Figure 2.

Gβγ is alone a weak activator of PLCβ yet a potent stimulator of PLCβ under Gαq_GTPbackground.A, upon addition of 10 μM norepinephrine (Norepi), α2AR expressing HeLa cells failed to show PIP2 hydrolysis but exhibited a profound Gγ9 translocation from PM to IMs. PM (white arrows) and IMs (yellow arrows). The corresponding plot shows the dynamics of mCh–g9 (IMs) and PIP2 sensor, Venus–PH (cytosol) upon NE addition. B, HeLa cells expressing the α2AR and mCh–PH independently exposed to different concentrations of NE also failed to show significant PIP2 hydrolysis. C, HeLa cells expressing the GRPR, α2AR–CFP, and mCh–PH were first exposed to 1 μM bombesin. After hydrolysis and partial recovery of PIP2, the same HeLa cells were exposed to 100 mM NE. The addition of NE induced rehydrolysis followed by the second recovery of PIP2. Note: Black and yellow arrows indicate the level of PIP2 recovery. D, An experiment similar to that in panel C was performed in HeLa cells expressing M3R, in place of the GRPR. When carbachol- and NE-exposed cells were treated with 25 mM atropine, PIP2 recovered completely. E, generation of DAG upon PLCβ activation was examined upon α2AR activation under Gαq_GTP background. F, HeLa cells expressing β1AR–CFP in place of the α2AR showed PIP2 rehydrolysis and recovery after stimulation with 50 mM isoproterenol. The scale bar represents 10 mm. Average curves plotted using n ≥10 cells from ≥3 independent experiments. The error bars represent the SEM. α2AR, α2-adrenergic receptor; DAG, diacylglycerol; GRPRs, gastrin-releasing peptide receptors; IMs, internal membranes; M3R, M3-muscarinic receptor; mCh–PH, mCherry–PH; PIP2, phosphatidylinositol 4,5-bisphosphate; PLCβ, phospholipase C β.