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. 2014 Dec 1;171(24):5650–5664. doi: 10.1111/bph.12868

Figure 3.

Figure 3

Heteromerization of D1 receptors and CRF2α receptors. (A) Co-immunoprecipitation of D1 receptormyc/His and CRF2α receptorFlag from HEK293T cells. Solubilized extracts from cells expressing D1 receptormyc/His (lane 1), CRF2α receptorFlag (lane 2), D1 receptormyc/His plus CRF2α receptorFlag (lane 3) or empty pcDNA 3.1 vector (lane 4) were immunoprecipitated with a rabbit anti-myc polyclonal antibody. Extracts (crude) and immunoprecipitates (IP) were analysed using a mouse anti-Flag and a rabbit anti c-myc antibodies. CRF2α receptorFlag immunoprecipitated with D1 receptormyc/His (IP, lane 3). The presence of D1 receptormyc/His was corroborated (lanes 1 and 3). (B) BRET saturation curve. BRET was measured in HEK293T cells co-expressing CRF2α receptorRluc plus D1 receptorYFP (blue squares), CRF2α receptorRluc plus D2 receptorYFP (red circles) or CRF2α receptorRluc plus GABAB2 receptorYFP (black circles). Co-transfections were performed with increasing amounts of the YFP-tagged vectors while the CRF2α receptorRluc was maintained constant. Plotted on the X-axis is the fluorescence value obtained from the YFP, normalized to the luminescence value of CRF2α receptorRluc 10 min after h-coelenterazine incubation. (C) Determination of the D1 and CRF2α receptor oligomerization by FRET experiments in living cells. D1 receptorYFP and CRF2α receptorCFP were expressed in HEK293T cells, and fluorescence images of CFP and YFP were recorded before (pre) and after (post) the YFP was photobleached by 5 min of exposure to light at 500 nm to corroborate the extent of acceptor photodestruction (left panel). Emission intensities of CRF2α receptorCFP (480 nm, blue) and D1 receptorYFP (535 nm, yellow) from single cells expressing both CRF2α receptorCFP and D1 receptorYFP were recorded before and after YFP photobleaching (right panel) to determine the FRET efficiency. Scale bar: 10 μm. (D) Quantification of the FRET efficiency of different FRET pairs: CFP plus D1 receptorYFP (n = 5), YFP plus CRH2αRCFP (n = 10), CRH2αRCFP (n = 5), D1RYFP plus CRF2α receptorCFP (n = 15) and D2 receptorYFP plus CRF2α receptorCFP (n = 10). The data indicate the mean ± SEM. An asterisk denotes the data that are significantly different from the control FRET pairs (i.e., CFP or YFP co-transfections). *P < 0.001, anova with a Bonferroni multiple comparison post hoc test.