Figure 4.

Optogenetic signaling control shows Gβγ plays a major role in the recovery of PIP2 hydrolysis in the Gαq-active background.A, GRPR and Gi/o-coupled light-sensing GPCR, blue opsin (bopsin), were activated together by adding bombesin and retinal while exposing cells to 445 nm blue light. Gβγ was abruptly removed from the Gαq_GTP–PLCβ–Gβγ complex at (I) 2-min and (II) 5-min intervals by terminating blue light and thereby ceasing bopsin activation. This caused a significantly faster PIP2 recovery than that during blue light (BL) exposure. The dose–response function was fitted to calculate the Hill slopes (the rate of PIP2 hydrolysis adaptation). III, the whisker box plot shows the calculated Hill slopes (H1, H2, and H3). The error bars represent the SD. B, turning ON and OFF of blue opsin activation exhibited a switch-like PIP2 response dynamics in pre-GRPR–activated HeLa cells. Note: Blue boxes indicate the duration of BL exposure. C, HeLa cells expressing the GRPR, α2AR–CFP, Venus–PH, and mCh-β1 exhibited robust PIP2 hydrolysis and subsequent partial recovery synergistically with Gβγ translocation upon simultaneous activation with 1 mM bombesin and 100 mM NE. The loss of Gβ from the PM and accumulation of Gβ1γ in IMs are indicated, respectively, by white and yellow arrows. The plot shows the interdependency of the dynamics of Gβγ loss from the PM and PIP2 hydrolysis adaptation. D, the correlation between percentage PIP2 hydrolysis adaptation and percentage loss of Gβγ from the PM. The red dashed line is the fitted regression line for the data points (R² = 0.97). The scale bar represents 10 μm. Average curves plotted using n ≥10 cells from ≥3 independent experiments. The error bars represent the SEM. α2AR, α2-adrenergic receptor; GRPRs, gastrin-releasing peptide receptors; IM, internal membrane; NE, norepinephrine; PIP2, phosphatidylinositol 4,5-bisphosphate; PLCβ, phospholipase C β.