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

Islam, A. F. M. T. and Yue, Haicen and Scavello, Margarethakay and Haldeman, Pearce and Rappel, Wouter-Jan and Charest, Pascale G. (2018) The cAMP-induced G protein subunits dissociation monitored in live Dictyostelium cells by BRET reveals two activation rates, a negative effect of caffeine and potential role of microtubules. Cellular Signalling, 48 . pp. 25-37. ISSN 0898-6568. http://resolver.caltech.edu/CaltechAUTHORS:20180426-102225178

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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.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1016/j.cellsig.2018.04.005DOIArticle
https://www.sciencedirect.com/science/article/pii/S0898656818300913PublisherArticle
Additional Information:© 2018 Elsevier Inc. Received 5 February 2018, Revised 17 April 2018, Accepted 22 April 2018, Available online 24 April 2018.
Subject Keywords:Dictyostelium; Chemoattractant; Chemotaxis; cAMP; GPCR; Heterotrimeric G protein; Bioluminescence resonance energy transfer
Record Number:CaltechAUTHORS:20180426-102225178
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:20180426-102225178
Official Citation:A.F.M. Tariqul Islam, Haicen Yue, Margarethakay Scavello, Pearce Haldeman, Wouter-Jan Rappel, Pascale G. Charest, The cAMP-induced G protein subunits dissociation monitored in live Dictyostelium cells by BRET reveals two activation rates, a negative effect of caffeine and potential role of microtubules, Cellular Signalling, Volume 48, 2018, Pages 25-37, ISSN 0898-6568, https://doi.org/10.1016/j.cellsig.2018.04.005. (http://www.sciencedirect.com/science/article/pii/S0898656818300913)
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:86058
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:26 Apr 2018 17:33
Last Modified:03 May 2018 22:58

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