Persistent cAMP-Signals Triggered by Internalized G-Protein–Coupled Receptors
Figure 14
Cell fractionation experiments.
The plasma membrane and the intracellular fractions of FRTL5 cells were obtained by separation with concanavalin A-coated magnetic beads. (A) Western blot analysis of subcellular markers in the obtained fractions. The following markers were used: Na+/K+ATPase for the plasma membrane, the early endosome antigen 1 (EEA1) for early endosomes, and Golgi 58K for the Golgi complex. 1, total homogenate. 2, first eluate from the magnetic beads, corresponding to the plasma membrane fraction. 3, postnuclear supernatant. 4, second eluate from the magnetic beads. 5, intracellular fraction. (B) Western blot for Gαs and adenylyl cyclase III (AC III) in the same fractions as in (A). (C) Effect of TSH stimulation on adenylyl cyclase activity in the subcellular fractions. FRTL5 cells were starved for 24 h in medium without TSH and either stimulated with 30 U/l TSH for 30 min or mock stimulated (control), followed by cell fractionation with concanavalin A-coated magnetic beads. The adenylyl cyclase activity in the plasma membrane and intracellular fractions was then determined in the absence of stimuli (−) or in the presence of either 30 U/l TSH (+TSH) or 10 µM forskolin. The results were normalized for the maximal adenylyl cyclase activity measured in the presence of forskolin. Shown are the data from three independent experiments. Error bars indicate SEM.