p>G protein-coupled receptors (GPCRs) show some level of basal activity even in the absence of an agonist, a phenomenon referred to as constitutive activity. Such constitutive activity in GPCRs is known to have important pathophysiological roles in human disease. The thromboxane A2 receptor (TP) is a GPCR that promotes thrombosis in response to binding of the prostanoid, thromboxane A2. TP dysfunction is widely implicated in pathophysiological conditions such as bleeding disorders, hypertension and cardiovascular disease. Recently, we reported the characterization of a few constitutively active mutants (CAMs) in TP, including a genetic variant A160T. Using these CAMs as reporters, we now test the inverse agonist properties of known antagonists of TP, SQ 29,548, Ramatroban, L-670596 and Diclofenac, in HEK293T cells. Interestingly, SQ 29,548 reduced the basal activity of both, WT-TP and the CAMs while Ramatroban was able to reduce the basal activity of only the CAMs. Diclofenac and L-670596 showed no statistically significant reduction in basal activity of WT-TP or CAMs. To investigate the role of these compounds on human platelet function, we tested their effects on human megakaryocyte based system for platelet activation. Both SQ 29,548 and Ramatroban reduced the platelet hyperactivity of the A160T genetic variant. Taken together, our results suggest that SQ 29,548 and Ramatroban are inverse agonists for TP, whereas, L-670596 and Diclofenac are neutral antagonists. Our findings have important therapeutic applications in the treatment of TP mediated pathophysiological conditions.</p></div