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

A. F.M. Tariqul Islam, Haicen Yue, Margarethakay Scavello, Pearce Haldeman, Wouter Jan Rappel, Pascale G Charest

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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.

Original languageEnglish (US)
Pages (from-to)25-37
Number of pages13
JournalCellular Signalling
Volume48
DOIs
StatePublished - Aug 1 2018

Fingerprint

Dictyostelium
Energy Transfer
Protein Subunits
Caffeine
GTP-Binding Proteins
Microtubules
Cyclic AMP
Heterotrimeric GTP-Binding Proteins
Chemotactic Factors
Actins

Keywords

  • Bioluminescence resonance energy transfer
  • cAMP
  • Chemoattractant
  • Chemotaxis
  • Dictyostelium
  • GPCR
  • Heterotrimeric G protein

ASJC Scopus subject areas

  • Cell Biology

Cite this

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. / Tariqul Islam, A. F.M.; Yue, Haicen; Scavello, Margarethakay; Haldeman, Pearce; Rappel, Wouter Jan; Charest, Pascale G.

In: Cellular Signalling, Vol. 48, 01.08.2018, p. 25-37.

Research output: Contribution to journalArticle

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AU - Rappel, Wouter Jan

AU - Charest, Pascale G

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