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Signaling efficiency of Gαq through its effectors p63RhoGEF and GEFT depends on their subcellular location.

Goedhart J, van Unen J, Adjobo-Hermans MJ, Gadella TW - Sci Rep (2013)

Bottom Line: These proteins can be activated by the heterotrimeric G-protein subunit Gαq.We show that p63RhoGEF is located at the plasma membrane, whereas GEFT is confined to the cytoplasm.A rapamycin dependent recruitment system was used to dynamically alter the subcellular location and concentration of GEFT, showing efficient signaling through GEFT only upon membrane recruitment.

View Article: PubMed Central - PubMed

Affiliation: Swammerdam Institute for Life Sciences, Section of Molecular Cytology, van Leeuwenhoek Centre for Advanced Microscopy, University of Amsterdam, P.O. Box 94215, NL-1090 GE Amsterdam, The Netherlands. j.goedhart@uva.nl

ABSTRACT
The p63RhoGEF and GEFT proteins are encoded by the same gene and both members of the Dbl family of guanine nucleotide exchange factors. These proteins can be activated by the heterotrimeric G-protein subunit Gαq. We show that p63RhoGEF is located at the plasma membrane, whereas GEFT is confined to the cytoplasm. Live-cell imaging studies yielded quantitative information on diffusion coefficients, association rates and encounter times of GEFT and p63RhoGEF. Calcium signaling was examined as a measure of the signal transmission, revealing more efficient signaling through the membrane-associated p63RhoGEF. A rapamycin dependent recruitment system was used to dynamically alter the subcellular location and concentration of GEFT, showing efficient signaling through GEFT only upon membrane recruitment. Together, our results show efficient signal transmission through membrane located effectors, and highlight a role for increased concentration rather than increased encounter times due to membrane localization in the Gαq mediated pathways to p63RhoGEF and PLCβ.

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Related in: MedlinePlus

The dynamics of the interaction between Gαq and p63RhoGEF.(A) mTurquoise-tagged Gαq was co-expressed with p63RhoGEF or p63RhoGEF-L475A tagged with YFP. (B) FRET ratio imaging reveals a substantial increase in YFP/CFP ratio for wild-type p63RhoGEF (n = 14 cells), but hardly any increase for the L475A mutant (n = 17 cells) upon addition of histamine. The interaction is readily reversed when the H1R antagonist pyrilamine is added. The graph shows the average YFP/CFP ratio change ±s.e.m. Width of the images corresponds to 230 μm.
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f5: The dynamics of the interaction between Gαq and p63RhoGEF.(A) mTurquoise-tagged Gαq was co-expressed with p63RhoGEF or p63RhoGEF-L475A tagged with YFP. (B) FRET ratio imaging reveals a substantial increase in YFP/CFP ratio for wild-type p63RhoGEF (n = 14 cells), but hardly any increase for the L475A mutant (n = 17 cells) upon addition of histamine. The interaction is readily reversed when the H1R antagonist pyrilamine is added. The graph shows the average YFP/CFP ratio change ±s.e.m. Width of the images corresponds to 230 μm.

Mentions: Although co-recruitment and co-localization strongly suggest interaction, it is not direct proof. To study the interaction more directly we made use of Förster Resonance Energy Transfer (FRET) which is highly distance dependent and is therefore suited to monitor proximity of molecules in living cells. To examine whether the interaction between Gαq and p63RhoGEF can be observed in living cells, a Gαq subunit tagged with mTurquoise, CFP-Gαq, and p63RhoGEF tagged with YFP were co-expressed. Both proteins are located at the plasma membrane (figure 5A). In case of interaction, it is expected that the YFP over CFP ratio increases. Upon addition of histamine, a significant increase in the YFP/CFP ratio was observed (figure 5B). When an H1R antagonist, pyrilamine, was added, the ratio decreased and returned to the baseline value. Kinetic analysis of the interaction yielded a kon of 0.23 s−1 and a koff of 0.045 s−1, corresponding to half-times of 3 and 15 seconds, respectively. To examine the specificity of the interaction, the mutant p63(L475A)-YFP was used. We hardly observed any change in YFP/CFP ratio when histamine receptors were activated (fig. 5B), indicating that the interaction between Gαq and the L475A mutant is strongly reduced.


Signaling efficiency of Gαq through its effectors p63RhoGEF and GEFT depends on their subcellular location.

Goedhart J, van Unen J, Adjobo-Hermans MJ, Gadella TW - Sci Rep (2013)

The dynamics of the interaction between Gαq and p63RhoGEF.(A) mTurquoise-tagged Gαq was co-expressed with p63RhoGEF or p63RhoGEF-L475A tagged with YFP. (B) FRET ratio imaging reveals a substantial increase in YFP/CFP ratio for wild-type p63RhoGEF (n = 14 cells), but hardly any increase for the L475A mutant (n = 17 cells) upon addition of histamine. The interaction is readily reversed when the H1R antagonist pyrilamine is added. The graph shows the average YFP/CFP ratio change ±s.e.m. Width of the images corresponds to 230 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3722567&req=5

f5: The dynamics of the interaction between Gαq and p63RhoGEF.(A) mTurquoise-tagged Gαq was co-expressed with p63RhoGEF or p63RhoGEF-L475A tagged with YFP. (B) FRET ratio imaging reveals a substantial increase in YFP/CFP ratio for wild-type p63RhoGEF (n = 14 cells), but hardly any increase for the L475A mutant (n = 17 cells) upon addition of histamine. The interaction is readily reversed when the H1R antagonist pyrilamine is added. The graph shows the average YFP/CFP ratio change ±s.e.m. Width of the images corresponds to 230 μm.
Mentions: Although co-recruitment and co-localization strongly suggest interaction, it is not direct proof. To study the interaction more directly we made use of Förster Resonance Energy Transfer (FRET) which is highly distance dependent and is therefore suited to monitor proximity of molecules in living cells. To examine whether the interaction between Gαq and p63RhoGEF can be observed in living cells, a Gαq subunit tagged with mTurquoise, CFP-Gαq, and p63RhoGEF tagged with YFP were co-expressed. Both proteins are located at the plasma membrane (figure 5A). In case of interaction, it is expected that the YFP over CFP ratio increases. Upon addition of histamine, a significant increase in the YFP/CFP ratio was observed (figure 5B). When an H1R antagonist, pyrilamine, was added, the ratio decreased and returned to the baseline value. Kinetic analysis of the interaction yielded a kon of 0.23 s−1 and a koff of 0.045 s−1, corresponding to half-times of 3 and 15 seconds, respectively. To examine the specificity of the interaction, the mutant p63(L475A)-YFP was used. We hardly observed any change in YFP/CFP ratio when histamine receptors were activated (fig. 5B), indicating that the interaction between Gαq and the L475A mutant is strongly reduced.

Bottom Line: These proteins can be activated by the heterotrimeric G-protein subunit Gαq.We show that p63RhoGEF is located at the plasma membrane, whereas GEFT is confined to the cytoplasm.A rapamycin dependent recruitment system was used to dynamically alter the subcellular location and concentration of GEFT, showing efficient signaling through GEFT only upon membrane recruitment.

View Article: PubMed Central - PubMed

Affiliation: Swammerdam Institute for Life Sciences, Section of Molecular Cytology, van Leeuwenhoek Centre for Advanced Microscopy, University of Amsterdam, P.O. Box 94215, NL-1090 GE Amsterdam, The Netherlands. j.goedhart@uva.nl

ABSTRACT
The p63RhoGEF and GEFT proteins are encoded by the same gene and both members of the Dbl family of guanine nucleotide exchange factors. These proteins can be activated by the heterotrimeric G-protein subunit Gαq. We show that p63RhoGEF is located at the plasma membrane, whereas GEFT is confined to the cytoplasm. Live-cell imaging studies yielded quantitative information on diffusion coefficients, association rates and encounter times of GEFT and p63RhoGEF. Calcium signaling was examined as a measure of the signal transmission, revealing more efficient signaling through the membrane-associated p63RhoGEF. A rapamycin dependent recruitment system was used to dynamically alter the subcellular location and concentration of GEFT, showing efficient signaling through GEFT only upon membrane recruitment. Together, our results show efficient signal transmission through membrane located effectors, and highlight a role for increased concentration rather than increased encounter times due to membrane localization in the Gαq mediated pathways to p63RhoGEF and PLCβ.

Show MeSH
Related in: MedlinePlus