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

Abstract

To study the dynamics and mechanisms controlling activation of the heterotrimeric G protein Gα2βγ 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 Gα2βγ. In addition, we found that treating cells with caffeine increases the potency of cAMP-induced Gα2βγ activation; and that disrupting the microtubule network but not F-actin inhibits the cAMP-induced dissociation of Gα2βγ. Thus, microtubules are necessary for efficient cAR1-mediated activation of the heterotrimeric G protein. Finally, kinetics analyses of Gα2βγ 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 Gα2βγ 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.

Caffeine inhibits PI3K and mTORC2 in Dictyostelium and differentially affects multiple other cAMP chemoattractant signaling effectors

“…In addition to the observations presented herein, in a previous study, we showed that caffeine also affects the cAR1-mediated heterotrimeric G protein activation, specifically increasing the potency of the cAMP-induced G protein subunit dissociation [47]. We don't think that the effect of caffeine on G protein subunit dissociation plays a role in the other effects of caffeine reported therein, since all the experiments were performed using saturating concentrations of the cAMP stimulus (1 μM) and that, at this concentration, caffeine displays no significant effect on G protein subunit dissociation.…”

Chemotaxis in uncertain environments: Hedging bets with multiple receptor types

“…Our work so far has not distinguished between truly different receptors and receptors that are phosphorylated or otherwise modified to change their K D [31,32], which play a role in adaptation to different signal levels in bacteria [33]. If receptor modification is fast compared to the environment's change in concentration c 0 , i.e., can occur before the cell samples a new concentration from P(c 0 ), hedging will be less effective.…”

An integrated, cross-regulation pathway model involving activating/adaptive and feed-forward/feed-back loops for directed oscillatory cAMP signal-relay/response during the development of Dictyostelium

“…During early development of Dictyostelium , secreted cAMP serves as a chemoattractant to organize cells at centers of aggregation ( Meima and Schaap, 1999 ; Jaiswal et al, 2012a ; Jaiswal et al, 2012b ; Fukujin et al, 2016 ; Scavello et al, 2017 ; Singer et al, 2019 ), The receptors for cAMP (CAR1) are surface chemoattractant GPCRs, coupled to Gα2 and Gβγ subunits, which transduce the extracellular signal for directed cell movement ( Janetopoulos et al, 2001 ; Xu et al, 2010 ; Tariqul Islam et al, 2018 ; Adhikari et al, 2021 ). In addition, CAR1 orchestrates a periodic activation/de-activation/re-activation pathway for cAMP generation and clearing, which relays the cAMP signal ( Louis et al, 1993 ; Insall et al, 1994b ; Hereld et al, 1994 ; Thomason et al, 1999 ; McMains et al, 2008 ; Kriebel and Parent, 2009 ; Xu et al, 2010 ; Brzostowski et al, 2013 ; Cao et al, 2014 ).…”

“…Many elements of the first phase have been described (see Figure 1 ). Activation of the G protein coupled receptor CAR1 induces the dissociation of the bound Gα2 and βγ subunits ( Janetopoulos et al, 2001 ; Xu et al, 2010 ; Tariqul Islam et al, 2018 ; Adhikari et al, 2021 ). Released βγ subunits stimulate Ras, mTORC2, and other pathways, which are collectively required to activate ACA ( Insall et al, 1994a ; Lee et al, 2005 ; McMains et al, 2008 ; Cai et al, 2010 ; Liao et al, 2010 ; Liao et al, 2013 ; Jaiswal et al, 2019 ).…”