Skip to main content
. 2021 Mar 31;10:e65620. doi: 10.7554/eLife.65620

Figure 2. Non-receptor guanine nucleotide exchange factors (GEFs) display different abilities to promote Gα-GTP and/or free Gβγ formation.

HEK293T cells expressing the components of the bioluminescence resonance energy transfer (BRET) biosensor for free Gβγ (top) or Gαi-GTP (bottom), the membrane-anchored FRB construct, and the indicated FKBP-fused G-protein regulators GIV GBA, AGS1*, Ric-8A*, or R12 GL were stimulated with rapamycin (0.5 μM) at the indicated time during kinetic BRET measurements. Stimulation of ectopically expressed M4 muscarinic receptor (M4R) with carbachol (100 μM) was done as a reference condition, and rapamycin stimulation of cells not expressing FKBP-fused constructs was done as a negative control. Bar graphs on the right summarize the BRET changes 90 s after addition of rapamycin or carbachol. Mean ± SD, n = 3–4. In the kinetic traces, the SD is displayed as bars of lighter color tone than data points and only in the positive direction for clarity.

Figure 2—source data 1. Numerical data used for the upper panel (free Gβγ biosensor).
elife-65620-fig2-data1.xlsx (243.6KB, xlsx)
Figure 2—source data 2. Numerical data used for the lower panel (Gαi-GTP biosensor).
elife-65620-fig2-data2.xlsx (228.8KB, xlsx)

Figure 2.

Figure 2—figure supplement 1. G-protein activation by a GPCR or by AGS1*, but not by GIV GBA, is inhibited by pertussis toxin (PTX).

Figure 2—figure supplement 1.

HEK293T cells expressing the components of the BRET biosensor for free Gβγ, M4R, the membrane-anchored FRB construct, and FKBP-GIV GBA, or FKBP-AGS1* were pretreated (red) or not (black) with PTX. Cells were stimulated with carbachol (100 μM) or rapamycin (0.5 μM) during kinetic BRET measurements as indicated. Mean ± SD, n = 4. SD is displayed as bars of lighter color tone than data points and only in the positive direction for clarity.
Figure 2—figure supplement 1—source data 1. Numerical data used for the graphs.
Figure 2—figure supplement 2. GIV-CT has the same G-protein activating properties as GIV GBA motif.

Figure 2—figure supplement 2.

HEK293T cells expressing the components of the BRET biosensor for free Gβγ (top) or Gαi-GTP (bottom), the membrane-anchored FRB construct, and FKBP-fused GIV GBA or GIV-CT were stimulated with rapamycin (0.5 μM) at the indicated time during kinetic BRET measurements. Stimulation of ectopically expressed M4R with carbachol (100 μM) was done as a reference condition, and rapamycin stimulation of cells not expressing FKBP-fused constructs was done as a negative control. Mean ± SD, n = 3–4. SD is displayed as bars of lighter color tone than data points and only in the positive direction for clarity. Representative immunoblots confirming the expression of similar amounts of FKBP-fused GIV GBA and GIV-CT and different G-proteins are shown on the right.
Figure 2—figure supplement 2—source data 1. Numerical data used for the upper panel (free Gβγ biosensor).
Figure 2—figure supplement 2—source data 2. Numerical data used for the lower panel (Gαi-GTP biosensor).