Skip to main content
. 2003 Feb 17;160(4):487–493. doi: 10.1083/jcb.200209105

Figure 4.

Figure 4.

Stimulation of the β2-AR with isoproterenol induces cell adhesion. (A) Isoproterenol induces adhesion to fibronectin. Ovcar3 cells transiently transfected with CMV- luciferase plasmid were treated with increasing concentrations of the β2-AR agonist isoproterenol, and cells adhering to fibronectin (2 μg/ml) were quantified as described in Materials and methods. (B) Isoproterenol induces activation of Rap1 and CREB. (Top) Ovcar3 cells were treated with increasing concentrations of isoproterenol for 5 min. Cells were lysed, and equal amounts of cell lysate were analyzed for activation of Rap1 (top) and CREB (bottom). Total levels of Rap1 in cell lysates are shown (middle blot). (Bottom) Cells were treated with 10 μM of isoproterenol for the indicated times. Cells were lysed, and equal amounts of cell lysate were analyzed for activation of Rap1 (top blot) and CREB (bottom blot). Total levels of Rap1 in cell lysates are shown (middle blot). (C) Isoproterenol-induced adhesion to fibronectin is independent of PKA. (Top) Ovcar3 cells were pretreated with H-89 as described in the legend to Fig. 1 C and seeded onto wells in the absence or presence of isoproterenol (100 μM). Cells were allowed to adhere for 1 h, and nonadherent cells were removed. The percentage of adherent cells was quantified and plotted relative to unstimulated cells (range from 2–10%). The plot shown is representative of two (long pretreatment) and four (short pretreatment) experiments, each in triplicate. Error bars represent SD. (Bottom) Cells were pretreated with either DMSO or H-89 for 30 min before trypsinization and during the recovery period (DMSO and long H-89 treatment, respectively) or during the last 30 min of recovery (short H-89 treatment). Then cells were stimulated with either 50 μM 8CPT-2Me-cAMP for 10 min or isoproterenol for 2 min, respectively. Cells were centrifuged, cell pellets were lysed, and equal amounts of cell lysate were incubated with precoupled GST-RalGDS-RBD, and activation of Rap1 was analyzed on Western blot using a Rap1 antibody. (D) Isoproterenol-induced adhesion to fibronectin is inhibited by Rap1GAPII. Ovcar3 cells were transfected with either mock DNA (Vector) or HA-Rap1GapII alone or in combination with a β2-AR expression vector where indicated. Adhesion of cells to fibronectin in the absence or presence of isoproterenol was quantified. The percentage of adherent cells was plotted relative to unstimulated, mock-transfected cells (range 5–15%). Summarizing data of four (for the left half of the plot) and two (for the right half of the plot) independent experiments performed in triplicate are shown with error bars representing SD.