The cAMP-induced G protein subunits dissociation monitored in live Dictyostelium cells by BRET reveals two activation rates, a positive effect of caffeine and potential role of microtubules

摘要

To study the dynamics and mechanisms controlling activation of the heterotrimeric G protein G alpha 2 beta gamma in Dictyostelium in response to stimulation by the chemoattractant cyclic AMP (cAMP), we monitored the G protein subunit interaction in live cells using bioluminescence resonance energy transfer (BRET). We found that CAMP induces the cAR1-mediated dissociation of the G protein subunits to a similar extent in both undifferentiated and differentiated cells, suggesting that only a small number of cAR1 (as expressed in undifferentiated cells) is necessary to induce the full activation of G alpha 2 beta gamma. In addition, we found that treating cells with caffeine increases the potency of cAMP-induced G alpha 2 beta gamma activation; and that disrupting the microtubule network but not F-actin inhibits the cAMP-induced dissociation of G alpha 2 beta gamma. Thus, microtubules are necessary for efficient cAR1-mediated activation of the heterotrimeric G protein. Finally, kinetics analyses of G alpha 2 beta gamma subunit dissociation induced by different cAMP concentrations indicate that there are two distinct rates at which the heterotrimeric G protein subunits dissociate when cells are stimulated with cAMP concentrations above 500 nM versus only one rate at lower cAMP concentrations. Quantitative modeling suggests that the kinetics profile of G alpha 2 beta gamma subunit dissociation results from the presence of both uncoupled and G protein pre-coupled cAR1 that have differential affinities for CAMP and, consequently, induce G protein subunit dissociation through different rates. We suggest that these different signaling kinetic profiles may play an important role in initial chemoattractant gradient sensing.